CN115128195B - LC-MS/MS detection method for pesticide and veterinary drug residues in animal oil and vegetable oil - Google Patents

LC-MS/MS detection method for pesticide and veterinary drug residues in animal oil and vegetable oil Download PDF

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CN115128195B
CN115128195B CN202211058741.6A CN202211058741A CN115128195B CN 115128195 B CN115128195 B CN 115128195B CN 202211058741 A CN202211058741 A CN 202211058741A CN 115128195 B CN115128195 B CN 115128195B
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邵兵
戚燕
靳玉慎
姚凯
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Beijing Center for Disease Prevention and Control
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract

The invention relates to an LC-MS/MS detection method for pesticide and veterinary drug residues in animal oil and vegetable oil, which comprises the following steps: (S1) extracting: mixing an animal oil and/or vegetable oil sample with a buffer solution, adding an extraction solvent, a water removing agent and a salting-out agent after shaking, centrifuging after vortex shaking, taking supernate, and purifying; (S2) purification: mixing the liquid to be purified obtained in the step (S1) with a purifying agent, performing vortex oscillation, centrifuging, taking supernatant, drying with nitrogen, redissolving, and filtering to obtain a liquid to be detected; the purifying agent is an amino-functionalized covalent organic framework material which is formed by orderly arranging a honeycomb porous structure and has a three-dimensional loose porous nanosphere structure; (S3) mass spectrum detection: measuring the solution to be measured in the step (S2) by liquid chromatography-tandem mass spectrometry; and quantifying by using an external standard method to obtain the content of the veterinary drug. The detection method has good detection effect on the pesticide and veterinary drug residues in most of animal oil and vegetable oil.

Description

LC-MS/MS detection method for pesticide and veterinary drug residues in animal oil and vegetable oil
Technical Field
The invention relates to the field of food safety, in particular to an LC-MS/MS detection method for pesticide and veterinary drug residues in animal oil and vegetable oil.
Background
The grease is an indispensable nutrient substance for the human body to maintain normal activities of life. Contains essential fatty acids which can not be synthesized by human body, participates in phospholipid synthesis, and can be combined with cholesterol to prevent arteriosclerosis, hypercholesterolemia and hyperlipidemia, and the essential fatty acids can only be obtained from food. The added grease can not only increase the flavor of the food, but also improve the mouthfeel. Therefore, with the continuous improvement of living standard, people can not eat animal oil and vegetable oil in life. During the cultivation of animals and plants, various pesticides and veterinary drugs are generally used to ensure the health of animals and plants in order to prevent diseases. Meanwhile, after animals eat feed containing pesticide residues, the pesticide in the organisms can be enriched. After eating the vegetable oil and the animal oil with pesticide and veterinary drug residues, human bodies can have serious influence on health.
At present, few researches are carried out on analysis methods of pesticide and veterinary drug residues in animal oil and vegetable oil, and the main focus is on animal muscle tissues and samples such as vegetables and fruits. The problem of pesticide residues in animal and vegetable oil samples is easily overlooked by people. The two samples have complex components and high fat content, which seriously affects the detection of pesticides and veterinary drugs, and how to effectively remove fat is the premise and difficulty for obtaining accurate determination results. The prior pretreatment method mainly adopts n-hexane and/or freezing method for degreasing, which not only has large usage amount of organic solvent, but also has long operation time and unsatisfactory purification effect.
The main pretreatment methods for removing oil include solid-phase extraction, liquid-liquid extraction and gel permeation chromatography for removing fat. However, the method consumes a large amount of organic solvent, is not long-lasting and complex to operate, and is not suitable for practical application and commercial production. More importantly, in fat-rich foods, lipophilic analytes are also lost due to non-specific adsorption, resulting in insufficient sensitivity of detection. In fat-rich foods, with many fats and free fatty acids, different carbon chain lengths, it is not practical to develop porous materials that cover the full size of the lipid. QuEChERS (Quick, easy, cheap, effective, rugged, safe) is of great interest to researchers because of its simplicity and rapidity of operation. QuEChERS is a novel food sample pretreatment method based on matrix solid phase dispersion, and is used for extracting components to be detected and removing redundant impurities simultaneously through extraction and purification. Has the advantages of rapidness, simplicity, low price, high efficiency, reliability and safety. However, there is a lack of suitable materials suitable for effective treatment of fats and oils in animal fats and vegetable oils.
The inventor's prior patent CN202210989648.0 discloses an amino ligand replacement covalent organic framework material, which is used as a QuEChERS purifying agent, can effectively remove grease in various foods, and provides technical dependence and support for sample pretreatment. The invention is based on the novel covalent organic framework material with functional group amino, which is used for adsorbing and removing animal oil and vegetable oil in food samples and efficiently and accurately detecting pesticide and veterinary drug residues in the animal oil and the vegetable oil.
Disclosure of Invention
The invention aims to provide an LC-MS/MS detection method for detecting pesticide and veterinary drug residues in animal oil and vegetable oil by replacing covalent organic framework materials with amino ligands as QuEChERS purificant aiming at the defect of lack of an effective method for detecting the pesticide and veterinary drug residues in the animal oil and the vegetable oil in the prior art.
The invention realizes the purpose through the following technical scheme:
an LC-MS/MS detection method for pesticide and veterinary drug residues in animal oil and vegetable oil comprises the following steps:
(S1) extracting: mixing an animal oil and/or vegetable oil sample with a buffer solution, adding an extraction solvent, a water removing agent and a salting-out agent after shaking, centrifuging after vortex shaking, and taking a supernatant for purification;
(S2) purification: and (3) mixing the liquid to be purified obtained in the step (S1) with a purifying agent, carrying out vortex oscillation, centrifuging, taking supernatant, drying with nitrogen, redissolving, and filtering to obtain the liquid to be detected.
(S3) mass spectrum detection: separating and measuring the liquid to be measured in the step (S2) by liquid chromatography-tandem mass spectrometry (LC-MS/MS); quantifying by an external standard method to obtain the content of the veterinary and agricultural medicines;
the purifying agent in the step (S2) is an amino functionalized covalent organic framework material, is formed by orderly arranging a honeycomb porous structure, has a three-dimensional loose porous nanosphere structure, simultaneously has macropores and mesopores, and has the average particle size of 500-1000nm; the amino functionalized covalent organic framework material is prepared by taking diamine and polyaldehyde as monomers, polymerizing the monomers in the presence of micelles formed by quaternary ammonium cationic surfactant to obtain three-dimensional nanospheres, adding excessive polyamine to replace the diamine by Building Block Exchange (BBE) and modify the diamine with amino functional groups, and finally washing the quaternary ammonium cationic surfactant to obtain the amino functionalized covalent organic framework material.
In the preparation process of the purifying agent, the quaternary ammonium salt cationic surfactant is used as a structure directing agent to form a micelle containing a hydrophobic chain, the added monomer diamine and the added polybasic aldehyde are polymerized around an alkane chain of the micelle under the action of the hydrophobic chain to form a loose and porous nanosphere structure with a three-dimensional structure, and if the quaternary ammonium salt cationic surfactant does not exist, the monomer polymerization can only obtain a two-dimensional structure. The resulting covalent organic framework is then used to introduce functional amino groups by adding an excess of polyamino compounds. The prepared amino functionalized covalent organic framework material has a loose and porous spherical shape, the average size is about 700 nm, and the material has a mesoporous/macroporous channel. The introduction of the cationic surface activity of the quaternary ammonium salt obviously increases the specific surface area of the material, and is beneficial to the promotion of the mass transfer rate in the adsorption process.
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Further, in the step (S1), the animal oil comprises one or more of lard, beef tallow, mutton tallow, fish oil, bone marrow, fat meat, fish liver oil and the like; the vegetable oil comprises one or more of soybean oil, rapeseed oil, palm oil, olive oil, peanut oil, sunflower seed oil and the like; the buffer solution is 0.1-0.3M of Na 2 EDTA-Mclvaine, acetic acid-sodium acetate, citric acid-sodium citrate and sodium citrate-disodium hydrogen citrate; the extraction solvent is at least one of acetonitrile, methanol, formic acid water, acetic acid acetonitrile and acetic acid methanol; the proportion of the sample containing the animal oil and/or the vegetable oil, the buffer solution and the extraction solvent is 1g:1-2mL:3-5mL; the mass ratio of the animal oil and/or vegetable oil sample to the water removing agent to the salting-out agent is 1:10-15:0.3-0.6; the water removing agent is selected from anhydrous sodium sulfate and/or anhydrous magnesium sulfate; the salting-out agent is selected from at least one of sodium chloride, sodium acetate, ammonium acetate and sodium citrate; centrifugal operation after vortex oscillationVortex and shake for 1-5 min, and centrifuge at 8000-12000 rpm at 0-4 deg.C for 10-30 min.
Further, in the step (S2), the volume-to-mass ratio of the liquid to be purified to the purifying agent is 1mL:15-25mg; the centrifugation after vortex oscillation is that the vortex oscillation time is 30 to 60 s, and then the centrifugation is carried out for 5 to 15min under the conditions of 0 to 4 ℃ and 14000 to 20000 rpm; the redissolving solvent is methanol/water with the volume ratio of 1-2 to 1-2, and a 0.22 mu m organic phase filter membrane is adopted for filtration.
Further, in the step (S3), the liquid chromatography is ultra high performance liquid chromatography (UPLC), and ACQUITY UPLC HSS T3 (2.1 mm. Times.100 mm,1.8 μm) is used for detecting the pesticide; the mobile phase adopted by the ultra-high performance liquid chromatography is as follows: 1-2 mM ammonium formate +0.01-0.02% (v/v) aqueous formic acid (A) and 1-2 mM ammonium formate +0.01-0.02% (v/v) methanolic formic acid solution (B); gradient elution procedure is 3% B (0-1.0 min), 3% -15% B (1.0-1.5 min), 15% -50% B (1.5-2.5 min), 50% -70% B (2.5-18.0 min), 70% -98% B (18.0-23.0 min), 98% B (23.0-27.0 min), 98% -3% B (27.0-27.1 min), 3% B (27.1-30.0 min); the flow rate is 0.1-0.3 mL/min; the column temperature is 40-45 ℃, and the sample injection amount is 2-4 mu L;
for the detection of veterinary drugs, ACQUITY UPLC BEH C18 (2.1 mm. Times.100 mm,1.7 μm) was used; the mobile phase adopted by the ultra-high performance liquid chromatography is as follows: 0.2-0.5 mM ammonium fluoride +0.1-0.2% (v/v) formic acid water (a) and acetonitrile/methanol (v/v = 1/1) (B); flow rate: 0.3 mL/min; gradient elution procedure is 3% B (0-2.0 min), 3% -15% B (2.0-5.0 min), 15% B (5.0-10.0 min), 15% -30% B (10.0-15.0 min), 30% -50% B (15.0-20.0 min), 50% -100% B (20.0-24.0 min), 100% B (24.0-28.0 min)), 3% B (28.5-29.0 min); the flow rate is 0.1-0.3 mL/min; the column temperature is 40-45 ℃, and the sample injection amount is 1-3 mu L.
Further, in the amino-functionalized covalent organic framework material, the quaternary ammonium cationic surfactant is cetyltrimethylammonium bromide (CTAB), the diamine is 1, 4-Phenylenediamine (PA), the polyaldehyde is 1,3, 5-tris (p-formylphenyl) benzene (TFPB), and the polyamine is diaminobenzidine (BD-NH) 2 ) Obtained byThe amino-functionalized covalent organic framework material of (a) is named CTAB @ TFBD-NH 2 (ii) a The molar ratio of the quaternary ammonium salt cationic surfactant to the diamine to the polyaldehyde is 0.8-1.2:1.5-2.0:1.0-1.2:10-15.
CTAB/TFBD-NH prepared by the invention 2 The nanospheres have appropriate mesoporous pore diameter (34.9A-46.1A) and functional groups (free amino groups) to remove lipid, and have good removal effect on lipid in animal and vegetable oil.
Further, the amino functionalized covalent organic framework material provided by the invention is a loose porous three-dimensional porous nanosphere structure with both mesopores and macropores, the average diameter is 500-1000nm, preferably 700-800 nm, and the specific surface area (BET) is 400-600 m 2 (ii)/g, the mesoporous pore diameter is from 20 to 50A, preferably from 30 to 50A; the pore diameter of the macropores is 50-300 nm, preferably 50-200 nm.
The resulting amino-functionalized covalent organic framework material has an ordered arrangement of high crystallinity, consisting of an ordered arrangement of cellular porous structures having pore diameters from 34.9 a to 46.1 a.
Further, the preparation method of the amino functionalized covalent organic framework material comprises the following steps:
(P1) adding a solution in which diamine, polyaldehyde and a catalyst are dissolved into a quaternary ammonium salt cationic surfactant aqueous solution, carrying out ultrasonic treatment on the mixture, carrying out circulating freeze-pumping, carrying out high-temperature reaction, and cooling to room temperature for later use after the reaction is finished;
and (P2) adding a solution of polyamine and a catalyst into the system obtained in the step (P1), carrying out ultrasonic treatment on the mixture, circularly freezing and pumping, heating for reaction, and centrifuging, extracting and drying the product to obtain the catalyst.
Further, in the step (P1), the concentration of the quaternary ammonium salt cationic surfactant in the aqueous solution of the quaternary ammonium salt cationic surfactant is 0.05 to 0.10 mol/L, and at this concentration, micelles having a suitable size are formed. Preferably, the concentration of the quaternary ammonium salt cationic surfactant is 0.06-0.08 mol/L.
In the step (P1), a solution in which a diamine, a polyaldehyde and a catalyst are dissolved, and in the step (S2), a solution in which a polyamine is dissolved in a solvent of at least one of 1, 4-dioxane, n-butanol, o-dichlorobenzene, 1,3, 5-trimethylbenzene, benzene, toluene and xylene, preferably 1, 4-dioxane and 1,3, 5-trimethylbenzene in a volume ratio of 1 to 2: 1-2.
The catalyst is acetic acid, and in the step (P1), the amount of the catalyst is 1 to 2 times, preferably 1.5 to 1.7 times, the amount of the monomer material; in step (P2), the amount of the catalyst used is 0.1 to 0.2 times, preferably 0.12 to 0.15 times the amount of the polyamine substance.
Further, in the steps (P1) and (P2), the number of times of freezing and pumping is 3-5 times in a circulating way; the operation of circulating freeze-thaw is well known in the art, i.e., circulating freeze-thaw-vacuum-thaw; in one embodiment of the invention, the circulating freezing and pumping is to put the glass tube filled with the mixture into liquid nitrogen for freezing, and vacuumize in the thawing process, wherein the vacuum degree is 0.1-10 kPa; the ultrasonic treatment time is 10-30 min, and the materials are uniformly mixed.
Further, in the step (P1), the high-temperature reaction temperature is 100-150 ℃, and the reaction time is 2-4 days; in the step (S2), the reaction temperature is heated to 30-60 ℃ and the reaction time is 2-4 days.
Further, in the step (P2), after the reaction is finished, centrifuging to remove the solvent, washing, performing Soxhlet extraction, and performing vacuum drying to obtain a product; the washing solvent is selected from water, ethanol, and acetone; the solvent for Soxhlet extraction is at least one of acetone, 1, 4-dioxane and tetrahydrofuran.
The purifying agent used by the invention is an amino functionalized covalent organic framework material, and is a three-dimensional nanosphere with macropores/mesopores, the average diameter is 500-1000nm, preferably 700-800 nm, and the specific surface area (BET) is 400-600 m 2 The specific surface area is increased by about 10 times compared to the polymer obtained in the absence of micelles, which is due to the loose and porous structure of the three-dimensional loose nanospheres obtained in the presence of micelles, the larger specific surface area providing more reaction sites and active adsorption sites.
Drawings
FIG. 1 is a schematic representation of the LC-MS/MS detection method for veterinary drug residues in animal and vegetable oils of the present invention;
FIG. 2 is a diagram of the synthesis of amino-functionalized covalent organic framework material CTAB @ TFBD-NH of the present invention 2 A schematic diagram of (a);
FIG. 3 shows TFBD-NH obtained in preparation example 2 CTAB @ TFPA and CTAB @ TFBD-NH 2 HRTEM images and SEM images of;
FIG. 4 shows TFPA, CTAB/TFPA, TFBD-NH obtained in example 1 2 And CTAB/TFBD-NH 2 Nitrogen adsorption-desorption isotherm.
Detailed Description
All reagents were purchased from commercial suppliers and used without further purification.
Preparation example 2 Synthesis of CTAB @ TFBD-NH
The preparation method is described in patent CN202210989648.0, and comprises the following specific steps:
cetyl trimethylammonium bromide (CTAB, 32.8 mg,0.09 mmol) was placed in a Schlenk tube, 1.3 mL of ultrapure water was added for ultrasonic dissolution, glacial acetic acid (0.6 mL, 6 mol/L), 1, 4-phenylenediamine (PA, 16.3 mg,0.15 mmol) and 1, 4-dioxane (1.5 mL) were added, and the mixture was stored in a refrigerator to prevent oxidation of PA. A solution containing 1,3, 5-tris (p-formylphenyl) benzene (TFPB, 39.0 mg,0.1 mmol) was added as a mixed solvent of 1.5 mL of 1, 4-dioxane and 3 mL of 1,3, 5-trimethylbenzene. The mixture is treated by ultrasonic for 10min to be mixed evenly, liquid nitrogen freezing-methanol unfreezing circulation degassing is carried out for three times, the mixture is heated for three days at 120 ℃ under the vacuum condition, and after the mixture is cooled to room temperature, a polymer which takes CTAB as a structure guiding agent and PA and TFPB as monomers is obtained, and the polymer is called CTAB @ TFPA. Subjecting Schlenk tube containing CTAB @ TFPA to ultrasonic treatment for 10min, and adding diaminobenzidine (BD-NH) 2 321.4 mg,1.5 mmol), 1, 4-dioxane (2.0 mL), 1,3, 5-trimethylbenzene (1.0 mL) and 6M acetic acid (0.3 mL), the mixture was sonicated for 10min, degassed by three liquid nitrogen freeze-methanol thaw cycles, heated at 40 ℃ for three days under vacuum sealed conditions to give a dark red precipitate, centrifuged to remove the solvent, washed with ultrapure water, extracted with THF in a Soxhlet extractor, then dried at 120 ℃ under vacuum for 12 hObtaining the amino-functionalized covalent organic framework material called CTAB @ TFBD-NH 2 . The synthetic scheme is shown in figure 2.
The invention further provides a synthesis route and researches and explains the morphological change of the amino functionalized covalent organic framework material with the three-dimensional structure. The invention also carries out the following synthesis:
synthesis of TFPA:
Figure 191567DEST_PATH_IMAGE001
to a 20 mL Schlenk tube were added PA (16.3 mg,0.15 mmol) and TFPB (39.0 mg,0.1 mmol), 1, 4-dioxane (3.0 mL), 1,3, 5-trimethylbenzene (3.0 mL), and 6M acetic acid (0.6 mL). The mixture was sonicated for 10 minutes to obtain a homogeneous dispersion, degassed by three liquid nitrogen freeze-pump-methanol thaw cycles, sealed under vacuum and heated at 120 ℃ for three days. The yellow precipitate obtained was centrifuged to remove the solvent, soxhlet extracted in THF for two days, then dried under vacuum at 120 ℃ for 12 h.
2 Synthesis of TFBD-NH by BBE:
to a 20 mL Schlenk tube were added PA (16.3 mg,0.15 mmol) and TFPB (39.0 mg,0.1 mmol), 1, 4-dioxane (3.0 mL), 1,3, 5-trimethylbenzene (3.0 mL), and 6M acetic acid (0.6 mL). The mixture was sonicated for 10 minutes to obtain a homogeneous dispersion, degassed by three liquid nitrogen freeze-pump-methanol thaw cycles, sealed under vacuum and heated at 120 ℃ for three days. After cooling to Room Temperature (RT), schlenk tubes containing TFPA were sonicated for 10 minutes. Then, BD-NH was added to the mixture 2 (321.4 mg,1.5 mmol), 1, 4-dioxane (1.5 mL), 1,3, 5-trimethylbenzene (1.5 mL) and 6M acetic acid (0.3 mL). The mixture was sonicated for an additional 10 minutes, degassed by three liquid nitrogen freeze-pump-methanol thaw cycles, sealed under vacuum and heated at 40 ℃ for three days. Separating the obtained dark red precipitateThe solvent was removed from the core, soxhlet extracted in THF for two days, then dried under vacuum at 120 ℃ for 12 h.
FIG. 3 shows the resulting TFBD-NH 2 CTAB @ TFPA and CTAB @ TFBD-NH 2 HRTEM image and SEM image of (A) of FIG. 3 is TFBD-NH 2 Fig. 3 (B) is an enlarged image of a selected region of fig. 3 (a), and fig. 3 (C) is a crystal structure observed by vertical projection of fig. 3 (B). As can be seen, TFBD-NH 2 Having an ordered arrangement of high crystallinity, a honeycomb-like porous structure can be observed under a high-resolution electron microscope ((C) of fig. 3). Porous Structure indicates TFBD-NH 2 The pitch of the holes in (a) is about 4 nm, which is very consistent with the pitch of 4.14 nm in (C) of the simulated structural model. FIG. 3 (D) is an SEM image of CTAB/TFPA, and FIG. 3 (E) is CTAB @ TFBD-NH 2 Fig. 3 (F) is a partially enlarged view of a selected region of fig. 3 (E). As can be seen, CTAB/TFPA and CTAB/TFBD-NH were added after initial CTAB addition in the material synthesis 2 The material presents a loose and porous 3D spherical shape, the average size is about 700 nm, and the material has obvious macroporous channels.
FIG. 4 shows TFPA, CTAB/TFPA, TFBD-NH 2 And CTAB/TFBD-NH 2 Nitrogen adsorption-desorption isotherms. TFPA and TFBD-NH 2 Respectively BET surface areas of 56 m 2 G and 43 m 2 (ii) in terms of/g. However, CTAB/TFPA and CTAB/TFBD-NH 2 Respectively increases the BET surface area by about ten times to 563 m 2 G and 455 m 2 G, which can be attributed to CTAB/TFPA and CTAB/TFBD-NH 2 Loose and porous structure in (1).
Application example
1.UHPLC-MS/MS working conditions:
for the pesticide-based standard, liquid chromatography analysis was performed using a Waters Acquity Ultra Performance LC high Performance liquid chromatograph. The analytes were chromatographed using an ACQUITY UPLC HSST 3 (2.1 mm. Times.100 mm,1.8 μm) column. The column temperature was 40 ℃ and the amount of sample was 2. Mu.L. Mobile phase: 2 mM ammonium formate +0.01% (v/v) aqueous formic acid (A) and 2 mM ammonium formate +0.01% (v/v) methanoic acid (B); flow rate: 0.3 mL/min. The gradient elution procedure is 3% B (0-1.0 min), 3% -15% B (1.0-1.5 min), 15% -50% B (1.5-2.5 min), 50% -70% B (2.5-18.0 min), 70% -98% B (18.0-23.0 min), 98% B (23.0-27.0 min), 98% -3% B (27.0-27.1 min), 3% B (27.1-30.0 min). The total cycle time for each sample was 30.0min.
Mass spectrometry was performed using a Waters Xevo TQ-S triple quadrupole mass spectrometer and operated in Multiple Reaction Monitoring mode (MRM). The main parameters are as follows, ion source: electrospray ion source (ESI); an ionization mode: a positive ion mode; ion source temperature: at 350 ℃.
For the veterinary drug standards, liquid chromatography analysis was performed using a Waters Acquity Ultra Performance LC high Performance liquid chromatograph. The analytes were chromatographed using an ACQUITY UPLC BEH C18 (2.1 mm. Times.100 mm,1.7 μm) chromatography column. The column temperature was 40 ℃ and the amount of sample was 3. Mu.L. Mobile phase: 0.5 mM ammonium fluoride +0.1% (v/v) formic acid water (a) and acetonitrile/methanol (v/v = 1/1) (B); flow rate: 0.3 mL/min. The gradient elution procedure is 3% B (0-2.0 min), 3% -15% B (2.0-5.0 min), 15% B (5.0-10.0 min), 15% -30% B (10.0-15.0 min), 30% -50% B (15.0-20.0 min), 50% -100% B (20.0-24.0 min), 100% B (24.0-28.0 min), 100% -3% B (28.0-28.5 min), 3% B (28.5-29.0 min). The total cycle time for each sample was 29.0min.
Mass spectrometry was performed using a Waters Xevo TQ-XS triple quadrupole mass spectrometer and operated in Multiple Reaction Monitoring mode (MRM). The main parameters are as follows, ion source: electrospray ion source (ESI); an ionization mode: a positive ion mode; ion source temperature: and (4) 400 ℃.
2. UPLC-MS/MS detection of pesticide and veterinary drug residues in animal oil and vegetable oil
(S1) extracting: 2 g animal oil/vegetable oil samples were mixed with 2mL 0.1M Na 2 Mixing EDTA-Mclvaine buffer solution, adding 10 mL acetonitrile, 20 g anhydrous sodium sulfate and 1g sodium chloride after 1 min vortex oscillation, taking 1mL supernatant after vortex oscillation for 1 min and centrifugation at 8000 rpm and 4 ℃ for 10min, and waiting for purification.
(S2) purification: and (2) mixing the liquid to be purified obtained in the step (1) with a purifying agent, and centrifuging for 10min at 14000 rpm after vortexing for 60 s. Take 0.5 mL of supernatant, blow dry under nitrogen, with 0.5 mL methanol/water =50: and (5) re-dissolving the solution, and filtering the solution by using a filter membrane of 0.22 mu m to obtain the solution to be detected.
(S3) mass spectrum detection: separating and measuring the solution to be measured in the step (2) by UPLC-MS/MS; and quantifying by using an external standard method to obtain the content of the veterinary drug.
3. The experimental results are as follows:
to evaluate the full recovery performance of the material, the accuracy and precision of the method was assessed by a recovery experiment at an addition level of 100 ppb (n = 3). The test tests carried out on 277 pesticides and 135 veterinary drugs, combined 412 pesticide and veterinary drug residues. The test results are shown in Table 1. Where recovery and Relative Standard Deviation (RSD) represent the accuracy and precision of the process, respectively. For the animal oil sample, among 412 kinds of agricultural and veterinary medicines, the recovery rate of 378 kinds is more than 50%; for the animal oil sample, 398 recovery rates out of 412 veterinary drugs were greater than 50%. Although the recovery rate of part of the veterinary drugs is low, all compounds can be detected, and the qualitative requirement of non-directional screening is met. In the animal oil and vegetable oil samples, the relative standard deviation of the 412 veterinary and agricultural medicines is 0.18% -19.79% and 0.43% -20.18%, respectively, which indicates that the method has high accuracy.
Table 1 information and standard recovery rate (100 mug/mL) of 412 kinds of agricultural and veterinary drugs
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The LC-MS/MS detection method disclosed by the invention is used for detecting the pesticide and veterinary drug residues (100 mug & lt/EN & gt) in most animal oil and plant oilmL) has good detection effect. Recovery rates for small quantities of veterinary drugs, particularly some veterinary drugs such as thiabendazole, tolfenpyrad, tricyclazole, triflumizole, cananolol, sulfamethizole, glyburide, brombuterol, are less than ideal, probably due to their size and CTAB/TFBD-NH 2 The pore size of the nanospheres is similar to that of CTAB/TFBD-NH 2 The amino groups on the nanospheres have strong hydrogen bonding interaction. Therefore, some other functional modifications may be made in the future to avoid this risk.

Claims (9)

1. An LC-MS/MS detection method for pesticide and veterinary drug residues in animal oil and vegetable oil is characterized by comprising the following steps:
(S1) extracting: mixing an animal oil and/or vegetable oil sample with a buffer solution, adding an extraction solvent, a water removing agent and a salting-out agent after shaking, centrifuging after vortex shaking, and taking a supernatant for purification; the buffer solution is Na 2 EDTA-Mclvaine, acetic acid-sodium acetate, citric acid-sodium citrate and sodium citrate-disodium hydrogen citrate, wherein the concentration is 0.1-0.3M; the extraction solvent is at least one of acetonitrile, methanol, formic acid water, acetic acid acetonitrile and acetic acid methanol;
(S2) purification: mixing the solution to be purified obtained in the step (S1) with a purifying agent, centrifuging after vortex oscillation, taking supernatant nitrogen for drying, redissolving and filtering to obtain a solution to be detected;
(S3) mass spectrum detection: separating and measuring the liquid to be measured in the step (S2) by liquid chromatography-tandem mass spectrometry; quantifying by an external standard method to obtain the content of the veterinary and agricultural medicines;
the purifying agent in the step (S2) is an amino functionalized covalent organic framework material, is formed by orderly arranging a honeycomb porous structure, has a three-dimensional loose porous nanosphere structure, simultaneously has macropores and mesopores, and has the average particle size of 500-1000nm; the amino-functionalized covalent organic framework material is prepared by taking diamine and polyaldehyde as monomers, polymerizing the monomers in the presence of micelles formed by quaternary ammonium salt cationic surfactant to obtain three-dimensional nanospheres, adding excessive polyamine to replace the diamine by building block exchange and modify the diamine with amino functional groups, and finally washing the quaternary ammonium salt cationic surfactant off to obtain the amino-functionalized covalent organic framework material;
the quaternary ammonium salt cationic surfactant is hexadecyl trimethyl ammonium bromide, the diamine is 1, 4-phenylenediamine, the polybasic aldehyde is 1,3, 5-tri (p-formylphenyl) benzene, the polybasic amine is diaminobenzidine, and the obtained amino functionalized covalent organic framework material is named as CTAB @ TFBD-NH 2 (ii) a The molar ratio of the quaternary ammonium salt cationic surfactant to the diamine to the polyaldehyde is 0.8-1.2:1.5-2.0:1.0-1.2:10-15.
2. <xnotran> 1 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 3- , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , -S- , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , </xnotran> <xnotran> , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , </xnotran> <xnotran> , , , , , , , , , , , , , , , , J, L, A, D, , - - , - - , , , , , , , , , , , , , , , , , , , , , , , , , , , , , - , - , , II, , , , FM-6-1, , , ; </xnotran>
<xnotran> , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 6- ,5- -1- -4- , 2- , , 2- -5- ,4- , , , , , , S1, , , , B, , , M1, , , , , , , , , , , , , , , , , , , , , , , , , , , , , </xnotran> Econazole, betamethasone, phenothiazine, glyburide, 2- (4-thiazolyl) benzimidazole, levamisole, 2-amino-5-propanesulfonylbenzimidazole, oxibendazole, ketoprofen, 2-aminofluoropyridazole, albendazole sulfoxide, N-acetaminosulfone, fenbendazole sulfone, phenylguanide, cimaterol, ceteroro, clenbuterol, chlorpropaline, clenbuterol, tulobuterol, clenbuterol, bromobuterol, clenbuterol, bambuterol, maprotide, cyproheptadine, methylacetylenone, progesterone, megestrol acetate, androstenedione, meindronol, canrenone, cloisonne, testosterone propionate, methyltestosterone, medroxyprogesterone, chlormadinone, acetylsalicylic acid, doramectin, or combinations of one or more thereof.
3. The method according to claim 1, wherein in the step (S1), the animal oil comprises one or more of lard, beef tallow, mutton tallow, fish oil, bone marrow, fat meat, and cod liver oil; the vegetable oil comprises one or more of soybean oil, rapeseed oil, palm oil, olive oil, peanut oil and sunflower seed oil.
4. The detection method according to claim 1, wherein in the step (S1), the sample containing the animal oil and/or the vegetable oil, the buffer solution and the extraction solvent are mixed in a ratio of 1g:1-2mL:3-5mL; animal oil and/or vegetable oil samples, and the mass ratio of the water removal agent to the salting-out agent is 1:10-15:0.3-0.6; the water removing agent is selected from anhydrous sodium sulfate and/or anhydrous magnesium sulfate; the salting-out agent is at least one selected from sodium chloride, sodium acetate, ammonium acetate and sodium citrate.
5. The detection method according to claim 1, wherein in the step (S2), the volume-to-mass ratio of the liquid to be purified to the purifying agent is 1mL:15-25mg.
6. The detection method according to claim 1, wherein the amino-functionalized covalent organic framework material has both mesopores and macropores in a loose porous structureThe three-dimensional porous nanosphere structure has an average diameter of 500-1000nm and a specific surface area of 400-600 m 2 The diameter of the mesopores is between 20 and 50A, and the diameter of the macropores is between 50 and 300 nm.
7. The detection method of claim 6, wherein the amino-functionalized covalent organic framework material has an average diameter of 700-800 nm, a mesoporous pore diameter of 30-50A, and a macroporous pore diameter of 50-200 nm.
8. The detection method according to claim 1, wherein the amino-functionalized covalent organic framework material is prepared by a method comprising the following steps:
(P1) adding a solution in which diamine, polyaldehyde and a catalyst are dissolved into a quaternary ammonium salt cationic surfactant aqueous solution, carrying out ultrasonic treatment on the mixture, carrying out circulating freeze-pumping, carrying out high-temperature reaction, and cooling to room temperature for later use after the reaction is finished;
(P2) adding a solution of polyamine and a catalyst into the system obtained in the step (S1), performing ultrasonic treatment on the mixture, performing circulating freeze-pumping, performing heating reaction, and centrifuging, extracting and drying the product to obtain the catalyst.
9. The detection method according to claim 8, wherein the concentration of the quaternary ammonium salt cationic surfactant in the aqueous solution of quaternary ammonium salt cationic surfactant in step (P1) is 0.05 to 0.10 mol/L; the catalyst is acetic acid, and in the step (S1), the amount of the catalyst is 1-2 times of the amount of the monomer substances; in the step (S2), the amount of the catalyst is 0.1 to 0.2 times of that of the polyamine substance; in the steps (P1) and (P2), the number of times of circulating freezing and pumping is 3-5 times;
and/or, in the step (P1), the high-temperature reaction temperature is 100-150 ℃, and the reaction time is 2-4 days; in the step (S2), the heating reaction temperature is 30-60 ℃, and the reaction time is 2-4 days.
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