Method for detecting herbicide and metabolite residues in livestock and poultry products

Abstract

The invention provides a method for detecting herbicide and metabolite residues in livestock and poultry products, which combines the advantages of high loading capacity, easy operation and high extract purity of rapid filtration type purification with high resolution of UHPLC-Q / Orbitrap HRMS, and takes beef, pork liver and egg as research objects. Extraction and purification conditions of livestock and poultry products and chromatographic conditions and mass spectrometry conditions of an ultra-high performance liquid chromatography-quadrupole rod / electrostatic field orbitrap mass spectrometer are optimized, and a high-flux qualitative and quantitative screening method for 85 herbicide and metabolite residues is established. The method is simple, convenient, rapid, sensitive and stable, can solve the problems of complex matrix and trace pesticide residue of livestock and poultry products, effectively improves the detection efficiency during high-throughput screening of pesticides in the livestock and poultry products, and has high practical application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of residual drug analysis, and particularly relates to a method for detecting herbicide and metabolite residues in poultry and livestock products. BACKGROUND

[0002] Herbicides are a class of substances that can selectively or completely cause weeds to die, inhibit plant growth or eliminate plants. Herbicides are widely used to control weeds in non-cultivated land such as rice, wheat, soybean, corn, rape fields and lawns. Most herbicides are not easily volatile and degrade slowly in the environment, thus persisting in water, crops and soil for a long time, further enriching in livestock and poultry animals through water bodies and livestock and poultry feed, and ultimately endangering human health through the food chain.

[0003] In order to achieve high levels of food safety protection, countries and regions such as the European Union and Japan have established a rigorous pesticide regulatory system. For example, the European Union's 396 / 2005 / EC regulation sets the maximum residue limits for more than 1300 pesticides, and a default limit of 0.01 mg / kg is applied to pesticides for which no limit has been set. Japan's "Positive List System for Residual Pesticides in Food" sets the use limits for more than 700 agricultural chemicals, and the maximum residue limit for agricultural chemicals not on the list is 0.01 mg / kg. China's current national food safety standard GB 2763-2021 sets the maximum residue limits for 564 pesticides, a total of 10092 items, of which only a small part of pesticide residue items have limited quantity provisions for poultry and livestock products. The coverage is much smaller than that of the European Union and Japan, and most of the items only have limit provisions without specifying corresponding detection standards and methods. In order to improve the level of food safety protection, reduce unnecessary economic losses and trade disputes, it is particularly important to establish a sensitive, rapid and reliable high-throughput pesticide residue detection method for poultry and livestock products.

[0004] The drug residues in poultry and livestock products are mostly extracted by homogenization extraction, oscillation extraction, accelerated solvent extraction, ultrasonic extraction and other technologies, and then purified by solid phase extraction, gel permeation chromatography, dispersive solid phase extraction, QuEChERS purification and other technologies. Multi-plug filtration cleanup (m-PFC) is a new type of rapid purification technology formed on the basis of QuEChERs purification, which does not need to be washed and activated, and is filtered by natural gravity or pressing, saving the operation steps such as oscillation and centrifugation. It has the advantages of traditional OuEChERs method, saves the pretreatment time, and has stronger purification capacity and higher sensitivity. It has been widely used in the detection of pesticide residues, organic pollutants and trace components in soil, oil, tea, poultry and livestock products, plant-derived foods and traditional Chinese medicinal materials, and has higher throughput and sensitivity. Ultrahigh performance liquid chromatography-quadrupole / orbitrap high resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) has higher resolution and selectivity than conventional triple quadrupole liquid chromatography-mass spectrometry, and has been widely used in residual analysis of food. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a method for detecting herbicide and metabolite residues in poultry and livestock products by combining rapid filtration purification technology with UHPLC-Q / Orbitrap HRMS, establishing a high-throughput qualitative and quantitative screening method for detecting herbicide and metabolite residues in poultry and livestock products, which can solve the problems of complex matrix and trace pesticide residues in poultry and livestock products.

[0006] To solve the above technical problems, the technical scheme adopted by the present application is: a method for detecting herbicide and metabolite residues in poultry and livestock products, comprising the following steps:

[0007] S1, sample pretreatment

[0008] The poultry and livestock product tissues are crushed and homogenized to obtain a test sample; an extraction solvent is added to the test sample, and after extraction, centrifugation is performed, and the supernatant is collected. The supernatant is purified by rapid filtration solid phase extraction, and the effluent is collected, which is the test solution;

[0009] S2, preparation of standard solution

[0010] Take the herbicide standard solution, dilute it with the extraction solvent to 10 mg / L mixed standard working solution;

[0011] S3, injecting the mixed standard working solution obtained in S2 into an ultra-high performance liquid chromatography-quadrupole / orbitrap high resolution mass spectrometer to make a standard curve; then injecting the sample solution obtained in S1 to calculate the content of herbicides and metabolites in the sample.

[0012] Preferably, the extraction solvent in S1 and S2 is acetonitrile, 0.2% formic acid acetonitrile, 0.5% formic acid acetonitrile, 1% formic acid acetonitrile, 2% formic acid acetonitrile, 4% formic acid acetonitrile, 2% formic acid-90% acetonitrile-water solution, 2% formic acid-80% acetonitrile-water solution or 2% formic acid-70% acetonitrile-water solution.

[0013] Preferably, the ratio of the sample to the extraction solvent in S1 is 2.00g:10mL.

[0014] Preferably, the extraction method in S1 is oscillation extraction or homogenization extraction.

[0015] More preferably, the rotation speed of the homogenization extraction is 13500r / min, and the extraction time is 0.8-1.2min.

[0016] Preferably, the rotation speed of the centrifugation in S1 is 4000r / min, and the centrifugation time is 2-3min.

[0017] Preferably, the extraction column of the rapid filtration type solid phase extraction in S1 is a functionalized polystyrene / divinylbenzene HLB solid phase extraction column.

[0018] Preferably, the column passing speed of the rapid filtration type solid phase extraction in S1 is 1-2 drops per second.

[0019] Preferably, the chromatographic conditions in S3 are as follows: Thermo Accucore aQ C18 chromatographic column, 2.6μm×2.1mm×150mm; flow rate: 0.4mL / min; injection volume: 5μL; mobile phase A: 2mmol / L ammonium acetate water containing 0.1% formic acid, mobile phase B: methanol; gradient elution program: 0-0.4min, 3%B; 0.4-0.7min, 3%-15%B; 0.7-1min, 50%B; 1-6min, 50%-70%B; 6-7min, 70%-97%B; 7-8min, 97%B; 8-8.1min, 97%-3%B; 8.1-10min, 3%B.

[0020] Preferably, the mass spectrometry conditions in S3 are: heating electrospray ion source, positive ion mode; spray voltage is 3500V; ion transmission tube temperature: 350 DEG C; sheath gas is N2, flow rate: 10.5 L / min; auxiliary gas is N2, flow rate: 3 L / min; acquisition mode: parallel reaction monitoring mode; secondary scanning resolution: 17500; secondary mass spectrum collision energy: 20, 40, 60 eV.

[0021] Compared with the prior art, the present application has the following remarkable technical effects:

[0022] 1. The present application provides a method for detecting herbicide and metabolite residues in poultry products, taking beef, pork liver and eggs as research objects, the advantages of rapid filtration type purification high load, easy operation and high purity of extract are combined with high resolution of UHPLC-Q / Orbitrap HRMS, the extraction and purification conditions of poultry products, the chromatographic conditions and mass spectrometry conditions of UHPLC-Q / Orbitrap HRMS are optimized, and a high-throughput qualitative and quantitative screening method for 85 kinds of herbicide and metabolite residues is established. The method is simple, fast, sensitive and stable, effectively improves the detection efficiency of high-throughput screening of pesticides in poultry products, solves the problems of complex poultry product matrix and trace pesticide residues, and promotes the establishment of related detection standards, provides reference basis and technical support for herbicide residue detection in poultry products, and has strong practical application value.

[0023] The present application will be further described in detail below in combination with the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is the influence of acetonitrile and acetic acid acetonitrile as extraction solvent in example 1 of the present application;

[0025] Figure 2 is the influence of acetic acid acetonitrile and acetic acid acetonitrile aqueous solution as extraction solvent in example 1 of the present application;

[0026] Figure 3 is the ion chromatogram of part of representative herbicides in example 1 of the present application. DETAILED DESCRIPTION

[0027] The instruments used in the present application are:

[0028] UHPLC-Q-Exactive ultra-high performance liquid chromatography-quadrupole / electrostatic field orbitrap high-resolution mass spectrometry system (Thermo Scientific, USA); TUP HLB solid-phase extraction column (functionalized polystyrene / divinyl benzene extraction column, 60 mg, 3 mL, Tianjin Chuanxuan Science and Technology Co., Ltd.); QuEChERS purification tube (50 mg PSA+50 mg C18+150 mg MgSO4, Xiamen Luja Polycore Biological Technology Co., Ltd.).

[0029] Reagents:

[0030] Acetonitrile and methanol (chromatographically pure, TEDIA Company, USA), formic acid (chromatographically pure, FISHER Company, USA); 0.2% formic acid acetonitrile refers to acetonitrile containing 0.2% formic acid, 0.5% formic acid acetonitrile refers to acetonitrile containing 0.5% formic acid, 1% formic acid acetonitrile refers to acetonitrile containing 1% formic acid, 2% formic acid acetonitrile refers to acetonitrile containing 2% formic acid, 4% formic acid acetonitrile refers to acetonitrile containing 4% formic acid, 2% formic acid-90% acetonitrile-water solution refers to 90% acetonitrile aqueous solution (containing 2% formic acid), 2% formic acid-80% acetonitrile-water solution refers to 80% acetonitrile aqueous solution (containing 2% formic acid), 2% formic acid-70% acetonitrile-water solution refers to 70% acetonitrile aqueous solution (containing 2% formic acid).

[0031] 85 kinds of herbicides and metabolites (including 80 kinds of herbicides, 2 kinds of flumioxazin metabolites: CSCD686480 and SYN503780, 1 kind of sulfonamide pyrazole metabolite: desmethyl-sulfonamide pyrazole, 2 kinds of clethodim metabolites: clethodim sulfone and clethodim sulfoxide) standard solution, concentration is 1000 mg / L, purchased from Beijing Manhag Biotechnology Co., Ltd., 85 kinds of herbicides and metabolites are respectively: CSCD686480 (flumioxazin metabolite), Flumetsulam (metribuzin), Metamitron (metribuzin), Florasulam (metribuzin), SYN503780 (flumioxazin metabolite), Pyrasulfotole-Desmethyl (desmethyl-sulfonamide pyrazole, sulfonamide pyrazole metabolite), Pyrasulfotole (sulfonamide pyrazole), Cyanazine (metribuzin), Cinosulfuron (metribuzin), Triasulfuron (metribuzin), Thifensulfuron-Methyl (metribuzin), Metribuzin (metribuzin), Metsulfuron-Methyl (metribuzin), Sulfentrazone (metribuzin), Simazine (metribuzin), Tebuthiuron (metribuzin), Hexazinone (metribuzin), Amidosulfuron (metribuzin), Penoxsulam (metribuzin), Simetryn (metribuzin), Mesosulfuron-Methyl (metribuzin), Atrazine (metribuzin), Metazachlor (metribuzin), Chlortoluron (metribuzin), Propachlor (metribuzin), Tribenuron-Methyl (metribuzin), Orthosulfamuron (metribuzin), Clethodim Sulfone (clethodim metabolite), Diclofop-Methyl (metribuzin), Isoproturon (metribuzin), Bicyclopyrone (metribuzin), Ametryn (metribuzin), Phenmedipham (metribuzin), Saflufenacil (metribuzin), Clethodim Sulfoxide (clethodim metabolite),Fluthiacet-methyl (fluthiacet-methyl), Anilofos (saxon), Pyraflufen-ethyl (pyraflufen-ethyl), Oxadiargyl (propanil), Clethodim (clethodim), Dimepiperate (dimepiperate), Pretilachlor (propanil), Diflufenican (diflufenican), Prosulfocarb (prosulfocarb), Fenoxaprop-P-ethyl (fenoxaprop-P-ethyl), Fluazifop (fluazifop), Oxaziclomefone (oxadiazon), Sethoxydim (sethoxydim), Metamifop (metamifop), Lactofen (lactofen), Pyribenzoxim (pyribenzoxim), Oxadiazon (oxadiazon), Butachlor (butachlor), Picolinafen (picolinafen), Tralkoxydim (tralkoxydim), Triallate (triazolopyrimidine), Pendimethalin (pendimethalin), Quizalofop-ethyl (quizalofop-ethyl).

[0032] Example 1

[0033] The embodiment is a method for detecting herbicide and metabolite residues in livestock products, comprising the following steps:

[0034] S1, sample pretreatment

[0035] Beef, pork liver, and eggs were used as research objects. Beef and pork liver samples were ground into a slurry with a knife grinder at 4000 r / min, and eggs were homogenized after being shelled. 2.00 g of the prepared sample was weighed into a 50 mL polypropylene centrifuge tube, 10 mL of 80% acetonitrile aqueous solution (containing 2% formic acid) was added, and homogenization extraction was performed at 13500 r / min for 1 min, followed by centrifugation at 4000 r / min for 2 min. 1.2 mL of the supernatant was absorbed into an HLB solid-phase extraction column with a 0.22 μm organic filter membrane, and the column was squeezed with a syringe push rod at a speed of about 1 drop / s. The effluent was vortexed for 10 s, and the beef, pork liver, and egg matrix solutions were obtained, which were used as the test solution for ultra-high performance liquid chromatography-quadrupole / electrostatic field orbitrap high-resolution mass spectrometry determination.

[0036] S2, preparation of standard solution

[0037] The herbicide standard solution was diluted with 80% acetonitrile aqueous solution (containing 2% formic acid) to prepare a 10 mg / L mixed standard working solution.

[0038] S3, the mixed standard working solution obtained in S2 was injected into the ultra-high performance liquid chromatography-quadrupole / electrostatic field orbitrap high-resolution mass spectrometer to prepare a standard curve. Then, the test solution obtained in S1 was injected to calculate the content of herbicides and metabolites in the test sample.

[0039] The chromatographic conditions were as follows: Thermo Accucore aQ C18 column (2.6 μm x 2.1 mm x 150 mm); flow rate: 0.4 mL / min; injection volume: 5 μL; mobile phase A: 2 mmol / L ammonium acetate water (containing 0.1% formic acid); mobile phase B: methanol; gradient elution program: 0-0.4 min, 3% B; 0.4-0.7 min, 3%-15% B; 0.7-1 min, 50% B; 1-6 min, 50%-70% B; 6-7 min, 70%-97% B; 7-8 min, 97% B; 8-8.1 min, 97%-3% B; 8.1-10 min, 3% B.

[0040] Mass spectrometry conditions: heated electrospray ion source (HESI source), positive ion mode; spray voltage 3500V; ion transfer tube temperature: 350℃; sheath gas (N2): 10.5L / min; auxiliary gas (N2): 3L / min; acquisition mode: parallel reaction monitoring mode (PRM mode); secondary scan resolution: 17500; secondary mass spectrum collision energy is normalized collision energy (NCE): 20, 40, 60eV.

[0041] The present application also studies different extraction methods, extraction solvents, solid-phase extraction columns, chromatography and mass spectrometry conditions, and the results are as follows:

[0042] (1) Extraction method

[0043] The two methods of oscillation extraction and homogenization extraction were investigated. The oscillation extraction cannot effectively extract the pesticides from the inside of the tissue to the extraction solution because the protein denatures quickly and the pesticides are wrapped inside the tissue. The recovery rate of more than 50% of the herbicide residues is less than 40%, and the recovery rate of more than 80% of the herbicide residues is less than 70%. The recovery rate is greatly increased by using the homogenization extraction method, and most of the herbicides can meet the detection requirements. Finally, the homogenization extraction method is selected for extraction.

[0044] (2) Extraction solvent

[0045] Acetonitrile is often used as an extraction solvent for drug residues in animal food because it has a wide solubility and good chromatographic compatibility, and also has good protein precipitation ability, and can avoid excessive extraction of fat. Most herbicides are acidic pesticides, and adding an appropriate amount of formic acid to the extraction solvent can inhibit the dissociation of acidic pesticides and increase the distribution ratio of acidic pesticides in the organic phase, thereby improving the extraction recovery rate. The extraction effects of acetonitrile, 0.2% formic acid acetonitrile, 0.5% formic acid acetonitrile, 1% formic acid acetonitrile, 2% formic acid acetonitrile and 4% formic acid acetonitrile as extraction solvents were compared, and it was found that the average recovery rate of 2% formic acid acetonitrile was the best, and the proportion of recovery rate falling into the interval of 60%~110% and 80~110% was the highest, indicating that the pH value of the extraction solvent has a greater impact on the extraction efficiency. Finally, the extraction solvent is selected as 2% formic acid acetonitrile. Figure 1

[0046] ​Meanwhile, studies have shown that 80% acetonitrile aqueous solution can cause slow and complete denaturation of protein, release drug residues in the matrix, and less than 80% acetonitrile cannot cause complete denaturation of protein, and cannot completely precipitate protein, and more than 80% acetonitrile causes rapid aggregation of protein to wrap drugs, resulting in low extraction efficiency. Therefore, this embodiment compares the extraction efficiency when 2% formic acid acetonitrile, 90% acetonitrile aqueous solution (containing 2% formic acid), 80% acetonitrile aqueous solution (containing 2% formic acid), and 70% acetonitrile aqueous solution (containing 2% formic acid) are used as extraction solvents. With the addition of acetonitrile in the extraction solvent, the phenomenon of protein coagulation into a group is improved, and the extraction recovery is best when the extraction solvent is 80% acetonitrile aqueous solution (containing 2% formic acid), and the proportion of herbicides falling into the 60%-110% and 80-110% intervals is the highest, as shown in Table 1. Figure 2 Therefore, 80% acetonitrile aqueous solution (containing 2% formic acid) is finally selected as the extraction solvent.

[0047] (3) Purification method

[0048] The experiment compares the purification of HLB solid phase extraction column and QuEChERS (50mg PSA+50mg C18+150mg MgSO4). As shown in Table 1, for beef, pig liver and egg three kinds of matrix, the number of herbicide items falling into the 80%-120% interval after HLB column purification is better than that of QuEChERS purification method, which may be because the PSA filler in the QuEChERS purification tube selectively removes sugars, fatty acids and other impurities through polar interaction, but easily leads to non-specific adsorption of basic target substances, affecting the recovery rate; C18 filler depends on non-polar adsorption of lipids and non-polar interferents, and competes with target compounds with weak polarity, often accompanied by loss of target compounds. In contrast, HLB composite adsorbent realizes broad-spectrum retention of different polarity compounds through hydrophilic-lipophilic balance mechanism; in acetonitrile solvent system, HLB can selectively retain protein, fat and other impurities, while target compounds are efficiently eluted due to the difference in polarity matching, so as to remove interferents while ensuring the maximum recovery of target compounds, showing excellent purification selectivity and recovery rate balance advantage, so HLB column is finally selected for purification treatment.

[0049] Table 1 Recovery rate under different purification methods in beef, pig liver and egg matrix

[0050]

[0051]

[0052] (4) Liquid chromatography conditions

[0053] Four kinds of chromatographic columns, Thermo Accucore aQ C18 chromatographic column (2.6 μm x 2.1 mm x 150 mm), Thermo Syncronis C18 (1.9 μm x 2.1 mm x 100 mm) chromatographic column, Waters Acquity UPLC BEH C18 chromatographic column (1.7 μm x 2.1 mm x 100 mm), and Waters Acquity UPLC HSS T3 chromatographic column (1.8 μm x 2.1 mm x 100 mm), were selected to investigate their separation effects on 85 kinds of herbicides and metabolites. The experimental results show that some compounds have tailing on the traditional Waters Acquity UPLC BEH C18 and Thermo Syncronis C18 chromatographic columns; the separation effects on the Thermo Accucore aQ C18 chromatographic column and the Waters Acquity UPLC HSS T3 chromatographic column are better, and the peak shape and response are the best on the Thermo Accucore aQ C18 chromatographic column (2.1 mm x 150 mm x 2.6 μm). The polar end-capped C18 stationary phase of the chromatographic column provides excellent polar analyte retention and is fully compatible with aqueous mobile phases, which can be used for 100% aqueous mobile phases, and is very suitable for the analyzed herbicides. Finally, the Thermo Accucore aQ C18 chromatographic column is selected as the analysis column.

[0054] The effects of different mobile phase systems, acetonitrile-water, acetonitrile-0.2% formic acid water, methanol-water, methanol-0.1% formic acid water, methanol-0.2% formic acid water, and methanol-2 mmol / L ammonium acetate water (containing 0.1% formic acid), on the peak shape and signal response were investigated. The results show that when acetonitrile and methanol are compared as organic phases, the separation effects of 85 kinds of herbicides and metabolites are not as good as when methanol is used as the organic phase because acetonitrile has greater polarity and stronger elution ability. Moreover, the system pressure is relatively small when methanol is used as the mobile phase. Therefore, methanol is selected as the organic phase. The peak shape of some herbicides such as alachlor, acetochlor, propisochlor, butachlor, fluoroglycofen, SYN503780, and CSCD686480 appears front extension or tailing in the mobile phase system without formic acid, and the peak shape is obviously improved after adding formic acid to the mobile phase. In addition, the addition of an appropriate amount of acid can promote the target compounds to form [M+H] +The peak shape of all substances can reach a satisfactory state when the content of formic acid in the aqueous phase reaches 0.1%; the addition of formic acid has little effect on the peak shape and response. After determining that the mobile phase is methanol-0.1% formic acid water, the addition of an appropriate amount of ammonium acetate can reduce the formation of [M+Na]+ and [M+K]+, so that the response of most target compounds is enhanced, and the response of some target compounds, such as carfentrazone-ethyl, quizalofop-p-ethyl, lactofen and fluazifop-butyl, is obviously increased. Therefore, methanol-2 mmol / L ammonium acetate water (containing 0.1% formic acid) is finally selected as the mobile phase.

[0055] Finally, the chromatographic conditions are as follows: Thermo Accucore aQ C18 chromatographic column (2.6 μm x 2.1 mm x 150 mm); flow rate: 0.4 mL / min; injection volume: 5 μL; mobile phase A is 2 mmol / L ammonium acetate water (containing 0.1% formic acid), and mobile phase B is methanol; gradient elution program: 0-0.4 min, 3% B; 0.4-0.7 min, 3%-15% B; 0.7-1 min, 50% B; 1-6 min, 50%-70% B; 6-7 min, 70%-97% B; 7-8 min, 97% B; 8-8.1 min, 97%-3% B; 8.1-10 min, 3% B.

[0056] (5) Mass spectrometry conditions

[0057] The high resolution of the quadrupole rod / electrostatic field orbitrap can accurately determine the ion mass number to five decimal places, and the mass spectrometry peak width is significantly smaller than that of the triple quadrupole low-resolution mass spectrometry, and the resolution capacity is strong; in the accurate qualitative quantification, the PRM technology is more accurate than the MRM technology, and the ion interference is far less than that of the MRM technology. In the present application, the PRM mode of UPLC-Q-Orbitrap HRMS is used to detect 85 kinds of herbicides and metabolite mixed standard solutions, only the chemical formula, the accurate mass number of the parent ion of each compound in the inclusion list is input, the ion scanning mode is selected, and the scanning window is set, so that the mass spectrometry can be scanned and cracked in the specified range. In the present application, the PRM mode is used to detect 85 kinds of herbicides and metabolites, and it is found that all target compounds are suitable for detection in the positive ion mode, and the response in the positive ion mode is obviously higher than that in the negative ion mode, so the positive ion mode is finally selected for detection; in order to obtain better secondary fragmentation ion response, the normalized collision energy (NCE) settings of 10, 20, 30 eV, 20, 40, 60 eV and 30, 60, 90 eV are compared, and it is found that the overall response is optimal when the normalized collision energy (NCE) settings are 20, 40, 60 eV, and the normalized collision energy (NCE) settings are finally set to 20, 40, 60 eV.

[0058] Finally, the mass spectrometry conditions were as follows: heated electrospray ion source (HESI source), positive ion mode; spray voltage 3500 V; ion transfer tube temperature: 350℃; sheath gas (N2): 10.5 L / min; auxiliary gas (N2): 3 L / min; acquisition mode: parallel reaction monitoring mode (PRM mode); secondary scan resolution: 17500; secondary mass spectrum collision energy was normalized collision energy (NCE): 20, 40, 60 eV.

[0059] Under the above-mentioned optimized mass spectrometry conditions, the mass spectrometry analysis parameters of the herbicides and metabolites were shown in Table 2, and the ion chromatograms of some representative herbicides were shown in FIG. 2. Figure 3

[0060] Table 2 Mass spectrometry analysis parameters of 85 herbicides and metabolites

[0061]

[0062]

[0063]

[0064] Further analysis was made on the effect of the detection method of the present application:

[0065] (1) Influence of matrix effect

[0066] Compared with the conventional triple quadrupole mass spectrometry, the Q-Orbitrap HRMS has stronger anti-interference ability, but the matrix effect (ME) still exists, and there are many kinds of poultry products, and different types of matrixes may differ significantly, so it is necessary to evaluate the matrix effect. The matrix effect after purification was evaluated according to the following formula:

[0067] Matrix effect (ME) = [(slope of matrix matching calibration curve / slope of pure solvent standard curve) - 1] x 100%.

[0068] The evaluation results showed that the proportion of absolute matrix effect less than -20% in beef, pork liver and egg samples was 7.1%, 8.2% and 27%, which was the matrix inhibition effect, the proportion of absolute matrix effect greater than 20% in beef, pork liver and egg samples was 11.8%, 2.4% and 7.1%, which was the matrix enhancement effect, and a certain proportion of items in the three samples had moderate matrix effect, and the difference between different types of matrixes was significant. In order to more accurately quantify the target compounds, the matrix matching calibration method was used to compensate for the matrix effect.

[0069] (2) Methodological verification

[0070] ① Linear relationship and limit of quantification

[0071] ​The blank matrix solution was used to prepare 85 kinds of herbicide and metabolite matrix matching mixed standard working solution, and the standard working curve was drawn with the quantitative ion peak area of 85 kinds of herbicide and metabolite as the ordinate (Y) and the mass concentration of the standard working solution (X, μg / L) as the abscissa. The standard solution was added to the blank matrix solution in an appropriate amount, and the concentration was gradually reduced. The limit of quantification (LOQ) of the method was determined by S / N=10. The linear regression equation, correlation coefficient (r), linear range and limit of quantification of 85 kinds of herbicide and metabolite in pig liver matrix are shown in Table 3.

[0072] Table 3 Regression equation, correlation coefficient, linear range and limit of quantification of 85 kinds of herbicide and metabolite

[0073]

[0074]

[0075]

[0076] Note: Y: Quantitative ion peak area; X: Mass concentration, μg / L.

[0077] 2. Recovery rate and relative standard deviation

[0078] The blank samples of beef, pig liver and chicken eggs were added with standard samples at 5, 10 and 50 μg / kg, respectively. Each standard sample was determined in parallel for 6 times (n=6), and the recovery rate and relative standard deviation (RSD) of the determined value of 85 kinds of herbicide and metabolite were calculated.

[0079] The results show that the recovery rate of 85 kinds of herbicide and metabolite in the negative standard sample of beef is 81.7% to 109.2%, and the RSD is 2.0% to 9.8%; the recovery rate of 85 kinds of herbicide and metabolite in the negative standard sample of pig liver is 78.2% to 109.4%, and the RSD is 2.6% to 9.9%; the recovery rate of 85 kinds of herbicide and metabolite in the negative standard sample of chicken eggs is 73.7% to 106.7%, and the RSD is 3.7% to 10.8%; which shows that the method has good accuracy and precision.

[0080] 3. Detection of actual samples

[0081] The detection method of the application was used to detect 20 samples of commercially available beef, pig liver and chicken eggs. It was found that only one chicken egg sample was detected to contain atrazine with a detection value of 5.6 μg / kg, and the content was low, and the other samples were not detected.

[0082] The above merely describes preferred embodiments of the present application, and is not intended to limit the present application. Any simple modification, change and equivalent variation of the above embodiments according to the technical essence of the present application are still within the protection scope of the technical scheme of the present application.

Claims

1. A method for detecting residues of herbicides and metabolites in poultry products, characterized in that, The method comprises the following steps: S1, sample pretreatment The poultry product tissue is crushed and homogenized to obtain a sample; an extraction solvent is added to the sample, and after extraction, centrifugation is performed, the supernatant is collected, and the supernatant is purified through a rapid filtration type solid phase extraction, and the effluent is collected, which is the sample solution to be measured; S2, preparation of a standard solution The herbicide standard solution is diluted with an extraction solvent to obtain a 10 mg / L mixed standard working solution; S3, the mixed standard working solution obtained in S2 is injected into an ultra-high performance liquid chromatograph-quadrupole / orbitrap high-resolution mass spectrometer to prepare a standard curve; Then, the sample solution obtained in S1 is injected, and the content of the herbicide and metabolites in the sample to be measured is calculated.

2. The method of claim 1, wherein, The extraction solvent in S1 and S2 is acetonitrile, 0.2% formic acid acetonitrile, 0.5% formic acid acetonitrile, 1% formic acid acetonitrile, 2% formic acid acetonitrile, 4% formic acid acetonitrile, 2% formic acid-90% acetonitrile-water solution, 2% formic acid-80% acetonitrile-water solution, or 2% formic acid-70% acetonitrile-water solution.

3. The method of claim 1, wherein, The ratio of the sample to the extraction solvent in S1 is 2.00 g:10 mL.

4. The method of claim 1, wherein, The extraction method in S1 is oscillation extraction or homogenization extraction.

5. The method of claim 4, wherein, The rotation speed of the homogenization extraction is 13500 r / min, and the extraction time is 0.8-1.2 min.

6. The method of claim 1, wherein, The rotation speed of the centrifugation in S1 is 4000 r / min, and the centrifugation time is 2-3 min.

7. The method of claim 1, wherein, The extraction column for the rapid filtration type solid phase extraction in S1 is a functionalized polystyrene / divinylbenzene HLB solid phase extraction column.

8. The method of claim 1, wherein, The column passing speed for the rapid filtration type solid phase extraction in S1 is 1-2 drops per second.

9. The method of claim 1, wherein, The chromatographic conditions in S3 are as follows: a Thermo Accucorea Q C18 chromatographic column, 2.6 μm×2.1 mm×150 mm; a flow rate of 0.4 mL / min; an injection amount of 5 μL; a mobile phase A of 2 mmol / L ammonium acetate water containing 0.1% formic acid, and a mobile phase B of methanol; a gradient elution program of 0-0.4 min, 3% B; 0.4-0.7 min, 3%-15% B; 0.7-1 min, 50% B; 1-6 min, 50%-70% B; 6-7 min, 70%-97% B; 7-8 min, 97% B; 8-8.1 min, 97%-3% B; 8.1-10 min, 3% B. The mass spectrometry conditions in S3 are as follows: a heated electrospray ion source, a positive ion mode; a spray voltage of 3500 V; an ion transmission tube temperature of 350℃; a sheath gas of N2, a flow rate of 10.5 L / min; an auxiliary gas of N2, a flow rate of 3 L / min; an acquisition mode of parallel reaction monitoring; a secondary scan resolution of 17500; and a secondary mass spectrometry collision energy of 20, 40, and 60 eV.

10. The method of claim 1, wherein, ​

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