Method for detecting pesticides and their metabolites in animal-derived samples

Abstract

This invention provides a method for detecting pesticides and their metabolites in animal-derived samples. The method includes the following steps: mixing an extraction solvent with a homogenized animal-derived sample for extraction; adding salt for salting out and purifying with a purification agent; and then using a dual-platform approach of LC-MS / MS and GC-MS / MS to detect residues of multiple pesticides and their metabolites in the animal-derived sample. This method is adaptable to pesticide residue detection in various animal-derived matrices, is simple to operate, requires no repeated development of pretreatment methods, has a wide detection range, and provides accurate and stable quantitative results.

Description

Technical Field

[0001] This invention belongs to the field of pesticide detection technology, specifically relating to a method for detecting pesticides and their metabolites in animal-derived samples. Background Technology

[0002] Animal-derived foods refer to foods derived from animals, including common meats found on the dinner table (pork, beef, lamb, etc.), poultry (chicken, duck, etc.), eggs, aquatic products (fish, shrimp, crab, shellfish, etc.), and dairy products. As a core source of nutrition in the human diet, these foods provide high-quality complete protein (containing all eight essential amino acids), essential fatty acids (such as Omega-3 polyunsaturated fatty acids), minerals (calcium, iron, zinc), and fat-soluble vitamins (vitamins A, D, and B12), playing an irreplaceable role in maintaining muscle synthesis, bone development, and immune function.

[0003] As people's living standards improve and dietary structures shift towards "high-protein, high-nutrition," the safety of food has become an increasingly important social concern. Among these concerns, pesticide residues, a key indicator affecting the safety of animal-derived foods, pose a significantly higher risk than plant-derived foods due to their multi-stage migration characteristics involving the environment, feed, animals, and food. For a long time, public awareness of pesticide residues has focused primarily on plant-derived foods (such as spray residues on the surface of fruits and vegetables). However, due to factors such as improper pesticide use and migration from environmental media, animal-derived foods also face serious risks of pesticide contamination and exceeding standards.

[0004] Whether directly or indirectly sourced, pesticides undergo an "absorption-distribution-metabolism-excretion" process within animals: fat-soluble pesticides (such as fipronil and pyrethroids) preferentially accumulate in adipose tissue (egg yolks, subcutaneous fat in livestock and poultry), while polar pesticides and their metabolites (such as chlorpyrifos metabolites TCP and fipronil sulfone) are distributed in muscles and body fluids, ultimately remaining in the final product as parent products or metabolites. These residues, once entering the human body through the food chain, can interfere with the endocrine system (e.g., fipronil sulfone inhibits thyroid hormone synthesis), damage the central nervous system (e.g., organophosphates inhibit cholinesterase activity), and impair immune system function. Long-term low-dose exposure may also induce chronic harms such as liver tumors and reproductive toxicity, posing even higher risks to sensitive populations such as infants and pregnant women.

[0005] Animal-derived food matrices are characterized by high fat, high protein, and high levels of interfering components (e.g., poultry meat has a fat content of 5%-30%, and dairy products have a lactose content of 4%-6%). Pesticide residue detection in these matrices requires breakthroughs in three key technical aspects: efficient extraction, deep purification, and precise detection. However, existing methods have significant shortcomings. The core objective of pretreatment is to remove interfering substances such as fat, protein, and phospholipids from the matrix, while simultaneously achieving efficient recovery of pesticides and their metabolites. Solid phase extraction (SPE), commonly used for pretreatment of animal products, can more effectively enrich target analytes and remove key interfering substances such as fats due to the diversity of adsorbents available (such as C18, Florisil, PSA, etc.), ensuring the purity of subsequent analyses. However, it suffers from problems such as "high solvent consumption (10-15 mL of elution buffer per sample), long operation cycle (1.5-2 h per sample), limited adsorbent selectivity, and a limited number of compatible drugs." For example, an SPE scheme used for the detection of one type of drug in a product cannot be directly adapted to other products or drugs, requiring re-optimization of the adsorbent ratio and elution conditions, making it difficult to meet the needs of high-efficiency detection of batch samples and multiple drugs. At the instrumental analysis level, liquid chromatography-tandem triple quadrupole mass spectrometry (LC-MS / MS) has become the "gold standard" for qualitative and quantitative analysis of multiple residues due to its excellent sensitivity and selectivity. However, it cannot detect non-polar / volatile pesticides such as pyrethroids (cypermethrin, deltamethrin) and organochlorines (HCH, DDT). These pesticides have low solubility in the liquid mobile phase, making it difficult to effectively enter the chromatographic column for separation. Furthermore, the response value is extremely low in electrospray ionization (ESI) mode, hindering quantitative analysis. Poor accuracy; Gas chromatography-tandem mass spectrometry (GC-MS / MS) can efficiently detect volatile and semi-volatile pesticides (such as organophosphates and pyrethroids) through electron impact ionization (EI) mode, but its ability to detect polar pesticides and their metabolites (such as TCP and fipronil) is insufficient. These compounds have poor thermal stability and are easily decomposed at the GC injection port (decomposition rate exceeds 30%), and the detection response cannot be improved by derivatization, resulting in a limit of quantitation (LOQ) of only 0.05-0.1 mg / kg, which cannot meet the requirements of some limits in GB 2763-2021. Some studies adopt the "sample collection - separate pretreatment - dual-platform detection" model, but this requires more than two samples and the pretreatment conditions are not uniform (e.g. QuEChERS for LC-MS / MS, SPE for GC-MS / MS). This is not only cumbersome and consumes a lot of samples, but may also lead to deviations in detection results due to differences in pretreatment (e.g., the recovery rate of the same pesticide may differ by more than 15% on the two platforms), failing to meet the high-efficiency requirement of "using the same purification solution for dual-platform detection".

[0006] Existing methods for detecting pesticide and metabolite residues in plant-derived foods still face the following challenges: (1) Limitations of detection technology Existing technologies mostly employ a single detection platform, either GC-MS / MS or LC-MS / MS. Limited by platform characteristics (e.g., GC-MS / MS struggles to detect polar metabolites, while LC-MS / MS struggles to detect non-polar pesticides), they cannot accommodate pesticides and metabolites with different physicochemical properties. Furthermore, the pretreatment processes in existing technologies are often designed for a single detection platform (GC-MS / MS or LC-MS / MS), meaning the same pretreatment process cannot be adapted to both platforms simultaneously, hindering cross-platform compatibility.

[0007] (2) Poor adaptability of pretreatment methods

[0008] From the perspective of animal-derived matrix composition, the core components of livestock and poultry meat are protein and fat, with significant differences in fat content among different types of meat. Poultry eggs are mainly composed of protein, fat, and phospholipids. Offal, in addition to protein and fat, contains a relatively high amount of vitamins and minerals, while dairy products are rich in casein, whey protein, and lactose. The significant differences in matrix composition and the varying types and amounts of interfering substances among different animal-derived matrices make it difficult to universally apply existing pretreatment and detection systems, resulting in incompatibility with multiple matrices such as meat, eggs, milk, and offal. Furthermore, existing pretreatment methods are not adaptable to multi-platform detection. Pretreatment for GC-MS / MS often employs highly volatile solvents and high-temperature assisted extraction, which is incompatible with the requirements of LC-MS / MS regarding sample solvent polarity and impurity content. While pretreatment for LC-MS / MS focuses on polar solvent extraction and precise impurity removal, it cannot meet the requirements of GC-MS / MS regarding sample volatility and thermal stability. This means that the same pretreatment solution cannot be simultaneously introduced into both platforms for detection, requiring repeated development of pretreatment procedures for different platforms. This not only increases operating costs and workload but also hinders the simultaneous and efficient screening of pesticides and metabolites. (3) Limitations in the coverage of the detected target Existing technologies, during the method development stage, primarily include common pesticides such as neonicotinoids (e.g., imidacloprid, thiamethoxam), organophosphates (e.g., chlorpyrifos, dichlorvos), carbamates (e.g., carbofuran, methomyl), and pyrethroids (e.g., cypermethrin, deltamethrin) in their detection scope. However, detection methods for pesticides such as morpholines, triazoles, triazines, pyrazoles, sulfonylureas, pyridines, and acylurea are less common. Furthermore, in animal-derived foods, pesticides are metabolized into metabolites through liver metabolism and enzymatic hydrolysis, and some of these metabolites are more toxic and have higher residue levels. However, some metabolites (e.g., 2,4,6-trichlorophenol from prochloraz, UF from dinotefuran, and FM-6-1 from flufenoxuron) are not included in the target analytes list in existing technologies, making qualitative and quantitative analysis impossible. Therefore, it is difficult to meet the practical needs of simultaneous monitoring of multiple substrates and pesticide categories and their metabolites.

[0009] In summary, developing more adaptable and comprehensive detection technologies has become an urgent need to solve the current regulatory challenges of pesticide residues in animal-derived foods. Summary of the Invention

[0010] To address the aforementioned problems, the present invention aims to provide a method for detecting pesticides and their metabolites in animal-derived samples. This detection method is adaptable to pesticide residue detection in various animal-derived matrices, is simple to operate, requires no repeated development of pretreatment methods, and offers a wide detection range with accurate and stable quantitative results.

[0011] To achieve the above objectives, the present invention provides a method for detecting pesticides and their metabolites in animal-derived samples, comprising the following steps: (1) The extraction solvent is mixed with the homogenized animal sample and extracted to obtain the extraction system; (2) Add salt to the extraction system and perform salting-out treatment to obtain animal-derived sample extract: (3) The animal-derived sample extract was purified with a purification agent to obtain a first animal-derived sample extract purified solution for LC-MS / MS analysis and detection; After nitrogen blowing concentration and redissolution, the animal-derived sample extract was purified with a purification agent to obtain a second purified animal-derived sample extract for GC-MS / MS analysis and detection. (4) The first animal-derived sample extract and purified solution was analyzed and detected by LC-MS / MS, and the second animal-derived sample extract and purified solution was analyzed and detected by GC-MS / MS.

[0012] According to a specific embodiment of the present invention, preferably, the extraction solvent includes acetonitrile and / or acidified acetonitrile.

[0013] According to a specific embodiment of the present invention, preferably, the salt comprises one or a combination of two or more of sodium chloride, sodium acetate, magnesium sulfate, and citrate.

[0014] According to a specific embodiment of the present invention, preferably, the purifying agent includes PSA and / or C18.

[0015] According to a specific embodiment of the present invention, preferably, in step (1), when the animal source sample is a non-liquid animal source sample, water is added to the homogenized non-liquid animal source sample and then mixed with the extraction solvent.

[0016] According to a specific embodiment of the present invention, preferably, the detection method includes the following steps: (1) Weigh 0.5-5 g of homogenized animal source sample. When the animal source sample is a non-liquid animal source sample, add 0.5-5 mL of water to the non-liquid animal source sample, mix well, add 1-10 mL of extraction solvent, shake, and obtain a non-liquid animal source sample extraction system. When the animal source sample is a liquid animal source sample, add 1-10 mL of extraction solvent to the liquid animal source sample, shake, and obtain a liquid animal source sample extraction system. (2) Add salt to the non-liquid animal sample extraction system or the liquid animal sample extraction system, shake, centrifuge, and obtain the supernatant as the animal sample extract; (3) Take 1-2 mL of the animal-derived sample extract, add 50-100 mg of PSA and 100-200 mg of C18, vortex mix, filter through a microporous membrane to obtain the first animal-derived sample extract purified solution for LC-MS / MS analysis and detection. Take 4-8 mL of the animal-derived sample extract, concentrate it under nitrogen blowing at 30-60℃, make up to volume with acetonitrile and / or acidified acetonitrile, add 50-100 mg of PSA and 100-200 mg of C18, vortex mix, filter through a microporous membrane to obtain the second animal-derived sample extract purified solution for GC-MS / MS analysis and detection. (4) The first animal-derived sample extract and purified solution was analyzed and detected by LC-MS / MS, and the second animal-derived sample extract and purified solution was analyzed and detected by GC-MS / MS.

[0017] According to a specific embodiment of the present invention, preferably, the detection method includes the following steps: (1) Weigh 5g of homogenized animal source sample. When the animal source sample is a non-liquid animal source sample, add 5mL of water to the non-liquid animal source sample, mix well, add 10mL of acetonitrile, shake, and obtain a non-liquid animal source sample extraction system. When the animal source sample is a liquid animal source sample, add 10mL of acetonitrile to the liquid animal source sample, shake, and obtain a liquid animal source sample extraction system. (2) Add 3g of sodium chloride to the non-liquid animal sample extraction system or the liquid animal sample extraction system, shake, and centrifuge at 10000r / min for 5min at 4℃ to obtain the supernatant as the animal sample extraction solution; (3) Take 1 mL of the animal-derived sample extract, add 50 mg of PSA and 100 mg of C18, vortex mix, filter through a microporous membrane to obtain the first animal-derived sample extract purified solution for LC-MS / MS analysis and detection. Take 4 mL of the animal-derived sample extract, concentrate it under nitrogen blowing at 40 °C to a volume of <1 mL, make up to 1 mL with acetonitrile, add 50 mg of PSA and 100 mg of C18, vortex mix, filter through a microporous membrane to obtain the second animal-derived sample extract purified solution for GC-MS / MS analysis and detection. (4) The first animal-derived sample extract and purified solution was analyzed and detected by LC-MS / MS, and the second animal-derived sample extract and purified solution was analyzed and detected by GC-MS / MS.

[0018] According to a specific embodiment of the present invention, preferably, the chromatographic conditions of GC-MS / MS include: a (5%-phenyl)-methylpolysiloxane column (e.g., an HP-5MS UI column); more preferably, the chromatographic conditions include: an HP-5MS UI column (specifications: 0.25 mm × 30.0 m, 0.25 μm, Agilent Technologies); a splitless injection method; helium as the carrier gas with a purity ≥99.999%; and a carrier gas flow rate of 1.0 mL / min.

[0019] According to a specific embodiment of the present invention, preferably, the mass spectrometry conditions for GC-MS / MS include: an EI ion source, an ion source temperature of 200-300℃, and an electron impact source voltage of 50-80eV.

[0020] According to a specific embodiment of the present invention, preferably, the mass spectrometry conditions of GC-MS / MS include: an EI ion source, an ion source temperature of 280°C, and an electron impact source voltage of 70 eV; more preferably, the conditions include: an EI ion source, an EI ion source temperature of 280°C, an injection port temperature of 280°C, a transfer line temperature of 280°C, a solvent delay time of 3 min, and an electron impact source voltage of 70 eV.

[0021] According to a specific embodiment of the present invention, preferably, the chromatographic conditions of LC-MS / MS include: an octadecylsilane reversed-phase column (e.g., a ZORBAX Eclipse Plus C18 Rapid Resolution HD column); more preferably, the chromatographic conditions include: a ZORBAX Eclipse Plus C18 Rapid Resolution HD column (size: 3.0 mm × 150 mm, 1.8 μm, Agilent Technologies); a flow rate of 0.4 mL / min; mobile phase A being 0.05% formic acid aqueous solution with an ammonium formate concentration of 2 mmol / L, and mobile phase B being 0.05% formic acid methanol; a flow rate of 0.4 mL / min; and a column temperature of 40 °C.

[0022] According to a specific embodiment of the present invention, preferably, the mass spectrometry conditions of LC-MS / MS include: the ion source is an ESI source; the electrospray voltage in positive ion mode is 3000-4500V, and the electrospray voltage in negative ion mode is 3000-4500V; the nebulizer gas temperature is 300-350℃; and the sheath gas temperature is 280-350℃.

[0023] According to a specific embodiment of the present invention, preferably, the mass spectrometry conditions of LC-MS / MS include: an ion source of ESI; an electrospray voltage of 3500V for positive ion mode and 3500V for negative ion mode; atomizing gas temperature of 300℃; and sheath gas temperature of 300℃. More preferably, the conditions include: an ion source of ESI; scanning mode: simultaneous scanning in positive and negative ion modes; electrospray voltage: 3500V for both positive and negative ion modes; atomizing gas temperature of 300℃; and sheath gas temperature of 300℃.

[0024] According to a specific embodiment of the present invention, preferably, the chromatographic temperature program of GC-MS / MS includes: an initial temperature of 60°C, holding for 1 min, then increasing the temperature to 120°C at a rate of 40°C / min, then increasing the temperature to 180°C at a rate of 10°C / min, holding for 5 min, and then increasing the temperature to 310°C at a rate of 5°C / min, holding for 3 min.

[0025] According to a specific embodiment of the present invention, preferably, the mobile phase A used for chromatographic detection by LC-MS / MS is a 0.05% formic acid aqueous solution with an ammonium formate concentration of 2 mmol / L, and the mobile phase B is a 0.05% formic acid methanol solution; the flow rate is 0.4 ml / min.

[0026] According to a specific embodiment of the present invention, preferably, the chromatographic elution program of LC-MS / MS, based on the volume fraction of mobile phase A, includes: 0-0.5min: 97%; 3min: 50%; 9min: 30%; 21-26 min: 0%; 26.1-30min: 97%.

[0027] According to a specific embodiment of the present invention, preferably, the GC-MS / MS injection volume is 1 μL.

[0028] According to a specific embodiment of the present invention, preferably, the LC-MS / MS injection volume is 2 μL.

[0029] According to a specific embodiment of the present invention, preferably, the mass spectrometry acquisition mode of the GC-MS / MS and LC-MS / MS is dynamic multiple reaction ion monitoring mode (dMRM).

[0030] According to a specific embodiment of the present invention, the detection method includes the following specific steps: (1) For animal-derived samples of livestock meat, poultry meat, and offal, remove fat and fascia tissue, mince and homogenize; for animal-derived samples of poultry eggs, remove shells and stir to homogenize; for animal-derived samples of dairy products, mix well to homogenize. Weigh 5g of homogenized animal source sample. If the animal source sample is a non-liquid animal source sample, add 5mL of water to the non-liquid animal source sample, vortex for 1min, mix well, add 10mL of acetonitrile, and vortex for 1min to obtain the non-liquid animal source sample extraction system. If the animal source sample is a liquid animal source sample, add 10mL of acetonitrile to the liquid animal source sample, and vortex for 1min to obtain the liquid animal source sample extraction system. (2) Add 3g of sodium chloride to the non-liquid animal sample extraction system or the liquid animal sample extraction system, shake for 1 min, and centrifuge at 10000 r / min for 5 min at 4℃ to obtain the supernatant as the animal sample extraction solution; (3) Take 1 mL of the animal-derived sample extract, add 50 mg of PSA and 100 mg of C18, vortex mix for 1 min, filter through a microporous membrane to obtain the first animal-derived sample extract purified solution for LC-MS / MS analysis and detection. Take 4 mL of the animal-derived sample extract, concentrate it under nitrogen blowing at 40 °C to a volume of <1 mL, make up to 1 mL with acetonitrile, add 50 mg of PSA and 100 mg of C18, vortex mix for 1 min, filter through a microporous membrane to obtain the second animal-derived sample extract purified solution for GC-MS / MS analysis. (4) The first animal-derived sample extraction and purification solution was analyzed and detected by LC-MS / MS, and the second animal-derived sample extraction and purification solution was analyzed and detected by GC-MS / MS; The chromatographic conditions for GC-MS / MS are as follows: The chromatographic column was an HP-5MS UI column (specifications: 0.25 mm × 30.0 m, 0.25 μm, Agilent Technologies); the injection method was splitless injection; the carrier gas was helium with a purity ≥99.999%; the carrier gas flow rate was 1.0 mL / min. The mass spectrometry conditions for GC-MS / MS are as follows: The ion source was an EI ion source; the EI ion source temperature was 280℃; the injection port temperature was 280℃; the transfer line temperature was 280℃; the solvent delay time was 3 min; and the electron impact source voltage was 70 eV. The chromatographic conditions for LC-MS / MS are as follows: The chromatographic column was a ZORBAX Eclipse Plus C18 Rapid Resolution HD column (3.0 mm × 150 mm, 1.8 μm, Agilent Technologies); the flow rate was 0.4 mL / min; mobile phase A was 0.05% formic acid aqueous solution with ammonium formate concentration of 2 mmol / L, and mobile phase B was 0.05% formic acid methanol; the flow rate was 0.4 mL / min; the column temperature was 40℃. The mass spectrometry conditions for LC-MS / MS are as follows: The ion source is an ESI source; scanning mode: simultaneous scanning in positive ion mode and negative ion mode; electrospray voltage: 3500V for positive ion mode and 3500V for negative ion mode; atomizing gas temperature: 300℃; sheath gas temperature: 300℃.

[0031] According to a specific embodiment of the present invention, preferably, the animal source sample includes one or more combinations of livestock meat, poultry meat, offal, poultry eggs, and dairy products.

[0032] According to a specific embodiment of the present invention, preferably, the pesticides and their metabolites include terbufos, butachlor, butachlor, butachlor, cyazofamid, pyraclostrobin, chlorpyrifos, parathion, paclobutrazol, oxadiazon, oxazolidinone, oxadiazon, diphenylamine, pendimethalin, diazinon, fenpyroximate, furazolidone, fipronil, fenpyroximate, flufenoxuron, flufenoxuron sulfone, flufenoxuron sulfide, flusilazole, flutriafol, flutriafol, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, clopyralid, clopyralid, cyproconazole, cyproconazole, fenpyroximate, fenpyroximate. Sulfone, sulfonium thion, cyclopyrimethanil, hexaconazole, phorate, phorate sulfonium, phorate sulfone, metolachlor, methyl chlorpyrifos, methyl parathion, methyl thiophanate, methyl pyrimiphos, methyl isofenphos, cypermethrin, carbofuran, fenpropathrin, pirimicarb, carbofuran, 3-hydroxycarbofuran, quinalphos, quinfenoxam, quinoxyfen, dimethoate, bifenazate, bifenthrin, bifenthrin, phosphamidon, α-endodole, β-endodole, endodole sulfate, thiocyclam, thionylphos, chlorfenapyr, chlorfenapyr, chlorfenapyr, benomyl, permethrin-trans, chlornitramine, chlorpyrifos, prochloraz and prochloraz manganese salt, imazalil, pyrimethanil, pyrimethanil, pyrimethanil Cyclofenac, Azoxystrobin, Pyrimethanil, Difenoconazole, Emethoate, Naphthaleneacetic Acid and Sodium Naphthaleneacetate, Piperazine, Prochloraz, Metazon, Cyfluthrin, Cypermethrin and S-Cypermethrin, Benziram, Acetylpyridinium, Procymidone, Bentazon, Thiamethoxam, O,P'-Trichlorfon, P,P'-Trichlorfon, Trichlorfon Sulfone, Triazole, Triazole Phosphate, Triadimefon, Amitraz, Fenitrothion, Chlorpyrifos, Methamidophos, Tetraflufenicol, Tetrachlorophthalide, Phenylphthalide, Tetrachloronitrobenzene, Tetrachlorfenapyr, Terbufos, Terbufos, Terbufos Sulfone, Pyriproxyfen, Carbendazim, Azoxystrobin, Pentachloronitrobenzene, Pentachloroaniline, Tebuconazole, Tebuconazole, Cymoxanil, Simazine Zinc, tebuconazole, octanoyl bromoxynil, bromofenac, bromodiphenyl ether, phosmet, imidacloprid, fenamidophos, fenamidophos, thiol-type systemic sulfoxide, acetochlor, ethion, ethoxyphos, acetochlor, pyrifluquinazon, ethirimol sulfonate, ethylene sclerotinia, acephate, ethoxysulfuron, ethoxyflufenoxam, acetochlor, metolachlor and S-metolachlor, isoprocarb, isoxaflutole, isoxaflutole, imazalil, cypermethrin, atrazine, atrazine, synergist, phosmet, tebuconazole, terbufos, terbufos, acetamiprid, acetamiprid, acetamiprid, acetamiprid, azoxystrobin, aldrin, 4,4'-DDT, 2,4'-DDT, 4,4'-DDT, 2,4'-DDT, 2,4'-DDT, Dildrin, Pyrazosulfuron, γ-HCH, α-HCH, β-HCH, δ-HCH, Cis-Chlordane, Trans-Chlordane, Oxychlordane, Isodrin, Isodrinaldehyde, Isodrinone, Nitraz-methyl, Ethylbutazone, Isofenphos, Azoxystrobin, Bromobenzylphos, Pyrazosulfuron, Defoliant, Trithion, Phoxim, Pyrimethanil, Pyrimethanil, Pyridaben, Cyprodinil, Chlorpyrifos, Isopropoxyfen, Cyprodinil, Phoxim, Dimethoate, Fonsophos, Pyraclostrobin, Deethylatrazine, Chlormethrin, Pyrimethanil, Prochloraz, Butylpyridinium, Bromothion, Cyfluthrin, Propiconazole, Benomyl, Thiophanate-methyl, Ethylpyridinium, Malaoxyfen, Phoxim, Phoxim, Pyrimethanil, Pyrimethanil, Pyrimethanil, Ethylbromothion, Propiconazole Phosphorus, chlorpyrifos, terbufos, isopropylfen, fenpropathrin, fluquinazole, thiophanate-methyl, dibromophos, flubutyroxyfen, herbicides, biphenyl, tebufenozide, chlorfenapyr, etoxazole, atrazine, dichlorvos, dicofol, trifluralin, ethyl ester, DDT, parabromophos, sulfadiazine, isopropylthiophanate-methyl, fluroxypyr, spirodiclofen, propyzoxystrobin, etoxazole, propyzoxystrobin, anthraquinone, abamectin, furazolidone, chlorpyrifos, dimethyl chlorphthalate, oat chlorpyrifos, fenpyroximate, dioxin, fluroxypyr, flufenoxuron, fludioxonil, chlorfenapyr, cyclohexane, fenpyroximate, fenpyroximate, cyclohexane, fenpyroximate, 2-methyl-4-chloroisooctyl ester, pyridaben, thiophanate-methyl, oxychlorpyrifos, methyl methacrylate, pyridaben, fenpyroximate, pyridaben, difenoconazole, quizalofop-p-ethyl, etoxazole Furazolidone, Heptenphos, Glyphosate, Flufenoxuron, Terbufos, Isoprothiolane, Procymidone, Prochloraz, Prochloraz, Dichlorvos, Dichlorvos, Difenoconazole, Difenoconazole, Butyral, Butachlor, Eutrimethoate, Acetamiprid, Pyrimethanil, Flufenoxuron, Carbendazim, Oxadiazon, Oxadiazon, Sulfonamide, Flufenoxuron, Flubendiamide, Flufenoxuron, Flupyrsulfuron, Pyrimethanil, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, 4-Fluoro-N-isopropylaniline, Hydroxychlorothalonil, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, 4-Nitrophenol Sodium, Silthiazamide, Cyprosulfuron, Cyclosulfuron, Emamectin benzoate Formate, mesosulfuron, mesosulfuron-methyl, sodium iodosulfuron, mesosulfuron-methyl, methyl thiocyclam, thiocarb, carbaryl, benzylfloxacin, metalaxyl and cymoxanil, methoxyfenozide, quizalofop-p-ethyl, phosmet, methyl pymetrozine, methyl-formylamino-pymetrozine, quizalofop-p-ethyl, bensulfuron-methyl, spirotetramat, spirotetramat-keto-hydroxy, spirotetramat-mono-hydroxy, spirodiclofen, chlorpyrifos, chlorpyrifos, chlorpyrifos, permethrin, chlorpyrifos, cypermethrin, chlorthiazoline, bensulfuron-methyl, ether bensulfuron-methyl, fensulfuron-methyl, fenpyroxime, pyrimethanil, pyrimethanil-4-hydroxy, pyrimethanil-5-hydroxy, 1-[2-chloro-4-(chlorophenoxy)-phenyl]-2-(1,2,4-Triazole)-1-yl-ethanol), Bensulfuron, Diflubenzuron, Demeton-methyl, Azoxystrobin, Pyrazosulfuron, Bensulfuron-methyl, Cyfluthrin, Cyazofamid, Cyazofamid metabolite, Cyazofamid, Lactobacillus, Thiaben, Thiamethoxam, Thiamethoxam, Thiamethoxam, 5-Hydroxyfen, Thiamethoxam, Thiazidone, Benzoylamidone, Thiamethoxam, Tricyclazole, Triflubenzuron, Benzoylphos, Benzoylphos sulfonate, Benzoylphos sulfoxide, Lufenuron, Lufenuron, Amitraz, Amitraz and Amitraz hydrochloride, Dimethomorph, Cymoxanil, Terbufos sulfoxide, Aldicarb, Aldicarb sulfonate, Aldicarb sulfoxide, Carbendazim sulfoxide, Oxycarb, Pentazon Flusulfanil, clethodim, clethodim sulfonium, clethodim sulfoxide, acetamiprid, clethodim, acetamiprid, imidacloprid, acetamiprid ester, dimethomorph, uniconazole, phoxim, bromobenzonitrile, bromocyanamide, deltamethrin, phosmet, oxammonium phosphate, quizalofop-P-ethyl and succinyl quizalofop-P-ethyl, sulfonium phosphate, methyl parathion, sulfonium parathion, omethoate, ivermectin, ethion sulfonium phosphate, ethionyl, ethoxysulfuron, acetamiprid, ethirimol, ethoxysulfuron, isoproturon, pyraclostrobin, (1-methyl-3-trifluoromethyl-1H-pyrazol-4-yl)formamide, indazolesulfonamide, azadirachtin, indoxacarb, rotenone, azoxystrobin, pyraclostrobin, azoxystrobin, pyraclostrobin, pyraclostrobin, pyraclostrobin Acetaminophen, pyraclostrobin, pyraclostrobin, methyl parathion, bensulfuron-methyl, ethyl parathion, oxyfenozide, oxyfenozide sulfone, fenozide sulfone, dextromethorphan, propoxur, fenoxam, oxazin, dioxamethoxam, chlorpyrifos, carbendazim, chlorpyrifos, flufenoxuron, chlorpyrifos, isofenphos, methamidophos, flupyridine, bifenthion, acetamiprid, trifluralin, fluthiazopyr, naphthylacetamide, 2-amino-4-methoxy-6-methyl-1,3,5-triazine, tetrazolium-sulfuron-methyl, bensulfuron-methyl, bromadiolone, butachlor, profenofos, carbaryl, phosphonocarboxylic acid, calcitriol, cyclone dichlorvos, indole-methyl, metolachlor, ether Azoxystrobin, terbufos, 4,6-dinitro-o-cresol, fenpicarbazole, pyrimisulfuron, chlorpyrifos, fluazinam, fenpyroxime, imidacloprid, iodobenzonitrile, isoprothiolane, tebufenozide, linuron, toluene-methyl, chlorpyrifos, fenpyroxime, pyrethroid I, pyrethroid II, methylbenzylthiazoline, sulfadiazine, metribuzin, pendimethalin, clethodim, fenpyroxime, flupyrfluthrin, cyclobenzyl, propoxyquin, bensulfuron-methyl, flusulfuron-methyl, methoxysulfuron-methyl, cyhalothrin, sulfadiazine, sulfonylsulfuron-methyl, benzoylsulfuron-methyl, cyhalothrin, fenpyroxime, fenpyroxime, trifluralin, warfarin, methomyl, oxadixyl, methyl ethyl sulfide, bensulfuron-methyl, dioxin, N,One or more combinations of N-dimethylamino-N-toluene, sulfothiamethoxam, ethoxybenzamide, fenvalerate, fenugreek, chlorfenapyr, terbufenozide, furazolidone, succinylcholine, methoxyfenozide, chlorfluazuron, methyl parathion, cyclophosphamide, dithion, pymetrozine, fluopyram, mancozeb, oxychlorpyrifos, chlorpyrifos, pymetrozine, pyrimethanil, isoprothiolane, abamectin, isoprothiolane, and diuron.

[0033] According to a specific embodiment of the present invention, preferably, the detection limit of the LC-MS / MS is ≤2 μg / kg, and the quantitation limit is 5-20 μg / kg.

[0034] According to a specific embodiment of the present invention, preferably, the detection limit of the GC-MS / MS is ≤2.5 μg / kg, and the quantitation limit is 5-20 μg / kg.

[0035] In this invention, the meat, poultry, offal, eggs, and dairy products mentioned are all edible animal-derived raw materials, specifically defined as follows: Livestock meat: refers to the edible muscle tissue and small amounts of attached fat, fascia, and other edible parts obtained from domestic or artificially raised livestock such as pigs, cattle, sheep, donkeys, deer, and horses after slaughter, bleeding, and removal of non-edible parts. This includes, but is not limited to, raw edible meat products from conventionally cut parts such as tenderloin, leg meat, rib meat, pork belly, and rump meat.

[0036] Poultry meat: refers to the edible muscle tissue and edible poultry skeleton attached meat obtained after slaughtering, plucking, removing internal organs and non-edible parts of domestic or artificially raised poultry such as chicken, duck, goose, turkey, quail, and pigeon, including but not limited to raw edible poultry meat parts such as chicken breast, chicken leg, duck breast, goose leg, and quail.

[0037] Offal: refers to the edible internal organs and tissues obtained after slaughtering the above-mentioned livestock and poultry, and after cleaning and removing inedible impurities and diseased tissues, including but not limited to the heart, liver, spleen, lungs, kidneys, stomach, intestines, abdomen, tongue and other edible offal raw materials of livestock and poultry.

[0038] Poultry eggs: refers to the edible eggs produced by poultry such as chickens, ducks, geese, quails, and pigeons, including whole fresh poultry eggs, as well as edible poultry egg raw materials such as egg liquid, yolk, and egg white after the eggshell has been removed, excluding poultry egg products that have undergone deep processing and whose core raw material properties have been altered.

[0039] Milk: refers to the raw milk secreted by artificially raised lactating mammals such as cows, sheep, buffalo, yaks, and camels, as well as edible milk raw materials that have undergone only physical treatments such as sterilization and homogenization without altering the core components and properties of the milk, including but not limited to raw cow milk, raw sheep milk, pasteurized milk, and raw camel milk.

[0040] This invention establishes a method for detecting pesticide residues and their metabolites in five major categories of animal-derived products: livestock meat, poultry meat, offal, eggs, and dairy products, based on a unified sample pretreatment technique and combined with liquid chromatography-tandem mass spectrometry (LC-MS / MS) and gas chromatography-tandem mass spectrometry (GC-MS / MS). This dual-platform detection method, which adapts to multiple matrices and uses a unified pretreatment, can achieve accurate quantification of at least 500 pesticides and their metabolites.

[0041] The present invention has the following beneficial effects: 1. Unified pretreatment method adaptable to multiple animal-derived matrices: The pretreatment method for pesticide residue detection constructed in this invention is applicable to five types of animal-derived matrices: poultry meat, livestock meat, offal, poultry eggs, and dairy products. Through a unified pretreatment process, it can be applied to these five types of animal-derived matrices without adjusting parameters, avoiding the need for repeated development of pretreatment schemes. It is also compatible with dual detection platforms, effectively simplifying operation steps, significantly improving detection efficiency, and significantly reducing sample volume and organic solvent consumption compared to traditional methods, thereby reducing detection costs and environmental burden. 2. Precise quantification of low-content pesticide metabolites: Addressing the characteristics of some pesticide metabolites that are present in extremely low concentrations and whose molecular structures change significantly after biological metabolism (e.g., the introduction of polar groups such as hydroxyl and sulfoxide groups), leading to substantial alterations in their physicochemical properties, this invention utilizes the advantages of dual-platform detection. It can match a dedicated detection platform based on the differences in metabolite polarity, thermal stability, and other properties, effectively overcoming matrix interference barriers associated with low-content metabolites and ensuring the accuracy and stability of the quantitative results. 3. Wide Detection Coverage: The unified sample pretreatment method adopted in this invention is compatible with both LC-MS / MS and GC-MS / MS dual-platform detection, and can be used in combination for the detection and analysis of pesticide residues in animal-derived samples. LC-MS / MS mainly covers polar targets, while GC-MS / MS mainly covers non-polar targets, simultaneously completing the analysis of pesticide parent compounds and metabolites. The detection range covers at least 500 pesticides and metabolites, significantly expanding the detection range compared to a single mass spectrometry platform. Simultaneously, it can complete the determination of target pesticide parent compounds and metabolites, such as carbofuran (detected by GC-MS / MS) and its metabolite 3-hydroxycarbofuran (detected by LC-MS / MS), ethion (detected by both GC-MS / MS and LC-MS / MS dual-platform) and its metabolite ethion sulfone (detected by LC-MS / MS), sulfonethiol-systemic sulfoxide (detected by both GC-MS / MS and LC-MS / MS dual-platform), and sulfone phosphonate (detected by LC-MS / MS), ensuring more comprehensive and accurate detection results. Attached Figure Description

[0042] Figure 1 This is a flowchart illustrating the detection method for pesticides and their metabolites in animal-derived samples. Figure 2 The GC-MS / MS chromatogram of a 100 ppb solvent standard is shown. Figure 3 The LC-MS / MS chromatogram is for a solvent standard of 100 ppb. Detailed Implementation

[0043] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0044] Example 1

[0045] This embodiment provides a method for detecting multiple pesticide and metabolite residues in animal-derived samples using a dual-platform LC-MS / MS and GC-MS / MS approach with a unified pretreatment adapted to multiple matrices. The animal-derived samples tested in this embodiment are meat (pork, beef, and mutton). A schematic flowchart of the method is shown below. Figure 1 As shown, the specific steps are as follows: 1. Sample preparation Take blank samples of fresh or thawed pork, beef and mutton, mince and homogenize them.

[0046] 2. Preparation of standard solutions

[0047] Standard stock solutions (concentration of 1000 μg / mL): Accurately weigh 10 mg (accurate to 0.00001 g) of each pesticide (pesticides and their metabolites shown in Tables 1-2) into different 10 mL volumetric flasks. Dissolve the pesticides in methanol or acetonitrile according to the solubility of the standard and the requirements of the assay, and dilute to the mark to prepare standard stock solutions with a concentration of 1000 μg / mL. Store below -20℃. Shelf life is 1 year.

[0048] Mixed standard stock solution (each pesticide concentration is 50 μg / mL): Accurately measure 0.5 mL of each of the above standard stock solutions into the same 10 mL volumetric flask, dilute to the mark with acetonitrile, and prepare a mixed standard stock solution with each pesticide concentration of 50 µg / mL. Store below -20℃. Shelf life is 6 months.

[0049] Mixed standard working solution (each pesticide concentration is 5 μg / mL): Accurately measure 1 mL of the above mixed standard stock solution into a 10 mL volumetric flask, dissolve it in acetonitrile and dilute to the mark to prepare a mixed standard working solution with each pesticide concentration of 5 μg / mL. Store below -20℃. Shelf life is 1 month.

[0050] 3. Pretreatment methods

[0051] Weigh 5g of sample into a 50mL centrifuge tube, add 5mL of water, and vortex for 1min until well mixed. Add 10mL of acetonitrile solution to the centrifuge tube and vortex vigorously for 1min. Add 3g of sodium chloride and continue to vortex vigorously for 1min. Centrifuge at 10000r / min for 5min at 4℃. After centrifugation, accurately pipette 1mL of the supernatant and transfer it to a 5mL centrifuge tube. Add 50mg of PSA and 100mg of C18, vortex for 1min, and filter the supernatant through a 0.22μm filter membrane for LC-MS / MS analysis. Accurately pipette 4mL of the supernatant and transfer it to a 5mL centrifuge tube. Concentrate the supernatant under nitrogen at 40℃ to a volume less than 1mL, and bring the volume to 1mL with acetonitrile. Add 50mg of PSA and 100mg of C18 to the concentrate, vortex for 1min, and filter the supernatant through a 0.22μm filter membrane for GC-MS / MS analysis.

[0052] 4. On-machine testing

[0053] The pretreated samples were then tested under the following conditions: (a) Chromatographic conditions for GC-MS / MS analysis: Chromatographic column: HP-5MS UI column (0.25 mm × 30.0 m, 0.25 μm, Agilent Technologies); The heating program is as follows: the initial temperature is 60℃, held for 1 min, then increased to 120℃ at a rate of 40℃ / min, then increased to 180℃ at a rate of 10℃ / min, held for 5 min, and finally increased to 310℃ at a rate of 5℃ / min, held for 3 min.

[0054] Injection method: splitless; Carrier gas: helium, purity ≥99.999%, flow rate 1.0 mL / min; Injection volume: 1 μL; Quantitative analysis using the external standard method.

[0055] Mass spectrometry conditions for GC-MS / MS analysis: EI ion source temperature 280℃; inlet temperature 280℃; transfer line temperature 280℃; solvent delay time 3 min; electron impact source voltage 70 eV; detection method: dynamic multiple reaction monitoring (dMRM).

[0056] (b) Chromatographic conditions for LC-MS / MS analysis: Chromatographic column: ZORBAX Eclipse Plus C18 Rapid Resolution HD column (3.0 mm × 150 mm, 1.8 μm, Agilent Technologies); Column temperature: 40℃; The mobile phase consists of phase A and phase B. Phase A is a 0.05% aqueous formic acid solution (containing 2 mmol / L ammonium formate), and phase B is a 0.05% methanolic formic acid solution; the flow rate is 0.4 mL / min. The elution method was gradient elution, and the gradient elution process was as follows: From 0 to 0.5 min, mobile phase A was 97% and mobile phase B was 3%; from 0.5 to 3 min, mobile phase A decreased from 97% to 50% and mobile phase B increased from 3% to 50%; from 3 to 9 min, mobile phase A decreased from 50% to 30% and mobile phase B increased from 50% to 70%; from 9 to 21 min, mobile phase A decreased from 30% to 0% and mobile phase B increased from 70% to 100%; from 21 to 26 min, mobile phase A was 0% and mobile phase B was 100%; from 26 to 26.1 min, mobile phase A increased from 0% to 97% and mobile phase B decreased from 100% to 3%; from 26.1 to 30 min, mobile phase A was 97% and mobile phase B was 3%.

[0057] Injection volume: 2 μL Quantitative analysis using the external standard method.

[0058] Mass spectrometry conditions for LC-MS / MS analysis: ESI source; Scanning method: simultaneous scanning of positive and negative ions; Electrospray voltage: 3500V for positive ions, 3500V for negative ions; Atomizing gas temperature: 300℃; Sheath gas temperature: 300℃; Detection method: dynamic multiple reaction monitoring (dMRM).

[0059] 5. Quantitative analysis

[0060] Based on the GC-MS / MS analysis results, the contents of 262 pesticides and their metabolites in the sample, as shown in Table 1, were determined by external standard quantification. Based on the LC-MS / MS analysis results, the contents of 436 pesticides and their metabolites in the sample, as shown in Table 2, were determined by external standard quantification.

[0061] 6. Methodological Validation

[0062] (1) Standard working curve and linear range

[0063] The prepared mixed standard working solutions were diluted with acetonitrile, pork blank matrix solution, beef blank matrix solution, and mutton blank matrix solution to prepare mixed standard working solutions with pesticide concentrations of 2.5 μg / kg, 5 μg / kg, 10 μg / kg, 50 μg / kg, 100 μg / kg, 200 μg / kg, and 500 μg / kg, respectively. The solutions were then analyzed using an instrument, and a standard working curve was plotted with the concentration of the mixed standard working solution as the x-axis and the peak area of ​​the instrument's response value as the y-axis. Within the concentration range of 2.5–500 μg / kg, the correlation coefficient R > 0.99, indicating that the detection method of this invention has good linearity. The aforementioned pork blank matrix solution, beef blank matrix solution, and mutton blank matrix solution refer to the supernatant obtained from blank samples of pork, beef, and mutton that do not contain pesticide residues or their metabolites, processed according to the pretreatment method described in step 3 above.

[0064] (2) Specificity of the method

[0065] Blank sample matrices of pork, beef, and mutton, free of pesticide residues and their metabolites, were pretreated according to the above method before measurement. The baseline of the blank sample matrices was flat near the retention time, and did not interfere with the experimental results.

[0066] (3) Limit of detection and limit of quantitation

[0067] The target pesticide standards (i.e., the specific pesticide residues and their metabolites shown in Tables 1-2) and the matrix-spikened samples of pork, beef and mutton were tested. Matrix-matched standard solutions with concentration gradients of 1-50 ppb were prepared, and the tests were performed on the instrument and the signal-to-noise ratio was calculated. The limit of detection (LOD) and limit of quantitation (LOQ) of the method were determined using the signal-to-noise ratio (S / N) method: the LOD was calculated with a signal-to-noise ratio of 3 (S / N=3), and the LQ was calculated with a signal-to-noise ratio of 10 (S / N=10).

[0068] The results showed that when using GC-MS / MS to detect the 262 pesticides and their metabolites shown in Table 1 in pork, beef, and mutton, the limits of detection for all targets were 2.5 μg / kg, and the limits of quantitation were all between 5 and 20 μg / kg. When using LC-MS / MS to detect the 436 pesticides and their metabolites shown in Table 2 in pork, beef, and mutton, the limits of detection for all targets were 2 μg / kg, and the limits of quantitation were all between 5 and 20 μg / kg.

[0069] (4) Accuracy and precision of the method

[0070] The accuracy of a method refers to the degree to which the obtained results correspond to the true values. The accuracy of a drug residue detection method is generally evaluated by the recovery rate. Using animal-derived products with negative test results as blank samples, 5.00 g of blank sample (in this example, pork, beef, and mutton blank samples were selected) was placed in a centrifuge tube. The target analyte concentrations were LOD, 50 μg / kg, and 100 μg / kg (here, "spiking concentration" means the theoretical concentration in the final test solution after complete recovery of the standard; the target analyte is the specific pesticide residue and its metabolites shown in Tables 1-2). After pretreatment according to the above detection method, the contents of 262 pesticides and their metabolites shown in Table 1 were determined by gas chromatography-tandem mass spectrometry (GC-MS / MS), and the contents of 436 pesticides and their metabolites shown in Table 2 were determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The spiked recovery rate was calculated. The recoveries of 262 pesticides and their metabolites in pork, beef, and mutton detected by GC-MS / MS ranged from 63.36% to 119.45%, 60.22% to 119.48%, and 60.55% to 118.65%, respectively. The recoveries of 436 pesticides and their metabolites in pork, beef, and mutton detected by LC-MS / MS ranged from 60.76% to 119.83%, 66.04% to 119.96%, and 60.79% to 119.98%, respectively. The specific detection results are shown in Tables 1 and 2. Figure 2 and Figure 3 The images show the TIC chromatograms of 262 pesticides and their metabolites at a solvent concentration of 100 ppb detected by GC-MS / MS and 436 pesticides and their metabolites at a solvent concentration of 100 ppb detected by LC-MS / MS.

[0071] The relative standard deviations among six parallel samples were calculated to evaluate the precision of the detection method. The precision of GC-MS / MS in detecting 262 pesticides and their metabolites in pork, beef, and mutton ranged from 0.65% to 14.89%, 1.06% to 19.92%, and 0.08% to 19.84%, respectively. The precision of LC-MS / MS in detecting 436 pesticides and their metabolites in pork, beef, and mutton ranged from 0.24% to 19.95%, 0.37% to 19.26%, and 0.18% to 19.84%, respectively. Specific detection results are shown in Tables 1 and 2.

[0072]

[0073] Example 2

[0074] In this embodiment, pig liver was selected as a representative matrix of viscera, and the method of Example 1 was used for detection and analysis.

[0075] Method validation results showed that the method exhibited excellent specificity and good linearity within the concentration range of 2.5–500 μg / kg. Regarding accuracy, the GC-MS / MS platform achieved recoveries of 61.94%–119.74% for the 262 target analytes in porcine liver (Table 1), while the LC-MS / MS platform achieved recoveries of 60.36%–119.83% for the 436 target analytes in porcine liver (Table 2). In terms of precision, the GC-MS / MS platform achieved precision of 1.95%–18.36% for the target analytes, while the LC-MS / MS platform achieved precision of 2.4%–19.98%. The limits of detection (LOD) and limits of quantitation (LOQ) showed that: when GC-MS / MS was used to detect 262 target substances in pig liver, the LOD for all components was 2.5 μg / kg, and the LOQ range was 5-20 μg / kg; when LC-MS / MS was used to detect 436 target substances in pig liver, the LOD for all components was 2 μg / kg, and the LOQ range was 5-20 μg / kg.

[0076] Example 3

[0077] In this embodiment, eggs were selected as a representative matrix for poultry eggs, and the method of Example 1 was used for detection and analysis. The difference lies in the sample preparation steps: an appropriate amount of fresh or refrigerated eggs were taken, shelled, and stirred evenly.

[0078] Method validation results showed that the method exhibited excellent specificity and good linearity within the concentration range of 2.5–500 μg / kg. Regarding accuracy, the GC-MS / MS platform achieved recoveries of 60.17%–111.40% for the 262 target analytes in eggs (Table 1), while the LC-MS / MS platform achieved recoveries of 60.96%–118.73% for the 436 target analytes in eggs (Table 2). In terms of precision, the GC-MS / MS platform achieved precision of 2.22%–19.81% for the target analytes, while the LC-MS / MS platform achieved precision of 1.11%–19.61%. The limits of detection (LOD) and limits of quantitation (LOQ) showed that: when GC-MS / MS was used to detect 262 target substances in eggs, the LOD for all components was 2.5 μg / kg, and the LOQ range was 5-20 μg / kg; when LC-MS / MS was used to detect 436 target substances in eggs, the LOD for all components was 2 μg / kg, and the LOQ range was 5-20 μg / kg.

[0079] Example 4

[0080] In this embodiment, chicken meat was selected as a representative matrix for poultry meat, and the method of Example 1 was used for detection and analysis.

[0081] Method validation results showed that the method exhibited excellent specificity and good linearity within the concentration range of 2.5–500 μg / kg. Regarding accuracy, the GC-MS / MS platform achieved recoveries of 62.15%–119.44% for the 262 target analytes in chicken (Table 1), while the LC-MS / MS platform achieved recoveries of 60.24%–119.78% for the 436 target analytes in chicken (Table 2). In terms of precision, the GC-MS / MS platform achieved precision of 0.31%–18.03% for the target analytes, while the LC-MS / MS platform achieved precision of 0.28%–19.08%. The limits of detection (LOD) and limits of quantitation (LOQ) showed that: when GC-MS / MS was used to detect 262 target substances in chicken, the LOD for all components was 2.5 μg / kg, and the LOQ range was 5-10 μg / kg; when LC-MS / MS was used to detect 436 target substances in chicken, the LOD for all components was 2 μg / kg, and the LOQ range was 5-20 μg / kg.

[0082] Example 5

[0083] In this embodiment, milk was selected as the representative matrix of dairy products, and the method of Example 1 was used for detection and analysis. The difference lies in the sample preparation steps: an appropriate amount of fresh or frozen milk was taken, mixed evenly, and no additional water was required during the pretreatment process.

[0084] Method validation results showed that the method exhibited excellent specificity and good linearity within the concentration range of 2.5–500 μg / kg. Regarding accuracy, the GC-MS / MS platform achieved recoveries of 62.74%–118.99% for the 262 target compounds in milk (Table 1), while the LC-MS / MS platform achieved recoveries of 63.11%–115.37% for the 436 target compounds in milk (Table 2). In terms of precision, the GC-MS / MS platform achieved precision of 0.65%–16.36% for the target compounds, while the LC-MS / MS platform achieved precision of 0.18%–19.17%. The limits of detection (LOD) and limits of quantitation (LOQ) showed that: when GC-MS / MS was used to detect 262 target compounds in milk, the LOD for all components was 2.5 μg / kg, and the LOQ range was 5-10 μg / kg; when LC-MS / MS was used to detect 436 target compounds in milk, the LOD for all components was 2 μg / kg, and the LOQ range was 5-20 μg / kg.

[0085] As can be seen from the above, the detection method of the present invention has a unified pretreatment method that is compatible with multiple animal-derived matrices and dual detection platforms. The detection range covers at least 500 kinds of pesticides and metabolites, and the quantitative results are accurate and stable.

Claims

1. A method for detecting pesticides and their metabolites in animal-derived samples, comprising the following steps: (1) The extraction solvent is mixed with the homogenized animal sample and extracted to obtain the extraction system; (2) Add salt to the extraction system and perform salting-out treatment to obtain animal-derived sample extract: (3) The animal-derived sample extract was purified with a purification agent to obtain a first animal-derived sample extract purified solution for LC-MS / MS analysis and detection; After nitrogen blowing concentration and redissolution, the animal-derived sample extract was purified with a purification agent to obtain a second purified animal-derived sample extract for GC-MS / MS analysis and detection. (4) The first animal-derived sample extract and purified solution was analyzed and detected by LC-MS / MS, and the second animal-derived sample extract and purified solution was analyzed and detected by GC-MS / MS.

2. The detection method according to claim 1, wherein, The extraction solvent includes acetonitrile and / or acidified acetonitrile; the salt includes one or more of sodium chloride, sodium acetate, magnesium sulfate, and citrate.

3. The detection method according to claim 1, wherein, The purifying agent includes PSA and / or C18.

4. The detection method according to claim 1, wherein, In step (1), when the animal source sample is a non-liquid animal source sample, water is added to the homogenized non-liquid animal source sample and then mixed with the extraction solvent.

5. The detection method according to claim 1, wherein, The detection method includes the following steps: (1) Weigh 0.5-5 g of homogenized animal source sample. When the animal source sample is a non-liquid animal source sample, add 0.5-5 mL of water to the non-liquid animal source sample, mix well, add 1-10 mL of extraction solvent, shake, and obtain a non-liquid animal source sample extraction system. When the animal source sample is a liquid animal source sample, add 1-10 mL of extraction solvent to the liquid animal source sample, shake, and obtain a liquid animal source sample extraction system. (2) Add salt to the non-liquid animal sample extraction system or the liquid animal sample extraction system, shake, centrifuge, and obtain the supernatant as the animal sample extract; (3) Take 1-2 mL of the animal-derived sample extract, add 50-100 mg of PSA and 100-200 mg of C18, vortex mix, filter through a microporous membrane to obtain the first animal-derived sample extract purified solution for LC-MS / MS analysis and detection. Take 4-8 mL of the animal-derived sample extract, concentrate it under nitrogen blowing at 30-60℃, make up to volume with acetonitrile and / or acidified acetonitrile, add 50-100 mg of PSA and 100-200 mg of C18, vortex mix, filter through a microporous membrane to obtain the second animal-derived sample extract purified solution for GC-MS / MS analysis and detection. (4) The first animal-derived sample extract and purified solution was analyzed and detected by LC-MS / MS, and the second animal-derived sample extract and purified solution was analyzed and detected by GC-MS / MS.

6. The detection method according to claim 1, wherein, The chromatographic conditions for GC-MS / MS include: The chromatographic column was a (5%-phenyl)-methylpolysiloxane column; The mass spectrometry conditions for GC-MS / MS include: The ion source is an EI ion source with a temperature of 200-300℃; the electron bombardment source voltage is 50-80eV. The chromatographic conditions for LC-MS / MS include: The chromatographic column is an octadecylsilane reversed-phase column; LC-MS / MS mass spectrometry conditions include: The ion source is an ESI source; the electrospray voltage in positive ion mode is 3000-4500V, and the electrospray voltage in negative ion mode is 3000-4500V; the atomizing gas temperature is 300-350℃; the sheath gas temperature is 280-350℃.

7. The detection method according to claim 1, wherein, The chromatographic temperature program for GC-MS / MS includes: an initial temperature of 60℃, holding for 1 min, then increasing the temperature to 120℃ at a rate of 40℃ / min, then increasing the temperature to 180℃ at a rate of 10℃ / min, holding for 5 min, and then increasing the temperature to 310℃ at a rate of 5℃ / min, holding for 3 min. For chromatographic detection by LC-MS / MS, mobile phase A was 0.05% formic acid aqueous solution with ammonium formate concentration of 2 mmol / L, and mobile phase B was 0.05% formic acid methanol; the flow rate was 0.4 ml / min. The chromatographic elution program for LC-MS / MS, expressed as a volume fraction of mobile phase A, includes: 0-0.5min: 97%; 3min: 50%; 9min: 30%; 21-26 min: 0%; 26.1-30min: 97%.

8. The detection method according to claim 1, wherein, The animal-derived samples include one or more of the following: meat, poultry, offal, eggs, and dairy products.

9. The detection method according to claim 1, wherein, The pesticides and their metabolites include terbufos, butachlor, butachlor, butifluzamide, cyazofamid, azoxystrobin, chlorpyrifos, parathion, paclobutrazol, oxadiazon, oxazolidinone, oxadiazon, diphenylamine, pendimethalin, diazinon, fenpyroximate, furazolidone, fipronil, fenpyroximate, flufenoxuron, flufenoxuron sulfonate, flusilazole, flutriafol, flutriafol, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, flufenoxuron, quizalofop-p-ethyl, quizalofop-p-ethyl, cyproconazole, cyproconazole, cyproconazole, cyproconazole sulfonate, cyproconazole sulfoxide, cyproconazole ether. Hexaconazole, phorate, phorate sulfoxide, phorate sulfone, metolachlor, methyl chlorpyrifos, methyl parathion, methyl thiophanate, methyl pyrimidin, methyl isofenphos, cypermethrin, carbofuran, fenfluramide, pirimicarb, carbofuran, 3-hydroxycarbofuran, quinalphos, quinfenoxam, quinoxal, dimethoate, bifenazate, bifenthrin, bifenthrin, phosphamidon, α-endodole, β-endodole, endodole sulfate, thiocyclam, thionyl, chlorfenapyr, chlorfenapyr, chlorfenapyr, chlorfenapyr-trans, chlornitramine, chlorpyrifos, prochloraz and prochloraz manganese salt, imazalil, pyrimethanil, pyrimethanil, pyrimethanil, pyrimethanil, pyrimethanil Methyl methamidophos, difenoconazole, ethoprophos, naphthaleneacetic acid and sodium naphthaleneacetate, piperazine, promethazine, cyhalofop-butyl, cypermethrin and S-cypermethrin, benzoyl methamidophos, propargite, fenthiamethoxam, thifluzamide, benzalkonium chloride, oxadiazon, o,p'-trichlorfon, p,p'-trichlorfon, trichlorfon sulfone, triazole alcohol, triazole phosphate, triazole ketone, amitraz, fenitrothion, chlorpyrifos, methamidophos, tetraflufenozide, tetrachlorophthalide, phthalide, tetrachloronitrobenzene, tetradifon, terbufos, terbufos sulfone, pyriproxyfen, carbendazim, azoxystrobin, pentachloronitrobenzene, pentachloroaniline, tebuconazole, tebuconazole, cymoxanil, simazine, tebuconazole , octanoyl bromoxynil, bromofenac, bromodiphenyl ether, phosmet, phosmet, phorate, thiophanate-methyl, acetochlor, ethion, ethoxyphos, acetochlor, ethoxyphos, acetochlor, pyrifluquinazon, ethirimol sulfonate, ethylene sclerotinia, acephate, ethoxysulfuron, ethoxyflufenoxam, acetochlor, metolachlor and succinyl-metolachlor, isoprocarb, isoxaflutole, isoxaflutole, imazalil, phosmet, atrazine, atrazine, synergist, phosmet, tebuconazole, terbufos, terbufos, acetochlor, azoxystrobin, chlorpyrifos, aldrin, 4,4'-DDT, 2,4'-DDT, 4,4'-DDT, 2,4'-DDT, 2,4'-DDT, Dildrin, Pyrazosulfuron, γ-HCH, α-HCH, β-HCH, δ-HCH, Cis-Chlordane, Trans-Chlordane, Oxychlordane, Isodrin, Isodrinaldehyde, Isodrinone, Nitraz-methyl, Ethylbutazone, Isofenphos, Azoxystrobin, Bromobenzylphos, Pyrazosulfuron, Defoliant, Trithion, Phoxim, Pyrimethanil, Pyrimethanil, Pyridaben, Cyprodinil, Chlorpyrifos, Isopropoxyfen, Cyprodinil, Phoxim, Dimethoate, Fonsophos, Pyraclostrobin, Deethylatrazine, Chlormethrin, Pyrimethanil, Prochloraz, Butylpyridinium, Bromothion, Cyfluthrin, Propiconazole, Benomyl, Thiophanate-methyl, Ethylpyridinium, Malaoxyfen, Phoxim, Phoxim, Pyrimethanil, Pyrimethanil, Pyrimethanil, Ethylbromothion, Propiconazole Phosphorus, chlorpyrifos, terbufos, isopropylfen, fenpropathrin, fluquinazole, thiophanate-methyl, dibromophos, flubutyroxyfen, herbicides, biphenyl, tebufenozide, chlorfenapyr, etoxazole, atrazine, dichlorvos, dicofol, trifluralin, ethyl ester, DDT, parabromophos, sulfadiazine, isopropylthiophanate-methyl, fluroxypyr, spirodiclofen, propyzoxystrobin, etoxazole, propyzoxystrobin, anthraquinone, abamectin, furazolidone, chlorpyrifos, dimethyl chlorphthalate, oat chlorpyrifos, fenpyroximate, dioxin, fluroxypyr, flufenoxuron, fludioxonil, chlorfenapyr, cyclohexane, fenpyroximate, fenpyroximate, cyclohexane, fenpyroximate, 2-methyl-4-chloroisooctyl ester, pyridaben, thiophanate-methyl, oxychlorpyrifos, methyl methacrylate, pyridaben, fenpyroximate, pyridaben, difenoconazole, quizalofop-p-ethyl, etoxazole Furazolidone, Heptenphos, Glyphosate, Flufenoxuron, Terbufos, Isoprothiolane, Procymidone, Prochloraz, Prochloraz, Dichlorvos, Dichlorvos, Difenoconazole, Difenoconazole, Butyral, Butachlor, Eutrimethoate, Acetamiprid, Pyrimethanil, Flufenoxuron, Carbendazim, Oxadiazon, Oxadiazon, Sulfonamide, Flufenoxuron, Flubendiamide, Flufenoxuron, Flupyrsulfuron, Pyrimethanil, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, 4-Fluoro-N-isopropylaniline, Hydroxychlorothalonil, Flufenoxuron, Flufenoxuron, Flufenoxuron, Flufenoxuron, 4-Nitrophenol Sodium, Silthiazamide, Cyprosulfuron, Cyclosulfuron, Emamectin benzoate Formate, mesosulfuron, mesosulfuron-methyl, sodium iodosulfuron, mesosulfuron-methyl, methyl thiocyclam, thiocarb, carbaryl, benzylfloxacin, metalaxyl and cymoxanil, methoxyfenozide, quizalofop-p-ethyl, phosmet, methyl pymetrozine, methyl-formylamino-pymetrozine, quizalofop-p-ethyl, bensulfuron-methyl, spirotetramat, spirotetramat-keto-hydroxy, spirotetramat-mono-hydroxy, spirodiclofen, chlorpyrifos, chlorpyrifos, chlorpyrifos, permethrin, chlorpyrifos, cypermethrin, chlorthiazoline, bensulfuron-methyl, ether bensulfuron-methyl, fensulfuron-methyl, fenpyroxime, pyrimethanil, pyrimethanil-4-hydroxy, pyrimethanil-5-hydroxy, 1-[2-chloro-4-(chlorophenoxy)-phenyl]-2-(1,2,4-Triazole)-1-yl-ethanol), Bensulfuron, Diflubenzuron, Demeton-methyl, Azoxystrobin, Pyrazosulfuron, Bensulfuron-methyl, Cyfluthrin, Cyazofamid, Cyazofamid metabolite, Cyazofamid, Lactobacillus, Thiaben, Thiamethoxam, Thiamethoxam, Thiamethoxam, 5-Hydroxyfen, Thiamethoxam, Thiazidone, Benzoylamidone, Thiamethoxam, Tricyclazole, Triflubenzuron, Benzoylphos, Benzoylphos sulfonate, Benzoylphos sulfoxide, Lufenuron, Lufenuron, Amitraz, Amitraz and Amitraz hydrochloride, Dimethomorph, Cymoxanil, Terbufos sulfoxide, Aldicarb, Aldicarb sulfonate, Aldicarb sulfoxide, Carbendazim sulfoxide, Oxycarb, Pentazon Flusulfanil, clethodim, clethodim sulfonium, clethodim sulfoxide, acetamiprid, clethodim, acetamiprid, imidacloprid, acetamiprid ester, dimethomorph, uniconazole, phoxim, bromobenzonitrile, bromocyanamide, deltamethrin, phosmet, oxammonium phosphate, quizalofop-P-ethyl and succinyl quizalofop-P-ethyl, sulfonium phosphate, methyl parathion, sulfonium parathion, omethoate, ivermectin, ethion sulfonium phosphate, ethionyl, ethoxysulfuron, acetamiprid, ethirimol, ethoxysulfuron, isoproturon, pyraclostrobin, (1-methyl-3-trifluoromethyl-1H-pyrazol-4-yl)formamide, indazolesulfonamide, azadirachtin, indoxacarb, rotenone, azoxystrobin, pyraclostrobin, azoxystrobin, pyraclostrobin, pyraclostrobin, pyraclostrobin Acetaminophen, pyraclostrobin, pyraclostrobin, methyl parathion, bensulfuron-methyl, ethyl parathion, oxyfenozide, oxyfenozide sulfone, fenozide sulfone, dextromethorphan, propoxur, fenoxam, oxazin, dioxamethoxam, chlorpyrifos, carbendazim, chlorpyrifos, flufenoxuron, chlorpyrifos, isofenphos, methamidophos, flupyridine, bifenthion, acetamiprid, trifluralin, fluthiazopyr, naphthylacetamide, 2-amino-4-methoxy-6-methyl-1,3,5-triazine, tetrazolium-sulfuron-methyl, bensulfuron-methyl, bromadiolone, butachlor, profenofos, carbaryl, phosphonocarboxylic acid, calcitriol, cyclone dichlorvos, indole-methyl, metolachlor, ether Azoxystrobin, terbufos, 4,6-dinitro-o-cresol, fenpicarbazole, pyrimisulfuron, chlorpyrifos, fluazinam, fenpyroxime, imidacloprid, iodobenzonitrile, isoprothiolane, tebufenozide, linuron, toluene-methyl, chlorpyrifos, fenpyroxime, pyrethroid I, pyrethroid II, methylbenzylthiazoline, sulfadiazine, metribuzin, pendimethalin, clethodim, fenpyroxime, flupyrfluthrin, cyclobenzyl, propoxyquin, bensulfuron-methyl, flusulfuron-methyl, methoxysulfuron-methyl, cyhalothrin, sulfadiazine, sulfonylsulfuron-methyl, benzoylsulfuron-methyl, cyhalothrin, fenpyroxime, fenpyroxime, trifluralin, warfarin, methomyl, oxadixyl, methyl ethyl sulfide, bensulfuron-methyl, dioxin, N,One or more combinations of N-dimethylamino-N-toluene, sulfothiamethoxam, ethoxybenzamide, fenvalerate, fenugreek, chlorfenapyr, terbufenozide, furazolidone, succinylcholine, methoxyfenozide, chlorfluazuron, methyl parathion, cyclophosphamide, dithion, pymetrozine, fluopyram, mancozeb, oxychlorpyrifos, chlorpyrifos, pymetrozine, pyrimethanil, isoprothiolane, abamectin, isoprothiolane, and diuron.

10. The detection method according to claim 1, wherein, The detection limit of the LC-MS / MS is ≤2 μg / kg, and the quantitation limit is 5-20 μg / kg; And / or, the detection limit of the GC-MS / MS is ≤2.5 μg / kg, and the quantitation limit is 5-20 μg / kg.

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