An extraction material for determining benzimidazole veterinary drug residues, its preparation method and application

Abstract

This invention relates to the field of magnetic solid-phase extraction (MSE) detection technology, specifically to an extraction material for determining benzimidazole veterinary drug residues, its preparation method, and its application. The preparation method includes the following steps: dispersing magnetic SiO2 / Fe3O4 particles in a mixed solution, adding aminopropyltriethoxysilane, stirring, heating, and washing to obtain APTES / SiO2 / Fe3O4 particles; dispersing the APTES / SiO2 / Fe3O4 particles in a mixture of ethanol and 2,5-dihydroxyterephthalaldehyde, stirring to obtain Dt / APTES / SiO2 / Fe3O4 particles; dispersing the Dt / APTES / SiO2 / Fe3O4 particles in a mixture of ethanol, 2,5-dihydroxyterephthalaldehyde, and 1,3,5-tris(4-aminophenyl)benzene, ultrasonically dispersing, then adding graphene oxide and acetic acid, stirring, washing, and drying to obtain the extraction material. The prepared magnetic composite material was used as an extractant to extract benzimidazole veterinary drug residues, and the residues were detected by LC-MS / MS. Compared with the detection scheme in the national food safety standard, the detection limit of the present invention was reduced by three orders of magnitude.

Description

Technical Field

[0001] This invention relates to the field of magnetic solid-phase extraction detection technology, specifically to an extraction material for determining benzimidazole veterinary drug residues, its preparation method, and its application. Background Technology

[0002] Benzimidazole veterinary drugs are a class of heterocyclic aromatic compounds characterized by the fusion of a benzene ring and an imidazole ring, forming a benzimazole core skeleton. These compounds possess a variety of biological activities, including antiparasitic, antiviral, antifungal, antitumor, anti-inflammatory, analgesic, and antihypertensive effects. In the veterinary field, benzimazole veterinary drugs are mainly used as anthelmintics, exerting their effects by targeting the microtubule formation of parasites. Commonly used benzimazole veterinary drugs include albendazole, flubendazole, mebendazole, oxybendazole, and oxendazole.

[0003] However, the widespread use of benzimidazole veterinary drugs in animal health has raised concerns about residues in food, as long-term exposure to these residues can lead to toxic effects, including embryonic death and teratogenicity. To mitigate these risks, regulatory agencies have established maximum residue limits for benzimidazole veterinary drugs in animal-derived foods. With increasing demands for food safety, more sensitive detection methods are needed to meet stricter testing standards.

[0004] Currently, detection techniques such as fluorescence spectroscopy, surface-enhanced Raman spectroscopy, immunoassay, and biosensors have been used to determine benzimidazole veterinary drug residues. However, given the complexity of food matrices and the typically low residue levels, liquid chromatography-tandem mass spectrometry (LC-MS / MS) has become the preferred method due to its high sensitivity, high selectivity, and ability to distinguish structurally similar analogs. Nevertheless, developing efficient and reliable sample pretreatment methods to separate the sample matrix and enrich the target analyte remains essential.

[0005] Traditional sample pretreatment methods, such as solvent extraction and conventional solid-phase extraction, are still widely used, but they suffer from drawbacks such as high organic solvent consumption, time-consuming operation, and high cost. To overcome these limitations, miniaturized sample pretreatment techniques, such as solid-phase microextraction (SPME), magnetic solid-phase extraction (MSPE), and QuEChERS, are increasingly being adopted. However, current magnetic solid-phase extraction materials exhibit poor adsorption performance when detecting benzimidazole veterinary drug residues, resulting in low sensitivity when coupled with liquid chromatography-tandem mass spectrometry (LC-MS / MS), making it impossible to accurately detect benzimidazole veterinary drug residues in food. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides an extraction material for determining benzimidazole veterinary drug residues, its preparation method, and its application. Through extensive experimentation, the inventors of this application have identified a magnetic composite material capable of extracting benzimidazole veterinary drug residues. The prepared magnetic composite material is used as an extractant to extract benzimidazole veterinary drug residues, and the residues are detected using LC-MS / MS. Compared to the detection scheme in the national food safety standard, this invention reduces the detection limit by three orders of magnitude.

[0007] Therefore, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides, in an optional embodiment, a method for preparing an extraction material for determining benzimidazole veterinary drug residues, comprising the following steps:

[0009] S1: Magnetic SiO2 / Fe3O4 particles are dispersed in a mixed solution, and aminopropyltriethoxysilane is added, stirred, heated and washed to obtain APTES / SiO2 / Fe3O4 particles;

[0010] S2: Disperse the APTES / SiO2 / Fe3O4 particles in a mixture of ethanol and 2,5-dihydroxyterephthalaldehyde and stir to obtain Dt / APTES / SiO2 / Fe3O4 particles;

[0011] S3: The Dt / APTES / SiO2 / Fe3O4 particles were dispersed in a mixture of ethanol, 2,5-dihydroxyterephthalaldehyde and 1,3,5-tris(4-aminophenyl)benzene by ultrasonic dispersion, and then graphene oxide and acetic acid were added, stirred, washed and dried to obtain the extraction material.

[0012] Preferably, in step S3, the mass ratio of the Dt / APTES / SiO2 / Fe3O4 particles, 1,3,5-tris(4-aminophenyl)benzene, 2,5-dihydroxyterephthalaldehyde and graphene oxide is 5:1.4:1:0.8-1.5.

[0013] Preferably, in step S1, the mass ratio of SiO2 to Fe3O4 in the magnetic SiO2 / Fe3O4 particles is 4:2-2.3; and / or, the mixed solution is a mixture of isopropanol and concentrated ammonia, with a volume ratio of isopropanol to concentrated ammonia of 35-40:1; and / or, the mass-to-volume ratio of the magnetic SiO2 / Fe3O4 particles to aminopropyltriethoxysilane is 2g:1mL; and / or, the stirring temperature is 30-40℃ for 0.5-1.5h; and / or, the heating temperature is 80-90℃ for 5-7h; and / or, the washing solution is deionized water and ethanol, and the washing is performed three times.

[0014] Preferably, in step S2, the mass ratio of the APTES / SiO2 / Fe3O4 particles to the 2,5-dihydroxyterephthalaldehyde is 100:17; and / or, the stirring time is 0.5-1.5 h.

[0015] Preferably, in step S3, the molar ratio of graphene oxide to acetic acid is 1g:0.0798mol, and the molar concentration of acetic acid is 6mol / L; and / or, the washing solution is ethanol, and the washing is performed 3 times; and / or, the drying method is vacuum drying at a temperature of 45-55℃.

[0016] Secondly, in an optional embodiment, the present invention provides an extraction material for determining benzimidazole veterinary drug residues, which is prepared by the above-described preparation method.

[0017] Thirdly, in an optional embodiment, the present invention provides an application of the above-mentioned extraction material in the determination of benzimidazole veterinary drug residues by magnetic solid-phase extraction.

[0018] Preferably, the application includes: mixing a sample containing benzimidazole veterinary drug residues with the extraction material described in claim 6, performing adsorption and elution, and analyzing the eluent by liquid chromatography-tandem mass spectrometry.

[0019] Preferably, the benzimidazole veterinary drug is one or more of the following: oxifendazole, fenbendazole, febantel, oxifendazole sulfone, albendazole, albendazole sulfoxide, albendazole sulfone, albendazole-2-aminosulfone, thiabendazole ester, thiabendazole, 5-hydroxythiabendazole, flubendazole, 2-aminoflubendazole, mebendazole, aminomebendazole, hydroxymebendazole, or oxybendazole; and / or, the volume-to-mass ratio of the sample containing benzimidazole veterinary drug residues to the extraction material is 10-75 mL:30 mg, preferably 75 mL:30 mg; and / or, the pH value of the sample containing benzimidazole veterinary drug residues is 3.5-4.5, preferably 4.0; and / or, the adsorption time is 5 min. The eluent used for elution is a 1% (v / v) formic acid methanol solution, and the volume of the eluent is 1 mL; and / or, the elution time is 10 min.

[0020] Compared with the prior art, the present invention has one of the following beneficial effects:

[0021] 1. Through extensive experiments, the inventors of this application have found a magnetic composite material with the ability to extract benzimidazole veterinary drug residues. The prepared magnetic composite material is used as an extractant to extract benzimidazole veterinary drug residues and is then detected by LC-MS / MS. Compared with the detection scheme in the national food safety standard, the detection limit of this invention is reduced by three orders of magnitude.

[0022] 2. This invention has conducted a detailed study on the experimental parameters of magnetic solid-phase extraction and phase chromatography-tandem mass spectrometry, and found detection conditions that can significantly improve detection sensitivity. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram showing the effect of sample pH on analyte adsorption when the extraction material TAPB-Dt&GO prepared in Example 1 of the present invention is used in conjunction with LC-MS / MS to determine benzimidazole veterinary drug residues.

[0025] Figure 2 This is a schematic diagram showing the effect of sample volume on analyte adsorption when the extraction material TAPB-Dt&GO prepared in Example 1 of the present invention is used in conjunction with LC-MS / MS to determine benzimidazole veterinary drug residues.

[0026] Figure 3 This is a schematic diagram showing the effect of the choice of eluent on the elution effect when the extraction material TAPB-Dt&GO prepared in Example 1 of the present invention is determined by LC-MS / MS in combination with LC-MS / MS for the determination of benzimidazole veterinary drug residues.

[0027] Figure 4 This is a schematic diagram showing the effect of formic acid concentration in methanol on the desorption effect when the extraction material TAPB-Dt&GO prepared in Example 1 of this invention is determined in conjunction with LC-MS / MS for the determination of benzimidazole veterinary drug residues.

[0028] Figure 5 This is a schematic diagram showing the effect of eluent volume on elution efficiency when the extraction material TAPB-Dt&GO prepared in Example 1 of the present invention is determined by LC-MS / MS in conjunction with LC-MS / MS for the determination of benzimidazole veterinary drug residues.

[0029] Figure 6This paper attempts to investigate the effect of ionic strength in the sample on the extraction efficiency when the extraction material TAPB-Dt&GO prepared in Example 1 of this invention is used in conjunction with LC-MS / MS to determine the residues of benzimidazole veterinary drugs.

[0030] Figure 7 The graph shows the adsorption efficiency of the TAPB-Dt material prepared in Comparative Example 1 of this invention for 17 kinds of benzimidazole veterinary drugs.

[0031] Figure 8 This is a comparison chart of the adsorption efficiency of the extraction material TAPB-Dt&GO prepared in Example 1 of the present invention and the material prepared in Comparative Example 2 for 17 kinds of benzimidazole veterinary drugs.

[0032] Figure 9 This is a TEM image of the extraction material TAPB-Dt&GO prepared in Example 1 of the present invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0034] In the following examples and comparative examples, the preparation method of magnetic SiO2 / Fe3O4 particles is as follows:

[0035] (1) Weigh 11.68g FeCl3·6H2O and 4.30g FeCl2·4H2O and dissolve them in 200mL of deionized water under high-purity nitrogen protection. Stir at 85℃ for 30 minutes. Then add 40mL of concentrated ammonia water with a volume fraction of 30% and stir gently to generate Fe3O4 nanoparticles. Wash with deionized water.

[0036] (2) 4.0 g of the prepared Fe3O4 nanoparticles were dispersed in a mixture of isopropanol (100 mL), deionized water (12 mL) and concentrated ammonia (7 mL), and then 8 mL of tetraethoxysilane was added. The mixture was stirred at room temperature for 12 hours, washed three times with 200 mL of deionized water and three times with 50 mL of ethanol to obtain magnetic SiO2 / Fe3O4 particles. The mass ratio of SiO2 to Fe3O4 in the magnetic SiO2 / Fe3O4 particles was 4:2.

[0037] The technical solution of the present invention will be described below with reference to embodiments.

[0038] Example 1

[0039] This embodiment provides a method for preparing an extractable material for determining benzimidazole veterinary drug residues, comprising the following steps:

[0040] S1: Disperse 4.0 g of SiO2 / Fe3O4 in a mixture of isopropanol (150 mL) and concentrated ammonia (4 mL), then add 2 mL of aminopropyltriethoxysilane. Stir at 35 °C for 1 hour, then heat to 85 °C and stir for 6 hours to prepare APTES / SiO2 / Fe3O4. Wash three times with 200 mL of deionized water and three times with 50 mL of ethanol to obtain APTES / SiO2 / Fe3O4 particles.

[0041] S2: Disperse 0.5g of APTES / SiO2 / Fe3O4 particles in a mixture of ethanol (20mL) and 85mg of 2,5-dihydroxyterephthalaldehyde (Dt), stir for 1 hour, and obtain Dt / APTES / SiO2 / Fe3O4 particles.

[0042] S3: Take 500 mg of Dt / APTES / SiO2 / Fe3O4 particles and disperse them in a mixture of 90 mL of ethanol, 100 mg of Dt and 140 mg of 1,3,5-tris(4-aminophenyl)benzene. After ultrasonic dispersion, add 100 mg of graphene oxide and 1.33 mL of acetic acid (molar concentration of 6 mol / L) and stir for 3 days. Finally, wash three times with 100 mL of ethanol and dry under vacuum at 50 °C to obtain the extraction material, denoted as TAPB-Dt&GO.

[0043] The TAPB-Dt&GO prepared in this embodiment was scanned by transmission electron microscopy, and the obtained TEM image is shown below. Figure 1 .

[0044] Comparative Example 1

[0045] To highlight the advantages of hybrid graphene oxide in the extraction material prepared by this invention in extracting benzimidazole veterinary drug residues, the applicant provides a method for preparing the extraction material. The difference between this method and Example 1 is that graphene oxide is not added in step S3. The specific preparation method is as follows:

[0046] S1: Disperse 4.0 g of SiO2 / Fe3O4 in a mixture of isopropanol (150 mL) and concentrated ammonia (4 mL), then add 2 mL of aminopropyltriethoxysilane. Stir at 35 °C for 1 hour, then heat to 85 °C and stir for 6 hours to prepare APTES / SiO2 / Fe3O4. Wash three times with 200 mL of deionized water and three times with 50 mL of ethanol to obtain APTES / SiO2 / Fe3O4 particles.

[0047] S2: Disperse 0.5g of APTES / SiO2 / Fe3O4 particles in a mixture of ethanol (20mL) and 85mg Dt, stir for 1 hour to obtain Dt / APTES / SiO2 / Fe3O4 particles.

[0048] S3: Take 500 mg of Dt / APTES / SiO2 / Fe3O4 particles and disperse them in a mixture of 90 mL of ethanol, 100 mg of Dt and 140 mg of 1,3,5-tris(4-aminophenyl)benzene. After ultrasonic dispersion, add 1.33 mL of acetic acid (6 mol / L) and stir for 3 days. Finally, wash three times with 100 mL of ethanol and dry under vacuum at 50 °C to obtain the extraction material, denoted as TAPB-Dt.

[0049] Comparative Example 2

[0050] To highlight that the extraction performance of the extraction material is related to the sum of the total mass of Dt and the mass of 1,3,5-tris(4-aminophenyl)benzene added in steps S2 and S3, and the mass of graphene oxide, the applicant provides a method for preparing the extraction material. The difference between this method and Example 1 is that the ratio of the mass of Dt added in steps S2 and S3 to the mass of graphene oxide is limited to 1:3. The specific preparation method is as follows:

[0051] S1: Disperse 4.0 g of SiO2 / Fe3O4 in a mixture of isopropanol (150 mL) and concentrated ammonia (4 mL), then add 2 mL of aminopropyltriethoxysilane. Stir at 35 °C for 1 hour, then heat to 85 °C and stir for 6 hours to prepare APTES / SiO2 / Fe3O4. Wash three times with 200 mL of deionized water and three times with 50 mL of ethanol to obtain APTES / SiO2 / Fe3O4 particles.

[0052] S2: Disperse 0.5g of APTES / SiO2 / Fe3O4 particles in a mixture of ethanol (20mL) and 85mg Dt, stir for 1 hour to obtain Dt / APTES / SiO2 / Fe3O4 particles.

[0053] S3: Disperse 500 mg Dt / APTES / SiO2 / Fe3O4 particles in a mixture of 90 mL ethanol, 100 mg Dt and 140 mg 1,3,5-tris(4-aminophenyl)benzene. After ultrasonic dispersion, add 300 mg graphene oxide and 1.33 mL acetic acid (6 mol / L) and stir for 3 days. Finally, wash three times with 100 mL ethanol and dry under vacuum at 50 °C to obtain the extraction material.

[0054] Experimental Example

[0055] To demonstrate the extraction performance of the extraction material prepared in Example 1 of this invention, the following experiments were conducted:

[0056] 1. Reagents and standard solutions

[0057] In this experiment, the benzimidazole veterinary drugs used were: oxifendazole, fenbendazole, febantel, oxifendazole sulfone, albendazole, albendazole sulfoxide, albendazole sulfone, albendazole-2-aminosulfone, thiabendazole ester, thiabendazole, 5-hydroxythiabendazole, flubendazole, 2-aminoflubendazole, mebendazole, aminomebendazole, hydroxymebendazole, and oxybendazole. All these benzimidazole veterinary drug standards were purchased from Shanghai Anpu Experimental Technology Co., Ltd. (China). All standards were stored according to the certificate recommendations.

[0058] All chemicals and reagents used were at least analytical grade. Ferric chloride hexahydrate (FeCl3·6H2O), ferrous chloride tetrahydrate (FeCl2·4H2O), ammonia (NH3·H2O), ethanol, and acetic acid were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd., China. 2,5-Dihydroxy-terephthalaldehyde (Dt), 1,3,5-tris(4-aminophenyl)benzene (TAPB), graphene oxide (GO), tetraethoxysilane (TEOS), and aminopropyltriethoxysilane (APTES) were purchased from Aladdin Reagent Company. High-purity deionized water obtained through the Milli-Q system was used throughout the work. HPLC-grade acetonitrile (ACN) and methanol (MeOH) were purchased from Sigma-Aldrich.

[0059] 2. Equipment

[0060] In this experiment, a liquid chromatography (LC) system (Abercrombie & Fitch, China) and a Kinetex C3000T system were used. 18 The target analyte was separated and detected using an LC column (2.6 µm, 100 × 2.1 mm, 100 Å). The column temperature was 35 °C. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile. The linear gradient was as follows: 0–1.0 min, 5% mobile phase B; 1.0–2.5 min, 30% B phase; 2.5–10.0 min, 60% B phase; 10.0–11.0 min, 95% B phase; 11.0–13.0 min, 95% B phase; 13.0–13.5 min, 5% mobile phase B; 13.5–16.0 min, 5% mobile phase B. The injection volume was 10 μL, and the flow rate was 0.30 mL / min.

[0061] The analytes were detected by connecting a liquid chromatography system to a QTRAP 6500 linear ion trap quadrupole mass spectrometer (AB Sciex, Singapore). The QTRAP 6500 was equipped with an electrospray ionization source and operated in positive ion mode. Data acquisition was performed using multiple reaction monitoring (MRM). The optimal detection conditions and MRM parameters for the analytes are shown in Table 1.

[0062]

[0063] 3. Sample preparation

[0064] Milk samples were purchased from the local market. The pH of the milk was adjusted to 4.6 with glacial acetic acid, followed by centrifugation to remove precipitated proteins. The milk supernatant was collected and its pH was adjusted to 4.0 with formic acid. 75 mL of the milk supernatant was transferred to a 100 mL polypropylene tube as the actual sample for MSPE.

[0065] Pork powder (quality control sample) was purchased from Beijing GRAND RM Technology Co., Ltd. Following the operating instructions, 15.0 g of high-purity deionized water was mixed with 5.0 g of pork powder and homogenized for 15 min to prepare the pork sample. The extraction of benzimidazole drugs from the pork sample was performed according to "GB / T 21324-2007 Method for the Determination of Benzimidazole Drug Residues in Muscle and Liver of Edible Animals". The operating steps are as follows: 5.0 g of pork sample was placed in a 50 mL polypropylene tube, and 20 mL of ethyl acetate, 0.15 mL of 50% (m / m) potassium hydroxide solution, 1 mL of 1% (m / v) BHT solution, and 1 g of anhydrous sodium sulfate were added. The mixture was homogenized for 30 s and centrifuged. The supernatant was collected, and the extraction was repeated once under the same conditions. The supernatants were combined and evaporated under reduced pressure to near dryness in a rotary evaporator at 38℃. The residue was redissolved in 50 mL of acetonitrile. The extract was diluted with formic acid-sodium formate buffer (pH 4.0), and 75 mL of the diluted solution was taken as the actual sample of MSPE.

[0066] To investigate the experimental parameters of MSPE, formate-sodium formate buffer solution (pH 4.0) was used as a blank sample.

[0067] 4. Magnetic solid phase extraction (MSPE) and detection analysis

[0068] MSPE comprises the following steps: 75 mL of sample and 30 mg of TAPB-Dt&GO magnetic material are transferred to a 100 mL centrifuge tube, and the mixture is vibrated for 5 minutes to allow adsorption. Then, an Nd-Fe-B strong magnet is used to attract the magnetic particles at the bottom of the centrifuge tube, and the supernatant is removed. Subsequently, 1.0 mL of 1% formic acid-methanol solution is added, and the target analyte is desorbed under vibration for 10 minutes. The eluent is filtered and then introduced into LC-MS / MS for quantitative analysis.

[0069] For many compounds, pH can affect their chemical properties and, consequently, the adsorption performance of the analyte on the adsorbent. To investigate the effect of pH on adsorption performance, studies were conducted using samples with pH values ​​ranging from 2.5 to 11. The results are as follows: Figure 1As shown in the figure, the adsorption efficiency of 17 benzimidazole drugs increased with increasing sample pH from 2.5 to 4; when the pH continued to increase from 4 to 11, the adsorption efficiency of albendazole sulfone, albendazole sulfoxide, and albendazole-2-aminosulfone decreased. The adsorption efficiency of thiabendazole ester decreased with increasing pH to 8; the adsorption efficiencies of flubendazole, hydroxymebendazole, oxybendazole, and albendazole also decreased with increasing pH to 11. The adsorption efficiency of the 17 benzimidazole drugs remained above 85% only in the pH range of 3.5-4.5. Therefore, pH 4.0 was selected as the sample pH.

[0070] The effect of sample volume, such as Figure 2 As shown, sample solutions of 10, 20, 25, 30, 40, 50, 75, 100, 125, 150, 200, and 250 mL were prepared, each containing 50 ng of each of the 17 benzimidazole drugs. The results showed that the extraction efficiency of the 17 benzimidazole drugs remained constant across sample solutions ranging from 10 to 75 mL. Therefore, 75 mL was used as the sample volume in the MSPE.

[0071] The effect of adsorption time (5-45 min) on adsorption efficiency was investigated. The adsorption efficiency of 17 benzimidazole drugs reached a plateau at 5 min, so 5 min was selected for adsorption.

[0072] The desorption efficiencies of acetonitrile, methanol, ethanol, and methanol / ethanol (1 / 1) for the target benzimidazole drug were investigated, and the results are as follows: Figure 3 As shown, acetonitrile exhibits poor desorption efficiency for albendazole sulfoxide, albendazole-2-aminosulfone, and thibendazole-5-hydroxyl. Methanol and ethanol show comparable desorption efficiencies.

[0073] pH analysis of the samples showed that the addition of either acid or alkali was beneficial for the elution of the target benzimidazole drug from the TAPB-Dt&GO magnetic material. Therefore, 1% (v / v) formic acid in methanol, methanol, 5% (v / v) ammonia in methanol, 1% (v / v) formic acid in ethanol, ethanol, and 5% (v / v) ammonia in ethanol were selected as eluents. The results showed that the addition of ammonia reduced the desorption efficiency of fenbendazole. Considering compatibility with LC-MS / MS detection, methanol containing formic acid was chosen as the desorption solvent.

[0074] The effect of formic acid concentration in methanol ranging from 0.1% to 2% (v / v) was then investigated, and the results are as follows: Figure 4As shown, the elution efficiency of benzimidazole drugs, especially oxifenedazolium sulfone, albendazole sulfoxide, albendazole-2-aminosulfone, 5-hydroxythiabendazole, 2-aminofluorobendazole, aminomebendazole, and oxybendazole, increases slightly with the formic acid concentration from 0.1% to 1% (v / v), and then tends to stabilize. Therefore, a 1% (v / v) formic acid methanol solution was chosen as the eluent for MSPE.

[0075] To determine the optimal elution volume, the effect of elution volume was investigated, and the results are as follows: Figure 5 As shown, when the elution volume is 1.0 mL, the elution efficiency of all analytes can reach over 90%. Therefore, an elution volume of 1.0 mL was chosen.

[0076] The elution time was investigated, and the results showed that the elution efficiency of all analytes reached over 90% when the elution time was 10 min. Therefore, an elution volume of 10 min was selected.

[0077] The amount of extractant used also affects the extraction efficiency of 17 benzimidazole drugs. The results showed that the extraction efficiency increased with the increase of extractant amount from 5 mg to 25 mg. To ensure quantitative extraction, the extract was 30 mg.

[0078] For animal-derived food samples, the extraction of target analytes may be affected by the presence of electrolytes. Therefore, the effect of ionic strength (NaCl concentration (m / v)) was investigated, and the results are as follows: Figure 6 As shown in the figure, for some target analytes, the extraction efficiency increases slightly with increasing NaCl concentration from 0 to 10%. For fenbendazole and nonbantel, the extraction efficiency decreases slightly with increasing NaCl concentration from 0 to 1%, and then decreases sharply with increasing NaCl concentration from 2% to 10%. For other benzimidazole drugs, the effect of NaCl concentration is negligible. Considering that the salt content in animal-derived matrix samples is usually no more than 2%, the effect of salt on extraction efficiency is not significant.

[0079] During the MSPE process, the TAPB-Dt&GO magnetic material is subjected to violent vibrations and immersion in acidic organic solvents, making its degradation inevitable. To investigate the lifespan of the TAPB-Dt&GO magnetic material, repeated adsorption and desorption of the target compound were performed. The results showed that after 10 MSPE cycles, the extraction efficiency of the TAPB-Dt&GO magnetic material could still reach over 85%, indicating that it exhibits good stability in aqueous, organic, and acidic environments.

[0080] Under optimal conditions, the analytical performance of this embodiment was investigated, and the results are listed in Table 2. The detection limit (3x signal-to-noise ratio) of this embodiment is 0.1-2 ng / L. -1 The relative standard deviation was 5.42%–12.8%; within the linear range, the r value was 0.9952–0.9999; the enrichment factor (the ratio of the slopes of the calibration curves before and after MSPE) was 63.3–74.6.

[0081]

[0082] To verify the applicability of the method of this invention in analyzing 17 benzimidazole veterinary drugs in actual samples, it was applied to determine the content of 17 benzimidazole veterinary drugs in milk and pork meal (quality control samples). The results and recoveries of actual samples and spiked samples are listed in Table 3.

[0083] As shown in the table, none of the 17 benzimidazole veterinary drugs were detected in milk. Under the conditions of low, medium and high concentrations of spiking in milk samples, the recovery rates of the 17 benzimidazole veterinary drugs were 71.2%-111%.

[0084] The concentrations of albendazole, albendazole sulfoxide, albendazole sulfone, and albendazole-2-aminosulfone detected in the pork meal supernatant were 0.190, 0.340, 0.530, and 0.116 mg / kg, respectively. -1 The detected content was consistent with the reference value on the quality control sample operation manual. To further verify the accuracy of the method in this embodiment, 100 ng / L of each of 17 benzimidazole veterinary drugs were spiked into the supernatant of the diluted pork powder, and the recovery rates were 78.3-99.1%. Among them, the recoveries of albendazole, albendazole sulfoxide, albendazole sulfone, and albendazole-2-aminosulfone were 90.5%, 78.3%, 90.7%, and 87.9%, respectively.

[0085] The above results indicate that this method is suitable for the detection of target benzimidazole veterinary drugs in milk and pork samples, and has the advantages of high sensitivity and accuracy.

[0086]

[0087] a: 250 times dilution.

[0088] ND indicates not detected.

[0089] 5. To highlight the advantages of hybrid graphene oxide in the extraction material prepared in this invention for extracting benzimidazole veterinary drug residues, the extraction efficiency of the TAPB-Dt magnetic material prepared in Comparative Example 1 for 17 benzimidazole veterinary drugs was compared. The results are as follows: Figure 7As shown in the figure, the results indicate that the TAPB-Dt magnetic material cannot achieve high-efficiency extraction of the 17 benzimidazole veterinary drugs in Example 1.

[0090] The extraction performance of 17 benzimidazole veterinary drugs under different pH conditions was investigated using TAPB-Dt magnetic materials. The results are as follows: For albendazole sulfone, albendazole-2-aminosulfone, and 5-hydroxythiabendazole, TAPB-Dt magnetic materials could not achieve an adsorption efficiency of over 85% within the pH range of 2.5-11. These results verify that the addition of graphene oxide plays a crucial role in improving the adsorption performance of the material for these 17 benzimidazole drugs, and also indicate that TAPB-Dt & GO magnetic materials possess unique advantages and superiority as solid-phase extraction materials for these 17 benzimidazole drugs.

[0091] 6. To highlight that the extraction performance of the extraction material is related to the sum of the total mass of Dt and the mass of 1,3,5-tris(4-aminophenyl)benzene added in steps S2 and S3, and the mass of graphene oxide, the extraction efficiency of the extraction material prepared in Comparative Example 2 for 17 benzimidazole drugs was investigated at the optimal pH of 4.0. The results are as follows: Figure 8 As shown in the results, for oxifenedazol, oxifenedazol sulfone, 2-aminofluorobendazole, and hydroxymethylbendazole, the high-GO TAPB-Dt&GO magnetic material exhibits a target analyte extraction efficiency below 60% at low concentrations (10 ng / L). These results confirm that a high proportion of graphene oxide has the opposite effect of improving the material's adsorption performance for 17 benzimidazole drugs.

[0092] Although the principles of the present invention have been described in detail above with reference to preferred embodiments, those skilled in the art should understand that the above embodiments are merely illustrative explanations of the implementation of the present invention and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Any obvious changes, such as equivalent transformations or simple substitutions, based on the technical solutions of the present invention without departing from the spirit and scope of the present invention fall within the protection scope of the present invention.

Claims

1. A method for preparing an extraction material for determining benzimidazole veterinary drug residues, characterized in that, Includes the following steps: S1: Magnetic SiO2 / Fe3O4 particles are dispersed in a mixed solution, and aminopropyltriethoxysilane is added, stirred, heated and washed to obtain APTES / SiO2 / Fe3O4 particles; S2: Disperse the APTES / SiO2 / Fe3O4 particles in a mixture of ethanol and 2,5-dihydroxyterephthalaldehyde and stir to obtain Dt / APTES / SiO2 / Fe3O4 particles; S3: The Dt / APTES / SiO2 / Fe3O4 particles were dispersed in a mixture of ethanol, 2,5-dihydroxyterephthalaldehyde and 1,3,5-tris(4-aminophenyl)benzene by ultrasonic dispersion, and then graphene oxide and acetic acid were added, stirred, washed and dried to obtain the extraction material.

2. The method for preparing the extraction material for determining benzimidazole veterinary drug residues according to claim 1, characterized in that, In step S3, the mass ratio of the Dt / APTES / SiO2 / Fe3O4 particles, 1,3,5-tris(4-aminophenyl)benzene, 2,5-dihydroxyterephthalaldehyde and graphene oxide is 5:1.4:1:0.8-1.

5.

3. The method for preparing the extraction material for determining benzimidazole veterinary drug residues according to claim 1, characterized in that, In step S1, the mass ratio of SiO2 to Fe3O4 in the magnetic SiO2 / Fe3O4 particles is 4:2-2.3; and / or, The mixed solution is a mixture of isopropanol and concentrated ammonia, wherein the volume ratio of isopropanol to concentrated ammonia is 35-40:1; and / or, The mass-to-volume ratio of the magnetic SiO2 / Fe3O4 particles to aminopropyltriethoxysilane is 2 g: 1 mL; and / or, The stirring temperature is 30-40℃, and the stirring time is 0.5-1.5h; and / or, The heating temperature is 80-90℃, and the time is 5-7 hours; and / or, The washing process uses deionized water and ethanol as the solution, and the washing is performed three times.

4. The method for preparing the extraction material for determining benzimidazole veterinary drug residues according to claim 1, characterized in that, In step S2, the mass ratio of the APTES / SiO2 / Fe3O4 particles to the 2,5-dihydroxyterephthalaldehyde is 100:17; and / or, The stirring time is 0.5-1.5 hours.

5. The method for preparing the extraction material for determining benzimidazole veterinary drug residues according to claim 1, characterized in that, In step S3, the molar ratio of graphene oxide to acetic acid is 1 g:0.0798 mol, and the molar concentration of acetic acid is 6 mol / L; and / or, The washing solution used is ethanol, and the washing is performed three times; and / or, The drying method is vacuum drying at a temperature of 45-55℃.

6. An extraction material for determining benzimidazole veterinary drug residues, characterized in that, It is prepared by the preparation method described in any one of claims 1-5.

7. The application of the extraction material according to claim 6 in the determination of benzimidazole veterinary drug residues by magnetic solid-phase extraction.

8. The application according to claim 7, characterized in that, The applications include: The sample containing benzimidazole veterinary drug residues was mixed with the extraction material described in claim 6, and then adsorbed and eluted. The eluent was then analyzed by liquid chromatography-tandem mass spectrometry.

9. The application according to claim 8, characterized in that, The benzimidazole veterinary drug is one or more of the following: oxifendazole, fenbendazole, febantel, oxifendazole sulfone, albendazole, albendazole sulfoxide, albendazole sulfone, albendazole-2-aminosulfone, thiabendazole ester, thiabendazole, 5-hydroxythiabendazole, flubendazole, 2-aminoflubendazole, mebendazole, aminomebendazole, hydroxymebendazole, or oxybendazole; and / or, The volume-to-mass ratio of the sample containing benzimidazole veterinary drug residues to the extraction material is 10-75 mL:30 mg, preferably 75 mL:30 mg; and / or, The sample containing benzimidazole veterinary drug residues has a pH value of 3.5-4.5, preferably 4.0; and / or, The adsorption time is 5 minutes.

10. The application according to claim 8, characterized in that, The eluent used for elution is a 1% (v / v) formic acid methanol solution, and the volume of the eluent is 1 mL; and / or, The elution time is 10 minutes.

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