Solid-phase extraction material for detecting caine anesthetics in livestock and aquatic products as well as preparation method and application of solid-phase extraction material

Abstract

The invention relates to a solid-phase extraction material for detecting caine anesthetics in livestock and aquatic products as well as a preparation method and application of the solid-phase extraction material, and belongs to the technical field of solid-phase extraction. The preparation method of the solid-phase extraction material for detecting the caine anesthetics in the livestock and aquatic products comprises the following steps: S1, preparing St-DVB copolymer microspheres; s2, preparing a sulfonic group functionalized St-DVB copolymer microsphere; s3, preparing a sulfonic acid group / C18 bifunctional functionalized St-DVB copolymer, and filling the sulfonic acid group / C18 bifunctional functionalized St-DVB copolymer into a solid-phase extraction column; and S4, accurately weighing the prepared livestock and aquatic products in a centrifuge tube, adding an acetic acid acetonitrile solution for extraction and centrifugation, transferring all the supernatant to a solid-phase extraction column, carrying out elution and elution, collecting the target object, carrying out nitrogen blowing concentration and redissolution, filtering through a membrane, and carrying out ultra-high performance liquid chromatography-electrostatic field orbitrap high-resolution mass spectrometry determination. According to the application, accurate detection of the caine anesthetics in livestock and aquatic products is successfully realized, and a promising technical scheme is provided for daily supervision and quality control of the caine anesthetics.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of solid phase extraction, in particular to a solid phase extraction material for detecting caine anesthetics in livestock and aquatic products and a preparation method and application thereof. BACKGROUND

[0002] There are various types of anesthetics, among which caine anesthetics are most widely used in the market due to their exact anesthetic effect, convenient operation, fast onset and rapid recovery. In the livestock and aquatic breeding and circulation links, some practitioners exist the situation of non-standard use of such drugs in order to pursue economic benefits or to ensure product quality. For example, before livestock and poultry are put on the market, caine anesthetics are used to make animals anesthetized for water injection weight gain. During the transportation of livestock and aquatic products, such anesthetics with fresh-keeping effect are also the first choice to reduce the stress response of animals, improve the survival rate and maintain the freshness of products.

[0003] Among various types of caine anesthetics, MS-222 is most widely used, especially in the field of live transportation, and it is also the only anesthetic approved for live animals in the European Union, Canada, the United States and other countries and regions, and strict use specifications and drug holiday requirements are formulated for it. However, caine anesthetics have central nervous toxicity and cardiovascular toxicity, and their residues in the animal body can accumulate into the human body through the food chain, and long-term intake may lead to drug accumulation and further harm human health. At present, there are no clear limit standards for most caine anesthetics in China, and the corresponding food safety detection methods and supervision system also need to be improved, which cannot effectively control the drug residue risk in the circulation link. In view of this, it is of great practical significance to establish a specific analysis method for caine anesthetics in livestock and aquatic products, and to develop a high selectivity and high sensitivity pretreatment technology is the core prerequisite for realizing the accurate qualitative and quantitative analysis of target substances.

[0004] In the analysis and detection of livestock and aquatic products, protein, fat and other matrix impurities in muscle tissue compete with target substances during ionization, directly inhibiting the response signal of target substances, and seriously affecting the accuracy of detection results. Therefore, effective enrichment of trace target substances, meeting the detection sensitivity and accurate purification of sample matrix, removing or reducing the interference of components on accurate detection of target substances, have become the core problems to be solved in sample pretreatment. Solid phase extraction (SPE) has become one of the mainstream methods for livestock and aquatic product sample pretreatment due to its simple operation and high enrichment efficiency. However, the commonly used solid phase extraction columns (such as HLB column, MCX column, amino column, silica gel column, etc.) have the limitations of single functional site or insufficient selectivity, and it is difficult to specifically separate target substances and matrix impurities in the purification process of caine anesthetics. The purification effect is not ideal, and it cannot meet the accurate detection at trace level.

[0005] Based on the above status quo, in order to improve the analysis efficiency of the caine anesthetics in livestock and aquatic products, it is urgent and necessary to develop a new type of solid phase extraction material with high selectivity and high adsorption capacity, and the core target is to simultaneously improve the extraction efficiency and matrix purification effect of the target substance, and to provide reliable technical support for subsequent qualitative and quantitative analysis.

[0006] From the physicochemical properties of caine substances, they have strong polarity, which provides a key basis for material design. In the material preparation process, a substance with high mechanical strength and resistance to organic phase is selected as the substrate to ensure that the material remains stable in complex pretreatment environments (such as organic phase elution); secondly, sulfonic acid groups are introduced on the surface of the substrate through chemical modification, and the specific ionic bond between the sulfonic acid groups and the amino groups in the protonated caine substances is used to realize the directional capture of the target substance, greatly improving the extraction selectivity; at the same time, C18 groups are further bonded to effectively adsorb fat, non-polar impurities and other interfering components in the matrix of livestock and aquatic products, optimizing the purification effect from two dimensions of targeted extraction and impurity removal. In the detection link, Q-Exactive method of electrostatic field orbitrap high resolution mass spectrometry is used to analyze the samples after extraction and purification. The core advantage of this method is that it can accurately provide the accurate mass number of the target compound within the set mass range, effectively distinguish the target substance from the matrix interference signal with ultra-high mass resolution, and significantly reduce the matrix effect caused by complex livestock and aquatic product matrix; at the same time, this method can also greatly improve the sensitivity, accuracy and precision of detection, and provide reliable technical support for accurate quantitative and qualitative analysis of caine anesthetics in livestock and aquatic products. SUMMARY

[0007] Therefore, the application provides a solid phase extraction material for detecting caine anesthetics in livestock and aquatic products, a preparation method and application thereof. The material selects a substance with high strength and resistance to organic phase as the substrate, introduces specific adsorption groups according to the characteristic ion properties of caine compounds, and bonds hydrophobic groups to selectively remove impurity interference, achieving the dual effects of targeted capture of target substances and selective removal of impurities, ensuring its suitability for detection of caine anesthetics in livestock and aquatic products. At the same time, the corresponding analysis and detection method is established, and is successfully applied to the detection of such products. By combining the high purification capacity of the new material with detection technology, the problems of strong matrix interference and suppressed target response in traditional methods are effectively solved, and finally the accurate detection of caine anesthetics in livestock and aquatic products is successfully realized, providing a practical technical solution for the daily supervision and quality control of such substances, which can effectively overcome the defects of the above existing technologies.

[0008] The first aspect of the application provides a preparation method of a solid phase extraction material for detecting caine anesthetics in livestock and aquatic products, comprising the following steps:

[0009] S1, dissolve styrene St, divinylbenzene DVB and initiator in a porogen, magnetically stir until completely dissolved to obtain a mixed solution; then add a dispersing agent to pure water, stir and dissolve, and cool to room temperature, then pour into the mixed solution to obtain a mixed solution; continuously stir the mixed solution at a constant speed and heat to make styrene St and divinylbenzene DVB react to form a St-DVB copolymer emulsion; after cooling to room temperature, separate by suction filtration, wash with ultrapure water and anhydrous ethanol in sequence, and vacuum dry to obtain St-DVB copolymer microspheres;

[0010] S2, ultrasonically disperse the dried St-DVB copolymer microspheres in dichloroethane, add a sulfonating agent dropwise after cooling, heat and stir to react, and control the pH during the reaction; after cooling, collect the microspheres by suction filtration, wash with ultrapure water, and vacuum dry to obtain sulfonic acid group functionalized St-DVB copolymer microspheres;

[0011] S3, add the sulfonic acid group functionalized St-DVB copolymer microspheres to a reaction solvent, ultrasonically disperse, then add a bonding agent and a catalyst, heat and stir to react, cool, separate the microspheres by suction filtration, wash with toluene and anhydrous ethanol in sequence, and vacuum dry to obtain sulfonic acid group / C18 bifunctional group functionalized St-DVB copolymer; pre-load the sulfonic acid group / C18 bifunctional group functionalized St-DVB copolymer in a solid-phase extraction empty column, tightly pack the filler in the solid-phase extraction empty column to obtain a solid-phase extraction column, and activate the solid-phase extraction column with methanol and formic acid water to obtain an activated solid-phase extraction column for standby use;

[0012] S4, accurately weigh the homogenate prepared from livestock and aquatic products in a centrifuge tube, add ethyl acetate solution, extract on a constant-speed shaker, and then centrifuge; transfer all the supernatant to the activated solid-phase extraction column, and collect the target product after elution and washing; concentrate the mobile phase by nitrogen blowing, re-dissolve, pass through a filter membrane, and collect in a sample vial for determination by ultra-high performance liquid chromatography-electrostatic field orbitrap high-resolution mass spectrometry.

[0013] Preferably, in step S1, the mass ratio of styrene St to divinylbenzene DVB is 6:1 to 3:1; the initiator is azobisisobutyronitrile AIBN, and the initiator accounts for 1-2% of the total mass of styrene St and divinylbenzene DVB; the dispersing agent is polyvinyl alcohol PVA, and the dispersing agent accounts for 3-8% of the total mass of the reaction system; and the porogen is toluene, which accounts for 30-50% of the total mass of the reaction system.

[0014] Preferably, in step S2, the sulfonating agent is chlorosulfonic acid; the mass ratio of the sulfonating agent to St-DVB copolymer microspheres is 1.5:1 to 1:1; the pH during the reaction is controlled to be less than 1; and the pH of the filtrate after washing with ultrapure water is 5-6.

[0015] Preferably, in step S3, the bonding agent is octadecyltrichlorosilane OTS, the mass ratio of the bonding agent to the sulfonic acid group functionalized St-DVB copolymer microspheres is 0.5:1-1:1; the catalyst is triethylamine, the amount of the catalyst is 10-25% of the total mass of the reaction system; the reaction solvent is anhydrous toluene; the temperature of the heating and stirring reaction is 80-100℃, and the time of the heating and stirring reaction is 5-8 hours.

[0016] Preferably, in step S4, the conditions of the liquid chromatography are as follows: a chromatographic column: an Atlantis T3 column, a column temperature: 30℃; a mobile phase A: 0.1% formic acid aqueous solution containing 5mmo / L ammonium formate, a mobile phase B: 0.1% formic acid methanol, a flow rate after mixing of the mobile phase A and the mobile phase B: 0.40 mL / min, and an injection volume: 10 μL.

[0017] The gradient elution program is as follows: 0-4min, 5-20%B; 4-5min, 20-40%B; 5-10min, 40-100%B; 10-13min, 100%B; 13-15min, 5%B.

[0018] Preferably, the size of the liquid chromatography column Atlantis T3 column is 100 mm column length x 2.1 mm inner diameter, and the particle size of the filler in the Atlantis T3 column is 3.0 μm.

[0019] Preferably, in step S4, the conditions of the mass spectrometry are as follows: an electrospray ion source: ESI, a positive ion scanning mode, a spraying voltage: 3200 V, an ion source temperature: 325℃, a sheath gas pressure: 40 arb, an auxiliary gas pressure: 30 arb, a capillary temperature: 350℃, an atomization gas: high-purity nitrogen, and a collision gas: high-purity helium.

[0020] Preferably, in step S4, the aquatic products are selected from at least one of freshwater fish, shrimp, crab, pork, and chicken manure.

[0021] The second aspect of the present application further provides a solid-phase extraction material for detecting a caine anesthetic in aquatic products, which is prepared by the above method.

[0022] The third aspect of the present application further provides application of the above solid-phase extraction material for detecting a caine anesthetic in aquatic products in detection of the caine anesthetic in aquatic products, wherein the caine anesthetic is selected from at least one of ethyl m-amino benzoate methanesulfonate MS-222, m-amino benzoic acid, benzocaine, p-amino benzoic acid, p-acetylamino benzoic acid, chloroprocaine, procainamide, lidocaine, cinchocaine, bupivacaine, prilocaine, and ropivacaine.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] 1、The solid phase extraction material of the present application is a copolymer matrix constructed with styrene (St) as a polymer monomer and divinylbenzene (DVB) as a crosslinking agent, sulfonic acid groups (-SO3H) are introduced on the surface of the matrix by sulfonation to give cation exchange function, and then C18 long chain bonding modification is performed to form reverse adsorption sites, so that a copolymer solid phase extraction material with dual functions of cation exchange and reverse adsorption is finally obtained. By controlling the key reaction conditions such as copolymer matrix preparation, sulfonation reaction and reverse group introduction, a solid phase extraction material with uniform particle size distribution, large effective adsorption capacity and strong specificity is prepared, and is packed into a solid phase extraction column. The column is used for detecting the residues of local anesthetics in aquatic products, and the sample extraction recovery, precision and repeatability meet the requirements of trace analysis of local anesthetics in aquatic products.

[0025] 2、The present application selects five typical aquatic product matrices of freshwater fish, shrimp, crab, pork and chicken manure, and establishes corresponding detection methods: after the sample is prepared, the target is extracted with 1% acetic acid acetonitrile solution, purified and removed by solid phase extraction (SPE) technology to remove matrix interference, and then subjected to qualitative and quantitative analysis by high-resolution mass spectrometry after nitrogen blowing concentration of the extract. This method can realize the synchronous detection of various local anesthetics in aquatic products, has the advantages of high sensitivity, strong selectivity, accurate and reliable quantitative results, and is suitable for rapid screening and accurate quantitative detection of local anesthetics in various aquatic products.

[0026] 3、The method of the present application is aimed at the basic structure of local anesthetics and the complex matrix characteristics of aquatic products, and a composite material with cation exchange and reverse adsorption functions is prepared. The material has three core advantages: ①St-DVB copolymer as the matrix, high mechanical strength and resistant to organic phase, which can adapt to the complex pretreatment environment of fish meat, pork and other aquatic products (such as high organic phase extract, repeated washing operation); ②The sulfonic acid group (-SO3H) on the surface can specifically capture the protonated local anesthetic compounds (containing basic amino groups), realizing the selective binding of the target; ③The bonded C18 long chain can adsorb the fat and non-polar impurities in the matrix through hydrophobic interaction, simultaneously completing the directional removal of interference components.

[0027] 4. The application provides a method of filling a copolymer material in a solid phase extraction column and using the column for solid phase extraction. During the sample loading process, the cocaine compounds in the aquatic product extract are specifically captured by the sulfonic acid group on the surface of the material through ionic bonds; during the elution process, the C18 group further adsorbs and removes fat and non-polar impurities, reducing matrix interference; finally, ammonia methanol is selected as the eluent to break the ionic bonds and achieve efficient elution of the target compounds. This method simultaneously improves the extraction efficiency and matrix purification effect of the target compounds, and is simple and specific to operate.

[0028] 5. Based on the above-mentioned solid phase extraction purification method, the application establishes a detection scheme for multiple cocaine anesthetics in aquatic products in combination with instrument analysis. The solid phase extraction technology effectively removes interfering components in the complex matrix, significantly reduces the matrix effect, provides high-purity samples for subsequent analysis, and ensures the accuracy of the detection results, which can meet the rapid screening and quantitative detection needs of cocaine anesthetics in various aquatic products.

[0029] 6. The application uses electrostatic field orbitrap high-resolution mass spectrometry for determination, matrix curve external standard method for quantification, and collects data in positive ion mode by multiple reaction monitoring (MRM) to complete qualitative and quantitative analysis. The method verification results show that: 12 kinds of cocaine anesthetics have good linear relationship in the matrix standard curve range of 1.0-100 μg / L, the correlation coefficient (r) is greater than 0.995, and the limit of quantification (LOQ) is 1.0-5.0 μg / kg; in five typical matrices of grass carp, green shrimp, crab, pork and chicken manure, the standard addition recovery experiments (6 parallel determinations) at three levels of 5, 10 and 50 μg / kg were carried out, the target recovery rates were 86.5-107.4%, 81.8-115.2%, 72.8-111.9%, 77.6-118.7%, and 73.8-112.5%, respectively, and the relative standard deviations (RSD) were ≤4.7%, ≤5.7%, ≤7.2%, ≤6.4%, and ≤6.1%, respectively, meeting the relevant technical requirements. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the application or the prior art, the following will briefly introduce the drawings needed to be used in the description of the application or the prior art. Obviously, the drawings described below are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creating any inventive labor.

[0031] Figure 1 The electron microscope image of the solid phase extraction material prepared for the detection of cocaine anesthetics in aquatic products in the application;

[0032] Figure 2Infrared spectrum of the solid-phase extraction material prepared in this application for the detection of cocaine-type anesthetics in livestock and aquatic products;

[0033] Figure 3 Particle size distribution of the solid-phase extraction material prepared in this application for the detection of cocaine-type anesthetics in livestock and aquatic products;

[0034] Figure 4 Thermogravimetric analysis of the solid-phase extraction material prepared in this application for the detection of cocaine-type anesthetics in livestock and aquatic products;

[0035] Figure 5 The chromatograms are of the 12 cocaine-type anesthetics described in this application. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0037] Unless otherwise specified, the experimental methods used in the embodiments of this application are all conventional methods.

[0038] In the following examples, unless otherwise specified, all raw materials can be obtained by commercial purchase or conventional methods.

[0039] This application provides a method for preparing a solid-phase extraction material for detecting cocaine-type anesthetics in livestock and aquatic products, comprising the following steps:

[0040] S1. Styrene (St), divinylbenzene (DVB), and azobisisobutyronitrile (AIBN) are dissolved in toluene and magnetically stirred until completely dissolved to obtain a toluene mixture. Polyvinyl alcohol (PVA) is then added to pure water, stirred at 40°C to dissolve, and cooled to room temperature before being poured into the toluene mixture to obtain a mixed solution. The mixed solution is continuously stirred at a constant rate and heated to allow styrene (St) and divinylbenzene (DVB) to react and form a St-DVB copolymer emulsion. After cooling to room temperature, the emulsion is separated by filtration, washed successively with ultrapure water and anhydrous ethanol, and then vacuum dried to obtain St-DVB copolymer microspheres.

[0041] S2. The dried St-DVB copolymer microspheres are ultrasonically dispersed in dichloroethane, cooled, and then chlorosulfonic acid is added dropwise. The mixture is heated and stirred to react, and the pH is controlled during the reaction. After cooling, the microspheres are collected by vacuum filtration, washed with ultrapure water, and vacuum dried to obtain sulfonic acid-functionalized St-DVB benzene copolymer microspheres.

[0042] S3. The sulfonic acid-functionalized St-DVB benzene copolymer microspheres are added to toluene, ultrasonically dispersed, and then octadecyltrichlorosilane (OTS) and triethylamine are added. The mixture is heated and stirred to react. After cooling, the microspheres are separated by filtration and washed successively with toluene and anhydrous ethanol. After vacuum drying, the sulfonic acid-functionalized / C18 bifunctionalized St-DVB copolymer is obtained. The sulfonic acid-functionalized / C18 bifunctionalized St-DVB copolymer is pre-packed into a solid-phase extraction (SPE) column to tightly encapsulate the packing material. The SPE column is then activated with methanol and 0.1% formic acid water to obtain an activated SPE column for later use.

[0043] S4. Accurately weigh the homogenized aquatic products into a centrifuge tube, add 1% acetic acid acetonitrile solution, extract on a shaker at a constant speed, centrifuge, transfer all the supernatant to the activated solid phase extraction column, elute and collect the target analyte, concentrate the mobile phase with nitrogen blowing, redissolve, filter through a 0.22 μm filter membrane, and collect in a vial for determination by ultra-high performance liquid chromatography-electrostatic field orbital trap high resolution mass spectrometry.

[0044] This application selects five typical livestock and aquatic product matrices—freshwater fish, shrimp, crab, pork, and chicken manure—and establishes corresponding detection methods. After sample preparation, the target analytes are extracted using a 1% acetic acid-acetonitrile solution. Solid-phase extraction (SPE) is then used to remove matrix interferences. The extract is concentrated by nitrogen blowing and then subjected to qualitative and quantitative analysis by high-resolution mass spectrometry. This method can simultaneously detect multiple cocaine-type anesthetics in livestock and aquatic products, exhibiting advantages such as high sensitivity, strong selectivity, and accurate and reliable quantitative results. It is suitable for rapid screening and precise quantitative detection of these anesthetics in various livestock and aquatic products.

[0045] Specifically, cocaine anesthetics include ethyl m-aminobenzoate methanesulfonate (MS-222), m-aminobenzoic acid, benzocaine, para-aminobenzoic acid, para-acetaminobenzoic acid, chloroprocaine, procainamide, lidocaine, cincocaine, bupivacaine, prilocaine, and ropivacaine.

[0046] Specifically, in step S1, St:DVB = 6:1~3:1 (mass ratio) to ensure a balance between pore size and strength; AIBN is the initiator, accounting for approximately 1~2% of the total monomer mass; PVA is the dispersant, accounting for approximately 3~8% of the total reaction system mass; and toluene is the porogen, accounting for approximately 30~50% of the total reaction system mass.

[0047] Specifically, in step S2, chlorosulfonic acid is used as a sulfonating agent, and its mass ratio with the microspheres obtained in step S1 is approximately 1.5:1 to 1:1. The pH is less than 1 throughout the reaction process. After the reaction, the solution is washed with ultrapure water until the pH of the filtrate is approximately 5 to 6.

[0048] Specifically, in step S3, OTS is the bonding agent, and its mass ratio with the sulfonated microspheres obtained in step S2 is approximately 0.5:1 to 1:1; triethylamine is the catalyst, and its amount is 10 to 25% of the total mass of the reaction system; anhydrous toluene is selected as the reaction solvent, the reaction temperature is controlled at 80 to 100°C, and the reaction is carried out with magnetic stirring for 5 to 8 hours.

[0049] Specifically, the liquid chromatography conditions were as follows: Column: Atlantis T3 column (100 mm length × 2.1 mm inner diameter, with a packing particle size of 3.0 μm); Column temperature: 30℃; Mobile phase A was a 0.1% formic acid aqueous solution containing 5 mmol / L ammonium formate, and mobile phase B was 0.1% formic acid in methanol. The flow rate of the mixture of mobile phases A and B was 0.40 mL / min, and the injection volume was 10 μL. Gradient elution program: 0–4 min, 5–20% B; 4–5 min, 20–40% B; 5–10 min, 40–100% B; 10–13 min, 100% B; 13–15 min, 5% B.

[0050] For example, the Atlantis T3 column has dimensions of 100 mm × 2.1 mm, and the packing particle size in the Atlantis T3 column is 3.0 μm.

[0051] Specifically, the mass spectrometry conditions were as follows: electrospray ionization source: ESI, positive ion scan mode, spray voltage: 3200 V, ion source temperature: 325℃, sheath gas pressure: 40 arb, auxiliary gas pressure: 30 arb, capillary temperature: 350℃, nebulizing gas: high-purity nitrogen, and collision gas: high-purity helium. The mass spectrometry data acquisition mode was full scan + automatic triggering of the secondary mass spectrometer in positive ion scan mode. Qualitative and quantitative ions, along with their corresponding voltages and collision energies, were optimized to the best possible level. Based on the name, CAS number, retention time, and precise mass number of fragment ions for each anesthetic agent, the specific mass spectrometry parameters are shown in Table 1.

[0052] Table 1. Multiple Response Monitoring Parameters for 12 Caine-Based Anesthetics

[0053]

[0054] Reagents and standards:

[0055] Styrene, divinylbenzene, chlorosulfonic acid, octadecyltrichlorosilane, azobisisobutyronitrile, and polyvinyl alcohol (analytical grade) were supplied by Shanghai Maclean Biochemical Co., Ltd.; toluene, dichloroethane, anhydrous ethanol, ammonia, triethylamine (analytical grade), and acetic acid (chromatographic grade) were supplied by Shanghai Anpu Experimental Technology Co., Ltd.; methanol and acetonitrile (chromatographic grade) were supplied by Supelco, Inc., USA; 12 standards of cocaine-type anesthetics (200 μg / mL, 1 mL / vial) were supplied by Manhag (Shanghai) Biotechnology Co., Ltd.; experimental water was provided by a Milli-Q Integral 5 ultrapure water system from Millipore, USA.

[0056] Preparation of standard curves: A mixed standard solution of 12 anesthetics—ethyl m-aminobenzoate methanesulfonic acid (MS-222), m-aminobenzoic acid, benzocaine, para-aminobenzoic acid, para-acetaminobenzoic acid, chloroprocaine, procainamide, lidocaine, cincocaine, bupivacaine, prilocaine, and ropivacaine—was prepared at a concentration of 1, 2, 5, 10, 20, 50, and 100 ng / mL. Grass carp, shrimp, crab, pork, and chicken manure matrices were extracted using the above pretreatment process and then prepared into mixed matrix standard curves with concentrations of 1, 2, 5, 10, 20, 50, and 100 ng / mL, to be used immediately after preparation.

[0057] Instruments and equipment:

[0058] Fourier transform infrared spectrometer Affinity-1, Shimadzu, Japan; laser particle size analyzer Mastersizer 3000E, Malvern, UK; field emission scanning electron microscope Sigma 500, ZEISS, Germany; thermogravimetric analyzer TG 209 F3, Netzsch, Germany; ion meter PXSJ-216, Shanghai Precision Instruments; ultra-high performance liquid chromatography-electrostatic field orbital trap high-resolution mass spectrometer Q-Exactive, Thermo Fisher, USA.

[0059] Example 1

[0060] A method for preparing a solid-phase extraction material for detecting cocaine-like anesthetics in livestock and aquatic products includes the following steps:

[0061] S1: Add 10g styrene (St), 2g divinylbenzene (DVB), 0.2g azobisisobutyronitrile (AIBN), and 10mL toluene to a three-necked flask and stir magnetically until the AIBN is completely dissolved. Add 200mL ultrapure water and 1.2g polyvinyl alcohol (PVA) to a beaker and heat to 40℃ while stirring until dissolved. Purge the three-necked flask with nitrogen for 15min to remove oxygen, then raise the temperature to 70℃ and start constant stirring (200r / min). Slowly add the PVA aqueous solution dropwise to the three-necked flask and react for 6h to form a white polymer microsphere emulsion. After cooling, filter to separate the microspheres, wash three times with ultrapure water, then wash twice with anhydrous ethanol, and dry under vacuum at 60℃ for 4h to obtain white St-DVB copolymer microspheres.

[0062] S2: Take 5g of the dried St-DVB copolymer microspheres from S1 and add them to 50mL of dichloroethane. Disperse the microspheres by sonication for 20min. After cooling to 0~5℃ in an ice bath, slowly add 5g of chlorosulfonic acid and stir the reaction. After the addition is completed, raise the temperature to 35℃ and continue stirring the reaction for about 2h, controlling the pH value (pH<1) during the process. After the reaction is completed, cool down and filter to separate the sulfonated microspheres. Wash with ultrapure water until the pH of the filtrate is 5~6, then wash once with 5% methanol water, and vacuum dry at 60℃ for 6h to obtain sulfonic acid-functionalized St-DVB benzene copolymer microspheres.

[0063] S3: Take 3g of dried sulfonic acid functionalized St-DVB benzene copolymer microspheres from S2, add 30mL of toluene and ultrasonically disperse, then add 3g of octadecyltrichlorosilane (OTS) and 1mL of triethylamine. Under nitrogen protection, heat to 80℃ and stir for 6h. After cooling, filter to separate the microspheres and wash them with toluene and anhydrous ethanol 3-5 times in sequence. After vacuum drying at 60℃ for 6h, the sulfonic acid / C18 bifunctional St-DVB copolymer is obtained.

[0064] S4: Pack the copolymer material obtained in S3 into a solid-phase extraction column. Take a 3mL syringe-type solid-phase extraction empty column pre-loaded with a hydrophobic sieve plate at the bottom, accurately weigh 60mg of the copolymer material obtained in S3 and add it into the column. Then, install the sieve plate on top of the packing material to obtain the packed solid-phase extraction column. Activate the column sequentially with 3mL of methanol and 3mL of 0.1% methanol-water solution, and set aside for use.

[0065] S5: Accurately weigh 5g of grass carp mince (accurate to 0.01g) into a 50mL centrifuge tube, add 10mL of 1% acetic acid acetonitrile solution, vortex mix for 2min until the meat is completely dispersed, extract by shaking for 10min, centrifuge at 6500r / min for 8min, take the supernatant and dilute with 0.1% methanol water to 20mL, then pass it through the solid phase extraction column activated in S4. After passing through, wash with 3mL of 0.1% methanol water and 3mL of 5% methanol water in sequence, and finally elute with 6mL of 2% ammonia water and methanol. Blow under nitrogen at 40℃ until nearly dry, redissolve with 1mL of mobile phase and filter through a 0.22μm filter membrane, and then determine by ultra-high performance liquid chromatography-electrostatic field orbital trap high resolution mass spectrometry. The liquid chromatography conditions were as follows: Column: Atlantis T3 column (100 mm length × 2.1 mm inner diameter, packing material particle size 3.0 μm); Column temperature: 30℃; Mobile phase A was 0.1% formic acid aqueous solution containing 5 mmol / L ammonium formate; Mobile phase B was 0.1% formic acid in methanol; The flow rate of the mixture of mobile phases A and B was 0.40 mL / min; Injection volume: 10 μL. Gradient elution program: 0–4 min, 5–20% B; 4–5 min, 20–40% B; 5–10 min, 40–100% B; 10–13 min, 100% B; 13–15 min, 5% B. The mass spectrometry conditions were as follows: electrospray ionization source: ESI, positive ion scan mode; spray voltage: 3200 V; ion source temperature: 325℃; sheath gas pressure: 40 arb; auxiliary gas pressure: 30 arb; capillary temperature: 350℃; nebulizer gas: high-purity nitrogen; collision gas: high-purity helium. Mass spectrometry data acquisition mode: full scan + automatic triggering of the secondary mass spectrometer in positive ion scan mode.

[0066] The copolymer material obtained in this embodiment, as observed by scanning electron microscopy, has a smooth surface, high sphericity, good dispersibility, no obvious agglomeration, and a certain particle size distribution (see...). Figure 1 The particle size distribution of the material measured by the laser particle size analyzer was mainly concentrated in the range of 100~110μm, with a diameter spacing of 0.437 and a diameter (D) [4,3] of 103.878μm (see [link to article]). Figure 2 ); the infrared spectrometer measured at 1190 cm⁻¹ -1 and 1040 cm -1 Nearby are asymmetric and symmetric stretching vibration peaks of sulfonate ions, 2920 cm⁻¹. -1 Characteristic peak of long-chain alkyl (C18) at 1730 cm⁻¹ -1 A stretching vibration peak of the ester group (C=O) appears nearby. The sulfonic acid group (-SO3H) introduced by sulfonation undergoes an esterification reaction with the C18 hydroxyl group (-OH) to form an ester bond structure (see...). Figure 3Thermogravimetric analysis showed that the mass remained essentially unchanged (weight loss <5%) at room temperature to 200℃, making it suitable as a material for subsequent solid-phase extraction. At 200–600℃, the C18 long-chain alkyl and sulfonic acid groups decomposed sequentially, while the St-DVB crosslinked framework remained largely stable (see...). Figure 4 ).

[0067] Quantification was performed using the external standard method based on matrix curves, such as Figure 5 As shown, the 12 cocaine-type anesthetics exhibited good linearity in the matrix solution standard curve within the range of 1.0–100 μg / L, with correlation coefficients (r) greater than 0.995. The limits of quantitation (LOQ) were 1.0–5.0 μg / kg. The recoveries in the matrix were 86.5–107.4%, with RSDs ≤4.7%.

[0068] Example 2

[0069] A method for preparing a solid-phase extraction material for detecting cocaine-like anesthetics in livestock and aquatic products includes the following steps:

[0070] S1: Add 8g styrene (St), 2g divinylbenzene (DVB), 0.1g azobisisobutyronitrile (AIBN), and 9mL toluene to a three-necked flask and stir magnetically until the AIBN is completely dissolved. Add 200mL ultrapure water and 1g polyvinyl alcohol (PVA) to a beaker and heat to 40℃ while stirring until dissolved. Purge the three-necked flask with nitrogen for 15min to remove oxygen, then raise the temperature to 70℃ and start constant stirring (200r / min). Slowly add the PVA aqueous solution dropwise to the three-necked flask and react for 6h to form a white polymer microsphere emulsion. After cooling, filter to separate the microspheres, wash three times with ultrapure water, then wash twice with anhydrous ethanol, and dry under vacuum at 60℃ for 4h to obtain white St-DVB copolymer microspheres.

[0071] S2: Take 5g of the dried St-DVB copolymer microspheres from S1 and add them to 50mL of dichloroethane. Disperse the microspheres by sonication for 20min. After cooling to 0-5℃ in an ice bath, slowly add 6g of chlorosulfonic acid and stir the reaction. After the addition is complete, raise the temperature to 35℃ and continue stirring the reaction for about 2h, controlling the pH value (pH<1) during the process. After the reaction is complete, cool down and filter to separate the sulfonated microspheres. Wash with ultrapure water until the pH of the filtrate is 5-6, then wash once with 5% methanol water, and vacuum dry at 60℃ for 6h to obtain sulfonic acid-functionalized St-DVB benzene copolymer microspheres.

[0072] S3: Take 3g of the dried sulfonic acid group-functionalized St-DVB benzene copolymer microspheres from S2, add 30mL of toluene and ultrasonically disperse, then add 2.1g of octadecyltrichlorosilane (OTS) and 1mL of triethylamine. Under nitrogen protection, heat to 80℃ and stir for 6h. After cooling, filter to separate the microspheres and wash them with toluene and anhydrous ethanol 3-5 times in sequence. After vacuum drying at 60℃ for 6h, the sulfonic acid group / C18 bifunctional group-functionalized St-DVB copolymer is obtained.

[0073] S4: Pack the copolymer material obtained in S3 into a solid-phase extraction column. Take a 3mL syringe-type solid-phase extraction empty column pre-loaded with a hydrophobic sieve plate at the bottom, accurately weigh 60mg of the copolymer material obtained in S3 and add it into the column. Then, install the sieve plate on top of the packing material to obtain the packed solid-phase extraction column. Activate the column sequentially with 3mL of methanol and 3mL of 0.1% methanol-water solution, and set aside for use.

[0074] S5: Accurately weigh 5g of shrimp meat (accurate to 0.01g) into a 50mL centrifuge tube, add 10mL of 1% acetic acid acetonitrile solution, vortex mix for 2min until the meat is completely dispersed, extract by shaking for 10min, centrifuge at 6500r / min for 8min, take the supernatant and dilute with 0.1% methanol water to 20mL, then pass it through the solid phase extraction column activated in S4. After passing through, wash with 3mL of 0.1% methanol water and 3mL of 5% methanol water in sequence, and finally elute with 6mL of 2% ammonia water and methanol. Blow under nitrogen at 40℃ until nearly dry, redissolve with 1mL of mobile phase and filter through a 0.22μm filter membrane, and then determine by ultra-high performance liquid chromatography-electrostatic field orbital trap high resolution mass spectrometry. The liquid chromatography conditions were as follows: Column: Atlantis T3 column (100 mm length × 2.1 mm inner diameter, packing material particle size 3.0 μm); Column temperature: 30℃; Mobile phase A was 0.1% formic acid aqueous solution containing 5 mmol / L ammonium formate; Mobile phase B was 0.1% formic acid in methanol; The flow rate of the mixture of mobile phases A and B was 0.40 mL / min; Injection volume: 10 μL. Gradient elution program: 0–4 min, 5–20% B; 4–5 min, 20–40% B; 5–10 min, 40–100% B; 10–13 min, 100% B; 13–15 min, 5% B. The mass spectrometry conditions were as follows: electrospray ionization source: ESI, positive ion scan mode; spray voltage: 3200 V; ion source temperature: 325℃; sheath gas pressure: 40 arb; auxiliary gas pressure: 30 arb; capillary temperature: 350℃; nebulizer gas: high-purity nitrogen; collision gas: high-purity helium. Mass spectrometry data acquisition mode: full scan + automatic triggering of the secondary mass spectrometer in positive ion scan mode.

[0075] In this embodiment, the obtained copolymer material, as observed by scanning electron microscopy, exhibits smooth microspheres, high sphericity, good dispersibility, no obvious agglomeration, and a certain particle size distribution. Laser particle size analyzer measurements show that the particle size is mainly concentrated in the range of 120-130 μm, with a diameter spacing of 0.618 and a diameter (D) of 125.034 μm. Infrared spectroscopy measurements show that the particle size is concentrated in the range of 1190 cm⁻¹. -1 and 1040 cm -1 Nearby are asymmetric and symmetric stretching vibration peaks of sulfonate ions, 2920 cm⁻¹. -1 Characteristic peak of long-chain alkyl (C18) at 1730 cm⁻¹ -1 The stretching vibration peak of the ester group (C=O) appears nearby. The sulfonic acid group (-SO3H) introduced by sulfonation undergoes esterification reaction with the hydroxyl group (-OH) of C18 to form an ester bond structure. Thermogravimetric analysis shows that the mass does not change significantly at room temperature to 200℃ (weight loss rate <5%), making it suitable as a material for subsequent solid phase extraction. At 200~600℃, the long-chain alkyl and sulfonic acid groups of C18 decompose sequentially, and the St-DVB crosslinked backbone is basically stable.

[0076] Quantification using the matrix curve external standard method revealed good linearity for 12 cocaine-type anesthetics within the matrix solution standard curve range of 1.0–100 μg / L, with correlation coefficients (r) greater than 0.995. The limits of quantitation (LOQ) ranged from 1.0 to 5.0 μg / kg. Spike recoveries in the matrix solution ranged from 81.8% to 115.2%, with RSDs ≤5.7%.

[0077] Example 3

[0078] A method for preparing a solid-phase extraction material for detecting cocaine-like anesthetics in livestock and aquatic products includes the following steps:

[0079] S1: Add 6g styrene (St), 2g divinylbenzene (DVB), 0.15g azobisisobutyronitrile (AIBN), and 6mL toluene to a three-necked flask and stir magnetically until the AIBN is completely dissolved. Add 200mL ultrapure water and 0.85g polyvinyl alcohol (PVA) to a beaker and heat to 40℃ while stirring until dissolved. Purge the three-necked flask with nitrogen for 15min to remove oxygen, then raise the temperature to 60℃ and start constant stirring (200r / min). Slowly add the PVA aqueous solution dropwise to the three-necked flask and react for 6h to form a white polymer microsphere emulsion. After cooling, filter to separate the microspheres, wash three times with ultrapure water, then wash twice with anhydrous ethanol, and dry under vacuum at 60℃ for 4h to obtain white St-DVB copolymer microspheres.

[0080] S2: Take 5g of the dried St-DVB copolymer microspheres from S1 and add them to 50mL of dichloroethane. Disperse the microspheres by sonication for 20min. After cooling to 0~5℃ in an ice bath, slowly add 7.5g of chlorosulfonic acid and stir the reaction. After the addition is completed, raise the temperature to 35℃ and continue stirring the reaction for about 2h, controlling the pH value (pH<1) during the process. After the reaction is completed, cool down and filter to separate the sulfonated microspheres. Wash with ultrapure water until the pH of the filtrate is 5~6, then wash once with 5% methanol water, and vacuum dry at 60℃ for 6h to obtain sulfonic acid-functionalized St-DVB benzene copolymer microspheres.

[0081] S3: Take 3g of the dried sulfonic acid group-functionalized St-DVB benzene copolymer microspheres from S2, add 30mL of toluene and disperse by ultrasonication, then add 1.5g of octadecyltrichlorosilane (OTS) and 0.8mL of triethylamine. Under nitrogen protection, heat to 80℃ and stir for 6h. After cooling, filter to separate the microspheres and wash them with toluene and anhydrous ethanol 3-5 times in sequence. After vacuum drying at 60℃ for 6h, the sulfonic acid group / C18 bifunctional group-functionalized St-DVB copolymer is obtained.

[0082] S4: Pack the copolymer material obtained in S3 into a solid-phase extraction column. Take a 3mL syringe-type solid-phase extraction empty column pre-loaded with a hydrophobic sieve plate at the bottom, accurately weigh 60mg of the copolymer material obtained in S3 and add it into the column. Then, install the sieve plate on top of the packing material to obtain the packed solid-phase extraction column. Activate the column sequentially with 3mL of methanol and 3mL of 0.1% methanol-water solution, and set aside for use.

[0083] S5: Accurately weigh 5g of crab meat (accurate to 0.01g) into a 50mL centrifuge tube, add 10mL of 1% acetic acid acetonitrile solution, vortex mix for 2min until the meat is completely dispersed, extract by shaking for 10min, centrifuge at 6500r / min for 8min, take the supernatant and dilute with 0.1% methanol water to 20mL, then pass it through the solid phase extraction column activated in S4. After passing through, wash with 3mL of 0.1% methanol water and 3mL of 5% methanol water in sequence, and finally elute with 6mL of 2% ammonia water and methanol. Blow under nitrogen at 40℃ until nearly dry, redissolve with 1mL of mobile phase and filter through a 0.22μm filter membrane, and then determine by ultra-high performance liquid chromatography-electrostatic field orbital trap high resolution mass spectrometry. The liquid chromatography conditions were as follows: Column: Atlantis T3 column (100 mm length × 2.1 mm inner diameter, packing material particle size 3.0 μm); Column temperature: 30℃; Mobile phase A was 0.1% formic acid aqueous solution containing 5 mmol / L ammonium formate; Mobile phase B was 0.1% formic acid in methanol; The flow rate of the mixture of mobile phases A and B was 0.40 mL / min; Injection volume: 10 μL. Gradient elution program: 0–4 min, 5–20% B; 4–5 min, 20–40% B; 5–10 min, 40–100% B; 10–13 min, 100% B; 13–15 min, 5% B. The mass spectrometry conditions were as follows: electrospray ionization source: ESI, positive ion scan mode; spray voltage: 3200 V; ion source temperature: 325℃; sheath gas pressure: 40 arb; auxiliary gas pressure: 30 arb; capillary temperature: 350℃; nebulizer gas: high-purity nitrogen; collision gas: high-purity helium. Mass spectrometry data acquisition mode: full scan + automatic triggering of the secondary mass spectrometer in positive ion scan mode.

[0084] In this embodiment, the obtained copolymer material, as observed by scanning electron microscopy, exhibits smooth microspheres, high sphericity, good dispersibility, no obvious agglomeration, and a certain particle size distribution. Laser particle size analyzer measurements show that the particle size is mainly concentrated in the range of 80-90 μm, with a diameter spacing of 0.325 and a diameter (D) of 88.257 μm. Infrared spectroscopy measurements at 1190 cm⁻¹... -1 and 1040cm -1 Nearby are asymmetric and symmetric stretching vibration peaks of sulfonate ions, 2920 cm⁻¹. -1 Characteristic peak of long-chain alkyl (C18) at 1730 cm⁻¹ -1The stretching vibration peak of the ester group (C=O) appears nearby. The sulfonic acid group (-SO3H) introduced by sulfonation undergoes esterification reaction with the hydroxyl group (-OH) of C18 to form an ester bond structure. Thermogravimetric analysis shows that the mass does not change significantly at room temperature to 200℃ (weight loss rate <5%), making it suitable as a material for subsequent solid phase extraction. At 200~600℃, the long-chain alkyl and sulfonic acid groups of C18 decompose sequentially, and the St-DVB crosslinked backbone is basically stable.

[0085] Quantification using the matrix curve external standard method revealed good linearity for 12 cocaine-type anesthetics within the matrix solution standard curve range of 1.0–100 μg / L, with correlation coefficients (r) greater than 0.995. The limits of quantitation (LOQ) ranged from 1.0 to 5.0 μg / kg. Spike recoveries in the matrix solution ranged from 72.8% to 111.9%, with RSDs ≤7.2%.

[0086] Example 4

[0087] A method for preparing a solid-phase extraction material for detecting cocaine-like anesthetics in livestock and aquatic products includes the following steps:

[0088] S1: Add 12g styrene (St), 2g divinylbenzene (DVB), 0.25g azobisisobutyronitrile (AIBN), and 13.5mL toluene to a three-necked flask and stir magnetically until the AIBN is completely dissolved. Add 200mL ultrapure water and 1.5g polyvinyl alcohol (PVA) to a beaker and heat to 40℃ while stirring until dissolved. Purge the three-necked flask with nitrogen for 15min to remove oxygen, then raise the temperature to 70℃ and start constant stirring (200r / min). Slowly add the PVA aqueous solution dropwise to the three-necked flask and react for 6h to form a white polymer microsphere emulsion. After cooling, filter to separate the microspheres, wash three times with ultrapure water, then wash twice with anhydrous ethanol, and dry under vacuum at 60℃ for 4h to obtain white St-DVB copolymer microspheres.

[0089] S2: Take 5g of the dried St-DVB copolymer microspheres from S1 and add them to 50mL of dichloroethane. Disperse the microspheres by sonication for 20min. After cooling to 0~5℃ in an ice bath, slowly add 6.5g of chlorosulfonic acid and stir the reaction. After the addition is completed, raise the temperature to 35℃ and continue stirring the reaction for about 2h, controlling the pH value (pH<1) during the process. After the reaction is completed, cool down and filter to separate the sulfonated microspheres. Wash with ultrapure water until the pH of the filtrate is 5~6, then wash once with 5% methanol water, and vacuum dry at 60℃ for 6h to obtain sulfonic acid-functionalized St-DVB benzene copolymer microspheres.

[0090] S3: Take 3g of the dried sulfonic acid group-functionalized St-DVB benzene copolymer microspheres from S2, add 30mL of toluene and disperse by ultrasonication, then add 2.4g of octadecyltrichlorosilane (OTS) and 1.2mL of triethylamine. Under nitrogen protection, heat to 80℃ and stir for 6h. After cooling, filter to separate the microspheres and wash them with toluene and anhydrous ethanol 3-5 times in sequence. After vacuum drying at 60℃ for 6h, the sulfonic acid group / C18 bifunctional group-functionalized St-DVB copolymer is obtained.

[0091] S4: Pack the copolymer material obtained in S3 into a solid-phase extraction column. Take a 3mL syringe-type solid-phase extraction empty column pre-loaded with a hydrophobic sieve plate at the bottom, accurately weigh 60mg of the copolymer material obtained in S3 and add it into the column. Then, install the sieve plate on top of the packing material to obtain the packed solid-phase extraction column. Activate the column sequentially with 3mL of methanol and 3mL of 0.1% methanol-water solution, and set aside for use.

[0092] S5: Accurately weigh 5g of minced pork (accurate to 0.01g) into a 50mL centrifuge tube, add 10mL of 1% acetic acid acetonitrile solution, vortex for 2min until the meat is completely dispersed, extract by shaking for 10min, centrifuge at 6500r / min for 8min, take the supernatant and dilute with 0.1% methanol water to 20mL, then pass it through the solid phase extraction column activated in S4. After passing through, wash with 3mL of 0.1% methanol water and 3mL of 5% methanol water in sequence, and finally elute with 6mL of 2% ammonia water and methanol. Blow under nitrogen at 40℃ until nearly dry, redissolve with 1mL of mobile phase and filter through a 0.22μm filter membrane, and then determine by ultra-high performance liquid chromatography-electrostatic field orbital trap high resolution mass spectrometry. The liquid chromatography conditions were as follows: Column: Atlantis T3 column (100 mm length × 2.1 mm inner diameter, packing material particle size 3.0 μm); Column temperature: 30℃; Mobile phase A was 0.1% formic acid aqueous solution containing 5 mmol / L ammonium formate; Mobile phase B was 0.1% formic acid in methanol; The flow rate of the mixture of mobile phases A and B was 0.40 mL / min; Injection volume: 10 μL. Gradient elution program: 0–4 min, 5–20% B; 4–5 min, 20–40% B; 5–10 min, 40–100% B; 10–13 min, 100% B; 13–15 min, 5% B. The mass spectrometry conditions were as follows: electrospray ionization source: ESI, positive ion scan mode; spray voltage: 3200 V; ion source temperature: 325℃; sheath gas pressure: 40 arb; auxiliary gas pressure: 30 arb; capillary temperature: 350℃; nebulizer gas: high-purity nitrogen; collision gas: high-purity helium. Mass spectrometry data acquisition mode: full scan + automatic triggering of the secondary mass spectrometer in positive ion scan mode.

[0093] In this embodiment, the obtained copolymer material, as observed by scanning electron microscopy, exhibits smooth microspheres, high sphericity, good dispersibility, no obvious agglomeration, and a certain particle size distribution. Laser particle size analyzer measurements show that the particle size distribution is concentrated between 110 and 120 μm, with a diameter spacing of 0.551 and a diameter (D) of 120.418 μm. Infrared spectroscopy measurements show that the particle size distribution is within the range of 1190 cm⁻¹. -1 and 1040cm -1 Nearby are asymmetric and symmetric stretching vibration peaks of sulfonate ions, 2920 cm⁻¹. -1 Characteristic peak of long-chain alkyl (C18) at 1730 cm⁻¹ -1 The stretching vibration peak of the ester group (C=O) appears nearby. The sulfonic acid group (-SO3H) introduced by sulfonation undergoes esterification reaction with the hydroxyl group (-OH) of C18 to form an ester bond structure. Thermogravimetric analysis shows that the mass does not change significantly at room temperature to 200℃ (weight loss rate <5%), making it suitable as a material for subsequent solid phase extraction. At 200~600℃, the long-chain alkyl and sulfonic acid groups of C18 decompose sequentially, and the St-DVB crosslinked backbone is basically stable.

[0094] Quantification using the matrix curve external standard method revealed good linearity for 12 cocaine-type anesthetics within the matrix solution standard curve range of 1.0–100 μg / L, with correlation coefficients (r) greater than 0.995. The limits of quantification (LOQ) ranged from 1.0 to 5.0 μg / kg. Spike recoveries in the matrix solution ranged from 77.6% to 118.7%, with RSDs ≤6.4%.

[0095] Example 5

[0096] A method for preparing a solid-phase extraction material for detecting cocaine-like anesthetics in livestock and aquatic products includes the following steps:

[0097] S1: Add 12g styrene (St), 3g divinylbenzene (DVB), 0.3g azobisisobutyronitrile (AIBN), and 15mL toluene to a three-necked flask and stir magnetically until the AIBN is completely dissolved. Add 200mL ultrapure water and 2g polyvinyl alcohol (PVA) to a beaker and heat to 40℃ while stirring until dissolved. Purge the three-necked flask with nitrogen for 15min to remove oxygen, then raise the temperature to 70℃ and start constant stirring (200r / min). Slowly add the PVA aqueous solution dropwise to the three-necked flask and react for 6h to form a white polymer microsphere emulsion. After cooling, filter to separate the microspheres, wash three times with ultrapure water, then wash twice with anhydrous ethanol, and dry under vacuum at 60℃ for 4h to obtain white St-DVB copolymer microspheres.

[0098] S2: Take 5g of the dried St-DVB copolymer microspheres from S1 and add 50mL of dichloroethane. Disperse the microspheres by sonication for 20min. After cooling to 0~5℃ in an ice bath, slowly add 5.5g of chlorosulfonic acid and stir the reaction. After the addition is completed, raise the temperature to 35℃ and continue stirring the reaction for about 2h, controlling the pH value (pH<1) during the process. After the reaction is completed, cool down and filter to separate the sulfonated microspheres. Wash with ultrapure water until the pH of the filtrate is 5~6, then wash once with 5% methanol water, and vacuum dry at 60℃ for 6h to obtain sulfonic acid-functionalized St-DVB benzene copolymer microspheres.

[0099] S3: Take 3g of dried sulfonic acid group-functionalized St-DVB benzene copolymer microspheres from S2, add 30mL of toluene and ultrasonically disperse, then add 1.8g of octadecyltrichlorosilane (OTS) and 1mL of triethylamine. Under nitrogen protection, heat to 80℃ and stir for 6h. After cooling, filter to separate the microspheres and wash them with toluene and anhydrous ethanol 3-5 times in sequence. After vacuum drying at 60℃ for 6h, the sulfonic acid group / C18 bifunctional group-functionalized St-DVB copolymer is obtained.

[0100] S4: Pack the copolymer material obtained in S3 into a solid-phase extraction column. Take a 3mL syringe-type solid-phase extraction empty column pre-loaded with a hydrophobic sieve plate at the bottom, accurately weigh 60mg of the copolymer material obtained in S3 and add it into the column. Then, install the sieve plate on top of the packing material to obtain the packed solid-phase extraction column. Activate the column sequentially with 3mL of methanol and 3mL of 0.1% methanol-water solution, and set aside for use.

[0101] S5: Accurately weigh 5g (accurate to 0.01g) of freeze-dried and ground chicken manure sample through a 0.25mm nylon sieve into a 50mL centrifuge tube, add 10mL of 1% acetic acid acetonitrile solution, vortex mix for 2min until the meat is completely dispersed, extract by shaking for 10min, centrifuge at 6500r / min for 8min, take the supernatant and dilute with 0.1% methanol-water to 20mL, then pass it through the solid phase extraction column activated in S4. After passing through, wash successively with 3mL of 0.1% methanol-water and 3mL of 5% methanol-water, and finally elute with 6mL of 2% ammonia-methanol solution. Blow under nitrogen at 40℃ until nearly dry, redissolve with 1mL of mobile phase and filter through a 0.22μm filter membrane, and then determine by ultra-high performance liquid chromatography-electrostatic field orbital trap high resolution mass spectrometry. The liquid chromatography conditions were as follows: Column: Atlantis T3 column (100 mm length × 2.1 mm inner diameter, packing material particle size 3.0 μm); Column temperature: 30℃; Mobile phase A was 0.1% formic acid aqueous solution containing 5 mmol / L ammonium formate; Mobile phase B was 0.1% formic acid in methanol; The flow rate of the mixture of mobile phases A and B was 0.40 mL / min; Injection volume: 10 μL. Gradient elution program: 0–4 min, 5–20% B; 4–5 min, 20–40% B; 5–10 min, 40–100% B; 10–13 min, 100% B; 13–15 min, 5% B. The mass spectrometry conditions were as follows: electrospray ionization source: ESI, positive ion scan mode; spray voltage: 3200 V; ion source temperature: 325℃; sheath gas pressure: 40 arb; auxiliary gas pressure: 30 arb; capillary temperature: 350℃; nebulizer gas: high-purity nitrogen; collision gas: high-purity helium. Mass spectrometry data acquisition mode: full scan + automatic triggering of the secondary mass spectrometer in positive ion scan mode.

[0102] In this embodiment, the obtained copolymer material, as observed by scanning electron microscopy, exhibits smooth microspheres, high sphericity, good dispersibility, no obvious agglomeration, and a certain particle size distribution. Laser particle size analyzer measurements show that the particle size distribution is concentrated between 90 and 100 μm, with a diameter spacing of 0.414 and a diameter (D) of 95.368 μm [4,3]. Infrared spectroscopy measurements at 1190 cm⁻¹ show a particle size distribution within this range. -1 and 1040 cm -1 Nearby are asymmetric and symmetric stretching vibration peaks of sulfonate ions, 2920 cm⁻¹. -1 Characteristic peak of long-chain alkyl (C18) at 1730 cm⁻¹ -1The stretching vibration peak of the ester group (C=O) appears nearby. The sulfonic acid group (-SO3H) introduced by sulfonation undergoes esterification reaction with the hydroxyl group (-OH) of C18 to form an ester bond structure. Thermogravimetric analysis shows that the mass does not change significantly at room temperature to 200℃ (weight loss rate <5%), making it suitable as a material for subsequent solid phase extraction. At 200~600℃, the long-chain alkyl and sulfonic acid groups of C18 decompose sequentially, and the St-DVB crosslinked backbone is basically stable.

[0103] Quantification using the matrix curve external standard method revealed good linearity for 12 cocaine-type anesthetics within the matrix solution standard curve range of 1.0–100 μg / L, with correlation coefficients (r) greater than 0.995. The limits of quantitation (LOQ) ranged from 1.0 to 5.0 μg / kg. Spike recoveries in the matrix solution ranged from 73.8% to 112.5%, with RSDs ≤6.1%.

[0104] As can be seen from the above experimental results, this application provides a method for preparing a solid-phase extraction material for the detection of cocaine-like anesthetics in livestock and aquatic products. The prepared copolymer packing material has the core advantages of suitable particle size, uniform distribution, and high effective adsorption capacity. Furthermore, the preparation process is simple, efficient, and cost-controllable, making it feasible for large-scale production. After packing the packing material into a solid-phase extraction column, spiked recovery tests on complex matrix samples verified that its extraction recovery rate for cocaine-like anesthetics in livestock and aquatic products is stable and reproducible, effectively meeting practical detection needs. In addition, based on the dual mechanism of cation exchange and reverse-phase adsorption, this copolymer material also exhibits good compatibility with other basic compounds such as ephedrine, clenbuterol, tetracyclines, and histamine. Therefore, it has broad application prospects and significant practical value in the detection of veterinary drug residues, alkaloids, and biogenic amines.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for the preparation of a solid phase extraction material for the detection of local anesthetics in aquatic products, characterized in that, Comprise the following steps: S1, the styrene St, divinylbenzene DVB, initiator is dissolved in the pore forming agent, magnetic stirring until completely dissolved, to obtain a mixed solution; then the dispersing agent is added to pure water, stirring and dissolving and cooling to room temperature, then pour into the mixed solution, to obtain a mixed solution; the mixed solution is continuously stirred at a constant speed and heated, and the styrene St and divinylbenzene DVB are reacted to form St-DVB copolymer emulsion; after cooling to room temperature, separation by suction filtration, sequentially washed with ultrapure water, anhydrous ethanol, and vacuum dried to obtain St-DVB copolymer microspheres; S2, the dried St-DVB copolymer microspheres are ultrasonically dispersed in dichloroethane, and after cooling, the sulfonating agent is added dropwise, heated and stirred, and the pH in the reaction process is controlled; after cooling, the microspheres are collected by suction filtration and washed with ultrapure water, and vacuum dried to obtain sulfonic acid group functionalized St-DVB copolymer microspheres; S3, the sulfonic acid group functionalized St-DVB copolymer microspheres are added to a reaction solvent, ultrasonically dispersed, and then a bonding agent and a catalyst are added, heated and stirred, and after cooling, the microspheres are separated by suction filtration, sequentially washed with toluene and anhydrous ethanol, and vacuum dried to obtain sulfonic acid group / C18 bifunctional functionalized St-DVB copolymer; the sulfonic acid group / C18 bifunctional functionalized St-DVB copolymer is preloaded in a solid phase extraction empty column, the filler is tightly packed in the solid phase extraction empty column to obtain a solid phase extraction column, and after activation with methanol and formic acid, an activated solid phase extraction column is obtained, which is ready for use; S4, accurately weigh the homogenate prepared from livestock and aquatic products in a centrifuge tube, add acetic acid acetonitrile solution, extract on a uniform speed shaker, and then centrifuge, transfer the supernatant to the activated solid phase extraction column, and after elution and washing, collect the target product, concentrate the mobile phase by nitrogen blowing, pass through a filter membrane, and collect in a sample vial for determination by ultra-high performance liquid chromatography-electron field orbitrap high resolution mass spectrometry.

2. The method for preparing solid-phase extraction material for detecting cocaine-like anesthetics in livestock and aquatic products according to claim 1, characterized in that, In step S1, the mass ratio of styrene St to divinylbenzene DVB is 6:1~3:1; the initiator is azobisisobutyronitrile AIBN, and the initiator accounts for 1~2% of the total mass of styrene St and divinylbenzene DVB; the dispersing agent is polyvinyl alcohol PVA, and the dispersing agent accounts for 3~8% of the total mass of the reaction system; the pore forming agent is toluene, and the pore forming agent accounts for 30~50% of the total mass of the reaction system.

3. The method for preparing solid-phase extraction material for detecting cocaine-like anesthetics in livestock and aquatic products according to claim 1, characterized in that, In step S2, the sulfonating agent is chlorosulfonic acid; the mass ratio of the sulfonating agent to St-DVB copolymer microspheres is 1.5:1~1:1; the pH in the reaction process is controlled to be less than 1; and the pH of the filtrate after washing with ultrapure water is 5~6.

4. The method for preparing solid-phase extraction material for detecting cocaine-like anesthetics in livestock and aquatic products according to claim 1, characterized in that, In step S3, the bonding agent is octadecyltrichlorosilane OTS, and the mass ratio of the bonding agent to sulfonic acid group functionalized St-DVB copolymer microspheres is 0.5:1~1:1; the catalyst is triethylamine, and the amount of the catalyst is 10~25% of the total mass of the reaction system; the reaction solvent is anhydrous toluene; the heating and stirring reaction temperature is 80~100℃, and the heating and stirring reaction time is 5~8 hours.

5. The method for preparing solid-phase extraction material for detecting cocaine-like anesthetics in livestock and aquatic products according to claim 1, characterized in that, In step S4, the conditions of the liquid chromatography are as follows: chromatographic column: Atlantis T3 column, column temperature: 30 DEG C; mobile phase A is 0.1% formic acid aqueous solution containing 5mmol / L ammonium formate, mobile phase B is 0.1% formic acid methanol, the flow rate of the mixture of mobile phase A and mobile phase B is 0.40 mL / min, injection volume is 10 μL; The gradient elution program is as follows: 0-4 min, 5-20%B; 4-5 min, 20-40%B; 5-10 min, 40-100%B; 10-13 min, 100%B; 13-15 min, 5%B.

6. The method for preparing a solid phase extraction material for detecting a caine anesthetic in aquaculture products according to claim 5 The method is characterized in that, The size of the liquid chromatography column Atlantis T3 column is 100 mm column length x 2.1 mm inner diameter, and the particle size of the filler in the Atlantis T3 column is 3.0 μm.

7. The method for preparing solid-phase extraction material for detecting cocaine-like anesthetics in livestock and aquatic products according to claim 1, characterized in that, In step S4, the conditions of the mass spectrometry are as follows: electrospray ion source: ESI, positive ion scanning mode, spray voltage: 3200 V, ion source temperature: 325 DEG C, sheath gas pressure: 40 arb, auxiliary gas pressure: 30 arb, capillary temperature: 350 DEG C, atomizing gas is high-purity nitrogen, and collision gas is high-purity helium.

8. The method for preparing solid-phase extraction material for detecting cocaine-like anesthetics in livestock and aquatic products according to claim 1, characterized in that, In step S4, the aquaculture products are selected from at least one of freshwater fish, shrimp, crab, pork, and chicken manure.

9. A solid phase extraction material for the detection of local anesthetics in aquatic products, characterized in that, The solid phase extraction material for detecting a caine anesthetic in aquaculture products is prepared by the method according to any one of claims 1-8.

10. Use of the solid phase extraction material for detection of caine-type narcotics in aquaculture products according to claim 9 for the detection of caine-type narcotics in aquaculture products, characterized in that, The caine anesthetic is selected from at least one of procaine penethamate mesylate MS-222, m-aminobenzoic acid, benzocaine, p-aminobenzoic acid, p-acetylamino benzoic acid, chloroprocaine, procainamide, lidocaine, cinchocaine, bupivacaine, prilocaine, and ropivacaine.

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