A magnetic hierarchical porous NH2-UiO-66 material and its application in the determination of quinolone antibiotics in animal food

Abstract

This invention, belonging to the field of chemical analysis, discloses a magnetic hierarchical porous NH2-UiO-66 material and its application in the determination of quinolone antibiotics in animal foods. The invention first prepares a metal-organic framework material (hierarchical porous NH2-UiO-66 material) with a hierarchical porous structure using molecular imprinting technology. This metal-organic framework material is then imbued with magnetic properties by treating it with trivalent iron salts, sodium citrate, and sodium acetate, resulting in the magnetic hierarchical porous NH2-UiO-66 material. The magnetic graded porous NH2-UiO-66 material prepared by the present invention is used for the extraction, separation, enrichment and detection of quinolone antibiotics in animal foods, achieving a high extraction rate (95.12-97.92%), a good linear range (5-500 μg / L), a low detection limit (milk: 0.16-1.18 μg / L, pork: 0.34-2.16 μg / L) and a low limit of quantification (milk: 0.54-3.95 μg / L, pork: 1.15-3.55 μg / L), as well as a satisfactory recovery rate (milk: 70.15-109.67%, pork: 71.48-93.84%). It can be used for the highly sensitive detection of quinolone antibiotics in complex matrix foods.

Description

Technical Field

[0001] The present invention belongs to the field of chemical analysis, and in particular relates to a magnetic graded porous NH2-UiO-66 material and an application thereof in the determination of quinolone antibiotics in animal food. Background Art

[0002] Antibiotics have excellent antimicrobial activity and are widely used to treat infectious diseases in humans and animals. In recent years, due to their extensive use and irrational discharge, they have been frequently detected in food and the environment, causing varying degrees of contamination and sparking widespread public concern. Long-term exposure to or heavy use of agricultural products contaminated with antibiotics can cause allergic reactions and potential carcinogenic, teratogenic, and mutagenic effects, inducing the production and spread of resistance genes and the formation of drug-resistant bacteria, posing a serious threat to human health. Many countries and organizations have restricted the use of various antibiotics and established maximum residue limits (MRLs) in animal tissues. Accurately testing antibiotic residue levels in food is crucial for conducting food quality and safety assessments.

[0003] Currently, methods for detecting antibiotics in food, both domestically and internationally, primarily include immunoassays, spectroscopy, chromatography, and chromatography-mass spectrometry. Chromatography and chromatography-mass spectrometry have been widely used for the detection of various trace antibiotics in food due to their high accuracy, selectivity, and short analysis times. However, these methods place high demands on sample pretreatment. Especially for animal-derived foods with complex sample matrices, sample pretreatment is required to remove matrix interference before detecting residual antibiotics. Therefore, it is crucial to develop sample pretreatment methods with excellent selectivity, high enrichment multiples, and high extraction efficiency, and to combine these with detection technologies to develop an antibiotic detection system that integrates sample separation, enrichment, and detection.

[0004] Metal-organic frameworks (MOFs) are a class of inorganic-organic hybrid materials that are self-assembled through coordination bonds using metal ions as nodes and organic ligands as connectors. MOFs have the characteristics of large specific surface area, high porosity, multiple unsaturated metal active sites, and adjustable structure. Their application as extraction and adsorption materials in the field of sample pretreatment has attracted widespread attention from researchers. For example, based on the large specific surface area and multiple active sites of ZIF-8, Li Jianhong's research group at Huazhong Agricultural University developed a magnetic composite adsorption material Fe3O4@APTES-GO / ZIF-8, which achieved effective extraction and separation of triazole fungicides from water, honey, and fruit juice. In order to improve the adsorption effect of MOFs materials on target substances, researchers often use soft / hard template methods, ligand replacement methods, and regulator-induced defect formation methods to flexibly control the specific surface area and pore size of MOFs.

[0005] Therefore, combining the characteristics of metal-organic framework compounds, it is worthwhile to further study the use of MOFs materials as extraction and adsorption materials in the detection of residual antibiotics in food. Summary of the Invention

[0006] In view of the problems and shortcomings in the prior art, the purpose of the present invention is to provide a magnetic graded porous NH2-UiO-66 material and its application in the determination of quinolone antibiotics in animal food.

[0007] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0008] A magnetic hierarchical porous NH2-UiO-66 material for detecting quinolone antibiotics in animal food is prepared from the hierarchical porous NH2-UiO-66 material, trivalent iron salt, sodium citrate and sodium acetate.

[0009] The preparation method of the magnetic graded porous NH2-UiO-66 material for detecting quinolone antibiotics in animal food comprises the following steps:

[0010] (1) adding a hierarchical porous NH2-UiO-66 material, a trivalent iron salt, sodium citrate, and sodium acetate to ethylene glycol to obtain a mixed solution, and stirring the mixed solution to form a colloidal solution; the hierarchical porous NH2-UiO-66 material is prepared by molecular imprinting technology, and the template molecule in the preparation process is a fluoroquinolone antibiotic or a fluoroquinolone antibiotic structural analog; further, the template molecule is pipemidic acid;

[0011] (2) reacting the colloidal solution at 180-220° C. for 8-12 hours, and cooling to room temperature after the reaction is completed;

[0012] (3) The product is separated by a magnet, and the product is washed and dried to obtain a magnetic graded porous NH2-UiO-66 material.

[0013] Furthermore, the amount of the graded porous NH2-UiO-66 material in step (1) is 50-300 mg, and the molar ratio of the trivalent iron salt, sodium citrate and sodium acetate is (2-6): (0.5-1): (2-12).

[0014] Furthermore, the washing process in step (3) is: washing the product with ethanol and water 2 to 3 times respectively.

[0015] Furthermore, the preparation process of the hierarchical porous NH2-UiO-66 material in step (1) is as follows:

[0016] S1: Add a metal salt to a solvent and dissolve it to obtain solution A; add a template molecule to a solvent and dissolve it to obtain solution B; pour solution B into solution A, stir, add an organic ligand, and dissolve it by ultrasonication to obtain solution C;

[0017] The metal salt is zirconium tetrachloride, the template molecule is pipemidic acid, and the organic ligand is 2-aminoterephthalic acid; the molar ratio of the zirconium tetrachloride, pipemidic acid and 2-aminoterephthalic acid is (2-10): (1-3): (1-5).

[0018] S2: Transfer solution C to a reactor and react at 120-180°C for 12-48 hours. After the reaction, cool the reaction solution to room temperature, centrifuge, and remove the supernatant to obtain an intermediate product.

[0019] S3: The intermediate product was washed with a methanol solution containing 1% acetic acid to remove the template molecules, and then washed with methanol three times and dried to obtain a hierarchical porous NH2-UiO-66 material.

[0020] The present invention also provides the use of the above-mentioned magnetic graded porous NH2-UiO-66 material in detecting quinolone antibiotics in animal food.

[0021] According to the application, further, the quinolone antibiotic is one or more of ciprofloxacin, enoxacin, enrofloxacin, gatifloxacin, norfloxacin and ofloxacin.

[0022] According to the application, further, the detection process includes the following steps:

[0023] (1) Magnetic solid phase extraction enrichment process

[0024] The magnetic graded porous NH2-UiO-66 material is added to a sample solution of an animal food to be tested, vortexed, and separated by magnetic adsorption using a magnet; the separated magnetic graded porous NH2-UiO-66 material is eluted, and the eluate is collected; the eluate is dried with nitrogen, and the residue is collected, redissolved, and filtered to obtain a filtrate;

[0025] (2) Determine the content of quinolone antibiotics in the subsequent filtrate using liquid chromatography-tandem mass spectrometry.

[0026] According to the application, further, the usage ratio of the magnetic graded porous NH2-UiO-66 material to the sample solution of the animal food to be tested is (1-5) mg:5 mL.

[0027] According to the application, further, the vortex time is 2 to 30 minutes.

[0028] According to the application, further, the pH of the sample solution of the animal food to be tested is 3-9.

[0029] According to the application, further, the solvent used for elution is a methanol solution containing NaOH, and the concentration of NaOH is greater than or equal to 0.01 mol / L.

[0030] According to the application, further, the solvent for re-dissolving in step (1) is methanol.

[0031] According to the application, further, the animal food to be tested includes milk and pork.

[0032] According to the application, further, before the magnetic solid phase extraction enrichment process, a sample pretreatment operation is also included, and the sample pretreatment operation is:

[0033] A certain amount of animal food sample is weighed into a conical flask, acetonitrile is added, vortexed, and centrifuged to obtain a supernatant; the supernatant is transferred to another conical flask, n-hexane is added, vortexed, and centrifuged, the supernatant is discarded, the remaining solution is rotary evaporated, and the residue is collected; the obtained residue is dissolved in an aqueous solution with a pH of 3 to 9, passed through a microporous filter membrane, and the filtrate is collected as the sample solution of the animal food to be tested.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The present invention first prepares a metal-organic framework material (hierarchical porous NH2-UiO-66 material) having a high specific surface area and a hierarchical porous structure through molecular imprinting technology, and then imparts magnetic properties to the metal-organic framework material by adding trivalent iron salt, sodium citrate, and sodium acetate, thereby preparing a magnetic hierarchical porous NH2-UiO-66 material. Based on the pore-forming effect of the template molecule (a structural analog of a quinolone antibiotic) and the high specific surface area of ​​the metal-organic framework substrate, the magnetic hierarchical porous NH2-UiO-66 material has a high selective adsorption ability and adsorption capacity for quinolone antibiotics and is suitable for detecting the content of quinolone antibiotics in animal foods.

[0036] (2) The present invention applies the prepared magnetic graded porous NH2-UiO-66 material to solid phase extraction. Under the action of an external magnetic field, the magnetic graded porous NH2-UiO-66 material can be quickly separated from the sample solution to be tested, thereby achieving efficient extraction, separation and enrichment of quinolone antibiotics, and can perform highly sensitive detection of quinolone antibiotics in complex matrix animal foods.

[0037] (3) The present invention has the advantages of simple operation, high extraction efficiency (95.12-97.92%), wide linear range (5-500 μg / L), low detection limit (milk: 0.16-1.18 μg / L, pork: 0.34-2.16 μg / L) and quantification limit (milk: 0.54-3.95 μg / L, pork: 1.15-3.55 μg / L) and satisfactory recovery rate (milk: 70.15-109.67%, pork: 71.48-93.84%), and can be used as a reliable method for detecting quinolone antibiotic residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a working curve diagram of the magnetic graded porous NH2-UiO-66 material of the present invention for detecting quinolone antibiotics in spiked milk samples;

[0039] Figure 2 This is a working curve diagram of the magnetic graded porous NH2-UiO-66 material of the present invention for detecting quinolone antibiotics in spiked pork samples. DETAILED DESCRIPTION

[0040] The following examples are intended only to further illustrate the present invention. It should be noted that all technical and scientific terms used herein have the same meanings as in the art to which the present invention pertains, unless otherwise specified. Experimental methods in the following examples, where specific conditions are not specified, were based on conventional techniques in the art or the conditions recommended by the manufacturer. Reagents and instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0041] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0042] (1) Application of magnetic graded porous NH2-UiO-66 material in magnetic solid phase extraction

[0043] In order to study the application of magnetic graded porous NH2-UiO-66 material in magnetic solid phase extraction, the present invention will explore the effects of extraction conditions such as adsorbent dosage, sample solution pH, extraction time and eluent type on the extraction effect of quinolone antibiotics by magnetic graded porous NH2-UiO-66 material, that is, carry out experiments in Examples 1 to 4, the specific contents of which are as follows:

[0044] Example 1: Optimization experiment of the amount of magnetic graded porous NH2-UiO-66 adsorbent

[0045] The present invention uses a standard solution of a mixture of six quinolone antibiotics as a sample solution to conduct a magnetic solid phase extraction experiment to study the effect of the dosage of the magnetic graded porous NH2-UiO-66 adsorbent on the extraction effect of the six quinolone antibiotics. The specific experimental contents are shown in Examples 1-1 to 1-5.

[0046] Example 1-1:

[0047] (1) Preparation of hierarchical porous NH2-UiO-66 materials

[0048] 240 mg of zirconium tetrachloride was dissolved in 30 mL of N,N-dimethylformamide (DMF) to obtain solution A; 234 mg of pipemidic acid was dissolved in 30 mL of DMF to obtain solution B; solution B was poured into solution A and stirred for 1 hour, and then 94 mg of 2-aminoterephthalic acid was added thereto, ultrasonically dissolved and reacted for 30 minutes to obtain solution C; solution C was transferred to a reactor and hydrothermally reacted at 120°C for 48 hours; after the reaction, the reaction solution was cooled to room temperature, centrifuged, and the supernatant was discarded to obtain an intermediate; the intermediate was washed with a methanol solution containing 1% acetic acid until no pipemidic acid was detected in the washing solution, and then washed three times with methanol. After drying, the product obtained a hierarchical porous NH2-UiO-66 material.

[0049] (2) Preparation of magnetic graded porous NH2-UiO-66 adsorbent

[0050] 300 mg of graded porous NH2-UiO-66 material, 3.9 g of FeCl3.6H2O and 1.2 g of sodium citrate dihydrate were added to 60 mL of ethylene glycol and ultrasonically dispersed for 10 minutes. Then, 7.2 g of sodium acetate was added and stirred continuously until a colloidal solution of uniform viscosity was generated. The colloidal solution was transferred to a reactor and subjected to a hydrothermal reaction at 200°C for 12 hours. After the reaction, the reaction solution was cooled to room temperature, the product was separated from the reaction solution by a magnet, and the unreacted materials were washed away with ethanol and water. The product was then dried in a vacuum drying oven at 60°C for 24 hours to obtain a magnetic graded porous NH2-UiO-66 adsorbent.

[0051] (3) Magnetic solid phase extraction process

[0052] Six quinolone antibiotic standards were accurately weighed and ultrasonically dissolved in pH 7 water to prepare a mixed quinolone antibiotic standard solution with a concentration of 0.5 mg / L. 1 mg of magnetic graded porous NH2-UiO-66 adsorbent was weighed and added to 5 mL of the mixed quinolone antibiotic standard solution. The solution was vortexed at room temperature for 30 min, and the magnetic adsorbent was separated and collected using an external magnetic force. 3 mL of a methanol solution containing 0.1 mol / L NaOH was added to the magnetic adsorbent, and ultrasonic elution was performed for 10 min. The eluate was collected. The eluate was dried with nitrogen at 50°C, and the residue was collected. The residue was re-dissolved in 500 μL of methanol, filtered through a 0.22 μm microporous filter membrane, and the filtrate was collected for subsequent analysis.

[0053] (4) Analysis and testing

[0054] After the extraction, the concentrations of the six quinolone antibiotics in the filtrate obtained in step (3) were detected using a liquid chromatography-tandem mass spectrometer. The liquid chromatography conditions were as follows: a Dikma Endeavorsil C18 column with a size of 100 mm × 2.1 mm and a particle size of 1.8 μm; a mobile phase consisting of water and methanol in a gradient mobile phase ratio as shown in Table 1; a flow rate of 0.25 mL / min; an injection volume of 1 μL; and a column oven temperature of 35°C. The mass spectrometry conditions were as follows: sheath gas temperature of 350°C, sheath gas flow rate of 12 L / min, nebulizer gas pressure of 35 psi, capillary voltage of 4000 V, and a multiple reaction monitoring scan mode.

[0055] Table 1 Gradient ratio of mobile phase

[0056] Time (min) water Methanol 0 90 10 1 90 10 10 55 45 13 0 100

[0057] The recoveries of the six quinolone antibiotics were calculated and the results are shown in Table 2.

[0058] Example 1-2

[0059] The content of Example 1-2 is basically the same as that of Example 1-1, except that the amount of the magnetic graded porous NH2-UiO-66 adsorbent used in step (3) is 2 mg.

[0060] Examples 1-3

[0061] The content of Example 1-3 is basically the same as that of Example 1-1, except that the amount of the magnetic graded porous NH2-UiO-66 adsorbent used in step (3) is 3 mg.

[0062] Examples 1-4

[0063] The content of Example 1-4 is basically the same as that of Example 1-1, except that the amount of the magnetic graded porous NH2-UiO-66 adsorbent used in step (3) is 4 mg.

[0064] Examples 1-5

[0065] The content of Example 1-5 is basically the same as that of Example 1-1, except that the amount of the magnetic graded porous NH2-UiO-66 adsorbent used in step (3) is 5 mg.

[0066] The recovery results of the six quinolone antibiotics in the subsequent filtrates of magnetic solid phase extraction of Examples 1-1 to 1-5 are shown in Table 2.

[0067] Table 2 Recovery rates of 6 quinolone antibiotics in Example 1-1 to Example 1-5

[0068]

[0069] As shown in Table 2, along with the consumption of adsorbent increased to 5mg by 1mg, the recovery of 6 kinds of quinolone antibiotics is significantly improved.When the adsorbent consumption was 4mg, the recovery of ciprofloxacin, enoxacin, enrofloxacin, gatifloxacin, norfloxacin and ofloxacin was respectively 97.51%, 97.37%, 97.30%, 95.12%, 97.92% and 95.26%, all more than 95%.When the adsorbent consumption continued to increase to 5mg from 4mg, the recovery of 6 kinds of quinolone antibiotics tended towards stability.Therefore, the present invention is preferably 4mg in the consumption of magnetic graded porous NH2-UiO-66 adsorbent in magnetic solid phase extraction process.

[0070] Example 2: Optimization experiment of sample solution pH in magnetic solid phase extraction

[0071] The present invention uses a standard solution of a mixture of six quinolone antibiotics as a sample solution to conduct a magnetic solid phase extraction experiment to study the effect of the sample solution pH on the extraction effect of the six quinolone antibiotics. The specific experimental contents are shown in Examples 2-1 to 2-4:

[0072] Example 2-1:

[0073] (1) Preparation of quinolone antibiotic mixed standard solution

[0074] Six quinolone antibiotic standards were accurately weighed and ultrasonically dissolved in water at pH 7 to prepare quinolone antibiotic stock solutions with a concentration of 5 mg / L. The stock solutions were then diluted with water at pH 7 and a certain amount of 0.01 mol / L HCl solution was added to obtain a mixed standard solution with a pH of 3 and a concentration of 0.5 mg / L for each of the six quinolone antibiotics. This mixed standard solution was used as the sample solution for the subsequent magnetic solid phase extraction process.

[0075] (2) Magnetic solid phase extraction process

[0076] 4 mg of the magnetic graded porous NH2-UiO-66 material prepared in Example 1 was added to 5 mL of the mixed standard solution of the above-mentioned quinolone antibiotics, vortexed at room temperature for 30 minutes, and the magnetic adsorbent was separated and collected by external magnetic force; 3 mL of a methanol solution containing 0.1 mol / L NaOH was added to the magnetic adsorbent, ultrasonically eluted for 10 minutes, and the eluate was collected; the eluate was blown dry with nitrogen at 50°C, and the residue was collected; the obtained residue was redissolved with 500 μL of methanol, filtered through a 0.22 μm microporous filter membrane, and the filtrate was taken for subsequent analysis.

[0077] (3) Analysis and testing

[0078] After the extraction was completed, the concentrations of the six quinolone antibiotics in the subsequent filtrate obtained in step (2) were detected by liquid chromatography tandem mass spectrometry, and the recovery rates of the six quinolone antibiotics were calculated respectively. The results are shown in Table 3.

[0079] Example 2-2

[0080] The content of Example 2-2 is basically the same as that of Example 2-1, except that the pH value of the mixed standard solution of quinolone antibiotics in step (1) is 5.

[0081] Example 2-3

[0082] The content of Example 2-3 is basically the same as that of Example 2-1, except that a certain amount of 0.01 mol / L NaOH solution is added in step (1) so that the pH value of the mixed standard solution of quinolone antibiotics is 9.

[0083] Examples 2-4

[0084] The content of Example 2-4 is basically the same as that of Example 2-1, except that a certain amount of 0.01 mol / L NaOH solution is added in step (1) so that the pH value of the mixed standard solution of quinolone antibiotics is 11.

[0085] The recovery results of the six quinolone antibiotics in the subsequent filtrates of magnetic solid phase extraction of Examples 2-1 to 2-4 are shown in Table 3.

[0086] Table 3 Recovery rates of 6 quinolone antibiotics in Example 2-1 to Example 2-4

[0087]

[0088] As shown in Table 3, when the pH value of the sample solution increases from 3 to 9, the recovery rates of the six quinolone antibiotics gradually increase to a stable state, and at pH = 7 and pH = 9, the recovery rates of the six quinolone antibiotics do not change much, all being above 95%. However, when the pH value of the sample solution is further increased to 11, the recovery rates of the six quinolone antibiotics drop sharply, all being less than 5%. This may be because the magnetic adsorbent and the six quinolone antibiotics all exist as negatively charged forms in a highly alkaline solution, and the two repel each other, resulting in a poor extraction effect of the quinolone antibiotics. Therefore, the present invention selects pH = 7 as the preferred sample solution pH in the magnetic solid phase extraction process.

[0089] Example 3: Optimization experiment of extraction time in magnetic solid phase extraction

[0090] The present invention uses a standard solution of a mixture of six quinolone antibiotics as a sample solution to conduct a magnetic solid phase extraction experiment to study the effect of extraction time on the extraction effect of the six quinolone antibiotics. The specific experimental content is shown in Example 3-1 to Example 3-4.

[0091] Example 3-1:

[0092] (1) Preparation of quinolone antibiotic mixed standard solution

[0093] Six quinolone antibiotic standards were accurately weighed and dissolved in water by ultrasonication to prepare a mixed standard solution with a pH of 7 and a concentration of 0.5 mg / L for each of the six quinolone antibiotics.

[0094] (2) Magnetic solid phase extraction process

[0095] 4 mg of the magnetic graded porous NH2-UiO-66 material prepared in Example 1 was added to 5 mL of the mixed standard solution of the above-mentioned quinolone antibiotics, vortexed at room temperature for 2 minutes, and the magnetic adsorbent was separated and collected by external magnetic force; 3 mL of a methanol solution containing 0.1 mol / L NaOH was added to the magnetic adsorbent, ultrasonically eluted for 10 minutes, and the eluate was collected; the eluate was blown dry with nitrogen at 50°C, and the residue was collected; the obtained residue was redissolved with 500 μL of methanol, filtered through a 0.22 μm microporous filter membrane, and the filtrate was taken for subsequent analysis.

[0096] (3) Analysis and testing

[0097] After the extraction, the concentrations of the six quinolone antibiotics in the subsequent filtrate obtained in step (2) were detected by liquid chromatography tandem mass spectrometry, and the recovery rates of the six quinolone antibiotics were calculated respectively. The results are shown in Table 4.

[0098] Example 3-2

[0099] The content of Example 3-2 is basically the same as that of Example 3-1, except that the extraction time in step (2) is 5 minutes.

[0100] Example 3-3

[0101] The content of Example 3-3 is basically the same as that of Example 3-1, except that the extraction time in step (2) is 10 minutes.

[0102] Examples 3-4

[0103] The content of Example 3-4 is basically the same as that of Example 3-1, except that the extraction time in step (2) is 20 minutes.

[0104] The recovery results of the six quinolone antibiotics in Examples 3-1 to 3-4 are shown in Table 4.

[0105] Table 4 Recovery rates of 6 quinolone antibiotics in Examples 3-1 to 3-4

[0106]

[0107] As shown in Table 4, when the extraction time was 30 min, the recoveries of the six quinolone antibiotics reached their peak values, all above 95%. Therefore, the extraction time was determined to be 30 min.

[0108] Example 4: Optimization experiment of elution solution composition in magnetic solid phase extraction

[0109] The present invention uses a standard solution of a mixture of six quinolone antibiotics as a sample solution to conduct a magnetic solid phase extraction experiment to study the effect of the elution solution composition on the extraction effect of the six quinolone antibiotics. The specific experimental content is shown in Examples 4-1 to 4-3.

[0110] Example 4-1

[0111] (1) Preparation of quinolone antibiotic mixed standard solution

[0112] Accurately weigh 6 quinolone antibiotic standards, dissolve them in water with a pH of 7 by ultrasonication, and prepare a mixed standard solution of quinolone antibiotics with a concentration of 0.5 mg / L.

[0113] (2) Magnetic solid phase extraction process

[0114] 4 mg of the magnetic graded porous NH2-UiO-66 material prepared in Example 1 was added to 5 mL of the mixed standard solution of the above-mentioned quinolone antibiotics, vortexed at room temperature for 30 minutes, and the mixed solution was separated by external magnetic force to collect the magnetic adsorbent; 3 mL of methanol solution was added to the magnetic adsorbent, ultrasonically eluted for 10 minutes, and the eluate was collected; the eluate was blown dry with nitrogen at 50°C, and the residue was collected; the obtained residue was redissolved with 500 μL of methanol, filtered through a 0.22 μm microporous filter membrane, and the filtrate was taken for subsequent analysis.

[0115] (3) Analysis and testing

[0116] After the extraction was completed, the concentrations of the six quinolone antibiotics in the subsequent filtrate obtained in step (2) were detected by liquid chromatography-tandem mass spectrometry, and the recovery rates of the six quinolone antibiotics were calculated respectively. The results are shown in Table 5.

[0117] Example 4-2

[0118] The content of Example 4-2 is basically the same as that of Example 4-1, except that in step (2), a methanol solution containing 0.01 mol / L NaOH is added to the magnetic adsorbent for elution.

[0119] Example 4-3

[0120] The content of Example 4-3 is basically the same as that of Example 4-1, except that in step (2), a methanol solution containing 0.05 mol / L NaOH is added to the magnetic adsorbent for elution.

[0121] The recovery results of the six quinolone antibiotics in Examples 4-1 to 4-3 are shown in Table 5.

[0122] Table 5 Recovery of 6 quinolone antibiotics in Examples 4-1 to 4-3

[0123]

[0124] As shown in Table 5, when eluting with pure methanol solution, the recovery rates of the six quinolone antibiotics were all lower. When eluting with methanol solution containing different concentrations of NaOH, the recovery effects of the six quinolone antibiotics were significantly improved. This is consistent with the result that the stronger alkaline solution was not conducive to the adsorption extraction of quinolone antibiotics in the sample solution pH optimization experiment (Example 2). As the concentration of NaOH in the methanol solution increased from 0.01mol / L to 0.1mol / L, the recovery rates of the six quinolone antibiotics also gradually increased. When the concentration of NaOH in the methanol eluent was 0.1mol / L, the recovery rates of the six quinolone antibiotics were all above 95%. Therefore, the methanol solution containing 0.1mol / L of NaOH was determined to be the elution solvent in the magnetic solid phase extraction experiment.

[0125] (2) Methodological validation

[0126] In order to demonstrate the application of the magnetic graded porous NH2-UiO-66 material provided by the present invention in the detection of quinolone antibiotics in animal food, based on the magnetic solid phase extraction conditions optimized in Examples 1 to 4, a series of blank matrix spiked working solutions with different quinolone antibiotic mixed standard concentrations were prepared using milk and pork blank matrix samples as actual samples for methodological verification. The specific experimental contents are shown in Examples 5 and 6.

[0127] Example 5:

[0128] The application of magnetic graded porous NH2-UiO-66 material in the determination of quinolone antibiotics in milk includes the following steps:

[0129] (1) Pretreatment of milk blank matrix sample solution

[0130] 30 mL of milk was added to a 250 mL conical flask, 120 mL of acetonitrile was added thereto, the mixture was vortexed for 5 minutes, and the mixture was centrifuged at 10,000 rpm for 10 minutes. The supernatant was transferred to another 250 mL conical flask, 75 mL of n-hexane was added thereto, the mixture was vortexed for 5 minutes, and the mixture was centrifuged again at 5,000 rpm for 5 minutes. The supernatant was discarded, and the remaining solution was evaporated at 50°C to collect the residue. The residue was dissolved in 30 mL of a pH 7 aqueous solution, filtered through a 0.22 μm microporous filter membrane, and the filtrate was taken for subsequent analysis.

[0131] (2) Preparation of milk blank matrix spiked working solution

[0132] The standard solutions of six quinolone antibiotics are weighed and added to the milk blank matrix sample solution pretreated in step (1) to prepare milk blank matrix spiked stock solutions with the six quinolone antibiotics having a concentration of 500 μg / L. Then, a stepwise dilution method is adopted, using the milk blank matrix sample solution obtained in step (1) as a solvent, to dilute the milk blank matrix spiked stock solutions by different multiples to obtain a series of milk blank matrix spiked working solutions with the six quinolone antibiotics having concentrations of 5, 10, 20, 50, 100, 200 and 500 μg / L, respectively.

[0133] (3) Magnetic solid phase extraction and analytical detection

[0134] 4 mg of the magnetic graded porous NH2-UiO-66 adsorbent prepared by the present invention was weighed and added to 5 mL of the above-mentioned milk blank matrix spiked working solution with different spiked concentrations, vortexed at room temperature for 30 minutes, and the magnetic adsorbent was separated and collected by external magnetic force; 3 mL of methanol solution containing 0.1 mol / L NaOH was added to the magnetic adsorbent, ultrasonically eluted for 10 minutes, and the eluate was collected; the eluate was blown dry with nitrogen at 50°C, and the residue was collected; the obtained residue was redissolved with 500 μL of methanol, filtered through a 0.22 μm microporous filter membrane, and the filtrate was collected; the signal intensity of the six quinolone antibiotics in the filtrate was determined by liquid chromatography tandem mass spectrometry, and the working curves of the six quinolone antibiotics were drawn, as shown in FIG. Figure 1 The relevant performance parameters of the detection method, including linear range, linear correlation coefficient, detection limit and quantification limit, are shown in Table 6.

[0135] Table 6 Related parameters of the detection method for the mixed standard solution of six quinolone antibiotics in milk

[0136]

[0137] The experimental data in Table 6 show that the six quinolone antibiotics in milk samples have a good linear relationship in the concentration range of 5-500 μg / L, R 2 The detection limit of quinolone antibiotics using the method proposed in the present invention, calculated at a signal-to-noise ratio (S / N) of 3, was 0.16 to 1.18 μg / L. The quantification limit of quinolone antibiotics using the method proposed in the present invention, calculated at a signal-to-noise ratio (S / N) of 10, was 0.54 to 3.95 μg / L. This indicates that the detection method proposed in the present invention has a wide linear range, low detection and quantification limits, and is suitable for the highly sensitive detection of quinolone antibiotics in complex milk matrix samples.

[0138] Example 6:

[0139] The application of magnetic graded porous NH2-UiO-66 material in the determination of quinolone antibiotics in pork includes the following steps:

[0140] (1) Pretreatment of pork blank matrix sample solution

[0141] 30 g of pork was added to a 250 mL conical flask, 30 mL of water and 120 mL of acetonitrile were added, the mixture was vortexed for 5 min, and the mixture was centrifuged at 10,000 rpm for 10 min. The supernatant was collected. The supernatant was transferred to another 250 mL conical flask, 75 mL of n-hexane was added, the mixture was vortexed for 5 min, and the mixture was centrifuged again at 5,000 rpm for 5 min. The supernatant was discarded, and the remaining solution was evaporated at 50°C to collect the residue. The residue was dissolved in 30 mL of a pH 7 aqueous solution, filtered through a 0.22 μm microporous filter membrane, and the filtrate was collected for subsequent analysis.

[0142] (2) Preparation of pork blank matrix spiked working solution

[0143] The standard solutions of six quinolone antibiotics are weighed and added to the pork blank matrix sample solution pretreated in step (1) to prepare pork blank matrix spiked stock solutions with the six quinolone antibiotics having a concentration of 500 μg / L. Then, a stepwise dilution method is adopted, using the pork blank matrix sample solution obtained in step (1) as a solvent, to dilute the pork blank matrix spiked stock solutions by different multiples to obtain a series of pork blank matrix spiked working solutions with the six quinolone antibiotics having concentrations of 5, 10, 20, 50, 100, 200 and 500 μg / L, respectively.

[0144] (3) Magnetic solid phase extraction and analytical detection

[0145] 4 mg of the magnetic graded porous NH2-UiO-66 adsorbent prepared by the present invention was weighed and added to 5 mL of the above-mentioned pork blank matrix spiked working solution with different spiked concentrations, vortexed at room temperature for 30 minutes, and the magnetic adsorbent was separated and collected by external magnetic force; 3 mL of methanol solution containing 0.1 mol / L NaOH was added to the magnetic adsorbent, ultrasonically eluted for 10 minutes, and the eluate was collected; the eluate was blown dry with nitrogen at 50°C, and the residue was collected; the obtained residue was re-dissolved with 500 μL of methanol, filtered through a 0.22 μm microporous filter membrane, and a filtrate was taken; the signal intensity of the six quinolone antibiotics in the filtrate was determined by liquid chromatography-tandem mass spectrometry, and the working curves of the six quinolone antibiotics in the pork blank matrix spiked working solution were drawn, as shown in FIG. Figure 2 The relevant performance parameters of the detection method, including linear range, linear correlation coefficient, detection limit and quantification limit, are shown in Table 7.

[0146] Table 7 Related parameters of the detection method for the mixed standard solution of six quinolone antibiotics in pork

[0147]

[0148] The experimental data in Table 7 show that the six quinolone antibiotics in pork samples have a good linear relationship in the concentration range of 5-500 μg / L, R 2 The detection limit of quinolone antibiotics using the method proposed in the present invention, calculated at a signal-to-noise ratio (S / N) of 3, was 0.34 to 2.16 μg / L. The limit of quantification of quinolone antibiotics using the method proposed in the present invention, calculated at a signal-to-noise ratio (S / N) of 10, was 1.15 to 3.55 μg / L. This indicates that the detection method proposed in the present invention has a wide linear range, low limits of detection and quantification, and is suitable for the highly sensitive detection of quinolone antibiotics in complex pork matrix samples.

[0149] (III) Detection of six quinolone antibiotics in animal foods

[0150] In order to verify the applicability of the method proposed in the present invention, the present invention selected three different brands of milk samples and two pork samples for analysis. Based on the working curves of the six quinolone antibiotics in the milk and pork blank matrix spiked working solutions obtained in Example 5 and Example 6, the concentrations of quinolone antibiotics in the actual milk and pork samples were determined. In addition, three different brands of milk actual samples and two different sources of pork actual samples were added with low (20 μg / L), medium (100 μg / L), and high (200 μg / L) concentrations of quinolone antibiotic mixed standard solutions to detect the spiked recovery of the six quinolone antibiotics. The specific experimental content is shown in Examples 7 and 8.

[0151] Example 7:

[0152] The application of magnetic graded porous NH2-UiO-66 material in the determination of quinolone antibiotics in milk samples of different brands includes the following steps:

[0153] (1) Pretreatment of actual milk sample solution

[0154] 30 mL of three different brands of milk were measured and added to three 250 mL conical flasks respectively. 120 mL of acetonitrile solution was added to each conical flask, vortexed for 5 minutes, and centrifuged at 10,000 rpm for 10 minutes. The supernatant was removed and transferred to two other 250 mL conical flasks respectively. 75 mL of n-hexane was added thereto, vortexed for 5 minutes, and centrifuged again at 5,000 rpm for 5 minutes. The supernatant was discarded, and the remaining solution was evaporated at 50°C to collect the residue; the obtained residue was dissolved with 30 mL of pH = 7 aqueous solution, passed through a 0.22 μm microporous filter membrane, and the filtrate was collected for subsequent analysis.

[0155] (2) Preparation of milk spiked sample solution

[0156] Six quinolone antibiotic standards were weighed and added to actual milk sample solutions of three different brands pretreated in step (1), to prepare milk spiked sample solutions with concentrations of 6 quinolone antibiotics of 20, 100 and 200 μg / L, respectively.

[0157] (3) Magnetic solid phase extraction and analytical detection

[0158] 4 mg of the magnetic graded porous NH2-UiO-66 adsorbent prepared by the present invention was weighed and added to 5 mL of actual milk sample solutions (unspiked) and milk spiked sample solutions of different brands, and vortexed at room temperature for 30 min. The magnetic adsorbent was separated and collected by external magnetic force; 3 mL of methanol solution containing 0.1 mol / L NaOH was added to the magnetic adsorbent, ultrasonic elution was performed for 10 min, and the eluate was collected; the eluate was blown dry with nitrogen at 50°C, and the residue was collected; the obtained residue was redissolved with 500 μL of methanol, filtered through a 0.22 μm microporous filter membrane, and the filtrate was collected; the concentrations of the six quinolone antibiotics in the filtrate were determined by liquid chromatography-tandem mass spectrometry, and their spiked recoveries were calculated respectively. The results are shown in Table 8.

[0159] Table 8 Detection of 6 quinolone antibiotics in milk samples

[0160]

[0161] As can be seen from the data in Table 8, no quinolone antibiotics were detected in the actual sample solutions of the three brands of milk; six quinolone antibiotics at three different concentrations, low, medium, and high, were added to the actual milk samples of the three brands and magnetic solid phase extraction experiments were performed. The results showed that the recoveries of the six quinolone antibiotics in these milk spiked samples at spiked concentrations of 20 μg / L, 100 μg / L, and 200 μg / L were between 70.15% and 109.67%, which met the analytical detection requirements and can achieve highly sensitive detection of quinolone antibiotics in milk samples.

[0162] Example 8:

[0163] The application of magnetic graded porous NH2-UiO-66 material in the determination of quinolone antibiotics in pork samples from different sources includes the following steps:

[0164] (1) Pretreatment of actual pork sample solution

[0165] 30 g of pork samples from two different sources were weighed separately and added to two 250 mL conical flasks. 30 mL of water and 120 mL of acetonitrile were added to each conical flask, vortexed for 5 minutes, and centrifuged at 10,000 rpm for 10 minutes. The supernatant was taken out and transferred to two other 250 mL conical flasks. 75 mL of n-hexane was added thereto, vortexed for 5 minutes, and centrifuged again at 5,000 rpm for 5 minutes. The supernatant was discarded, and the remaining solution was evaporated at 50°C to collect the residue; it was dissolved with 30 mL of pH = 7 aqueous solution, passed through a 0.22 μm microporous filter membrane, and the filtrate was collected for subsequent analysis.

[0166] (2) Preparation of pork spiked sample solution

[0167] Six quinolone antibiotic standards were weighed and added to the actual pork sample solutions from two different sources after pretreatment in step (1), to prepare pork spiked sample solutions with the six quinolone antibiotic concentrations of 20, 100, and 200 μg / L, respectively.

[0168] (3) Magnetic solid phase extraction and analytical detection

[0169] 4 mg of the magnetic graded porous NH2-UiO-66 adsorbent prepared by the present invention was weighed and added to 5 mL of actual pork sample solutions (unspiked) and pork spiked sample solutions from different sources, and the samples were vortexed at room temperature for 30 min. The magnetic adsorbent was separated and collected using an external magnetic force; 3 mL of a methanol solution containing 0.1 mol / L NaOH was added to the magnetic adsorbent, and ultrasonic elution was performed for 10 min, and the eluate was collected; the eluate was blown dry with nitrogen at 50°C, and the residue was collected; the obtained residue was re-dissolved with 500 μL of methanol, filtered through a 0.22 μm microporous filter membrane, and the filtrate was collected; the concentrations of the six quinolone antibiotics in the filtrate were determined by liquid chromatography-tandem mass spectrometry, and their spiked recoveries were calculated respectively. The results are shown in Table 9.

[0170] Table 9 Detection of 6 quinolone antibiotics in pork samples

[0171]

[0172] As shown in Table 9, no quinolone antibiotics were detected in the actual pork sample solutions from the two different sources. Six quinolone antibiotics at low, medium, and high concentrations were added to the actual pork samples from the two different sources, and magnetic solid-phase extraction experiments were performed. The results showed that the recoveries of the six quinolone antibiotics in these pork spiked samples at spiked concentrations of 20 μg / L, 100 μg / L, and 200 μg / L were between 71.48% and 93.84%, meeting the analytical detection requirements and achieving highly sensitive detection of quinolone antibiotics in pork samples.

[0173] In summary, the present invention effectively overcomes the deficiencies in the prior art and has high industrial application value.

[0174] The above embodiments are specific implementation methods of the present invention, but the implementation methods of the present invention are not limited to the above embodiments. Any other combination, change, modification, substitution, and simplification that does not exceed the design concept of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A magnetic graded porous NH2-UiO-66 material for detecting quinolone antibiotics in animal food, characterized in that: The hierarchical porous NH2-UiO-66 material is prepared by molecular imprinting technology, and the template molecule is pipemidic acid. The amount of the hierarchical porous NH2-UiO-66 material is 50-300 mg, and the molar ratio of the trivalent iron salt, sodium citrate and sodium acetate is (2-6): (0.5-1): (2-12).

2. The method for preparing the magnetic graded porous NH2-UiO-66 material according to claim 1, characterized in that: The following steps are involved: (1) adding a graded porous NH2-UiO-66 material, a trivalent iron salt, sodium citrate and sodium acetate to ethylene glycol to obtain a mixed solution, and stirring the mixed solution to form a colloidal solution; the amount of the graded porous NH2-UiO-66 material is 50 to 300 mg, and the molar ratio of the trivalent iron salt, sodium citrate and sodium acetate is (2 to 6): (0.5 to 1): (2 to 12); (2) reacting the colloidal solution at 180-220°C for 8-12 hours, and cooling to room temperature after the reaction is completed; (3) The product is separated by a magnet, and the product is washed and dried to obtain a magnetic graded porous NH2-UiO-66 material.

3. Use of the magnetic graded porous NH2-UiO-66 material according to claim 1 in detecting quinolone antibiotics in animal food.

4. The use according to claim 3, characterized in that The quinolone antibiotic is one or more of ciprofloxacin, enoxacin, enrofloxacin, gatifloxacin, norfloxacin and ofloxacin; The detection process includes the following steps: (1) Magnetic solid phase extraction enrichment process: adding the magnetic graded porous NH2-UiO-66 material to the sample solution of the animal food to be tested, vortexing, and using a magnet to separate the magnetic graded porous NH2-UiO-66 material; The separated magnetic graded porous NH2-UiO-66 material was eluted and the eluate was collected; the eluate was blown dry with nitrogen, and the residue was collected and redissolved and filtered to obtain a filtrate; (2) determining the content of quinolone antibiotics in the subsequent filtrate by liquid chromatography-tandem mass spectrometry; The dosage ratio of the magnetic graded porous NH2-UiO-66 material to the sample solution of the animal food to be tested is (1-5) mg: 5 mL; The pH of the sample solution of the animal food to be tested is 5 to 7, and the vortex time is 5 to 30 minutes; The elution solvent is a methanol solution containing NaOH, and the concentration of NaOH is 0.05-0.1 mol / L.

5. The use according to claim 4, characterized in that The animal foods to be tested include milk and pork.

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