A urea-based functionalized ternary magnetic composite material, a synthesis method, a kit and applications thereof

Abstract

The application discloses a urea-based functionalized ternary magnetic composite material, a synthesis method, a kit and application thereof, and the composite material is used for extracting and purifying propanil urea metabolites in urine and comprises a magnetic Fe3O4 core, a nitrogen-doped mesoporous carbon layer which is coated on the surface of the magnetic Fe3O4 core, a magnesium-aluminum hydrotalcite layer which is coated on the surface of the nitrogen-doped mesoporous carbon layer, and a urea-based functionalized oligomer layer which is covalently grafted on the surface of the magnesium-aluminum hydrotalcite layer. The composite material has a four-layer core-shell structure, the nitrogen-doped mesoporous carbon layer effectively prevents the magnetic particles from agglomerating and enhances the chemical stability, the magnesium-aluminum hydrotalcite layer provides high-density basic sites for loading urea-based functional groups, the urea-based functionalized oligomer on the outermost layer can specifically recognize the propanil urea metabolites in the urine through double hydrogen bond action, the adsorption capacity and selectivity are significantly improved, and meanwhile, the overall structure has excellent magnetic responsiveness, so that the composite material is convenient for rapid separation and recovery.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a urea-functionalized ternary magnetic composite material, its synthesis method, reagent kit, and applications. Background Technology

[0002] Imazalil, a broad-spectrum imidazole systemic fungicide, is widely used in agricultural production in my country, commonly for post-harvest preservation of fruits and vegetables such as citrus, bananas, and vegetables, as well as for the control of fungal diseases in field crops. Imazalil can enter the human body through various routes, including dietary intake, environmental respiration, and skin contact. It is metabolized by the hepatic cytochrome P450 enzyme system and primarily excreted in urine as two free urea primary metabolites: imazalil-deaminoimidazole (BTS 44595) and imazalil-deimidazole formamide (BTS 44596). Therefore, the concentration of these two urea metabolites in urine directly reflects the recent actual exposure level of the human body to imazalil and serves as a specific biomarker.

[0003] Currently, research on the detection of imazalil and its metabolites mainly focuses on matrices such as agricultural products, environmental water bodies, and soil, with relevant industry standards (such as SN / T5444-2022) primarily employing the QuEChERS method. However, research on the detection of human urine, a complex biological matrix, is relatively scarce, and existing technical solutions suffer from the following significant shortcomings: First, although traditional solid phase extraction (SPE) methods (such as those using HLB columns) can achieve a certain purification effect, they are expensive in terms of consumables, have complicated operation steps, require a large amount of organic solvent consumption, are difficult to process in batches, and have low pretreatment efficiency, making them unsuitable for large-scale population sample screening.

[0004] Second, most existing magnetic solid-phase extraction materials are Fe3O4@SiO2 and Fe3O4@C. 18 Such materials lack specific recognition sites and have poor selectivity for urea metabolites in complex urine matrices (such as high concentrations of salt, urea, uric acid, etc.), resulting in severe matrix effects and making it difficult to accurately quantify trace targets.

[0005] Third, existing detection methods use 2,4,6-trichlorophenol, another metabolite of imazalil, as the main analyte, requiring the use of β-glucuronidase. This results in a long pretreatment cycle, complex steps, easy degradation of urea metabolites, and poor stability. Summary of the Invention

[0006] The technical problem to be solved by the present invention is that the existing technology lacks a specific detection method for urea metabolites in urine, and the use of general methods has problems such as poor purification effect, low quantitative accuracy, and difficulty in detecting low concentrations of target substances.

[0007] To address the aforementioned technical problems, a first aspect of the present invention provides a urea-functionalized ternary magnetic composite material for extracting and purifying prochloraz urea metabolites from urine, the composite material comprising: Magnetic Fe3O4 core; A nitrogen-doped mesoporous carbon layer is coated on the surface of the magnetic Fe3O4 core; A magnesium aluminum hydrotalcite layer is coated on the surface of the nitrogen-doped mesoporous carbon layer; A urea-functionalized oligomer layer is covalently grafted onto the surface of the magnesium aluminum hydrotalcite layer.

[0008] The ternary magnetic composite material of this invention has a four-layer core-shell structure, with a hierarchical structure consisting of a magnetic core, a mesoporous carbon transition layer, a hydrotalcite functional layer, and a urea-based recognition layer. This solves the problems of structural instability and poor selectivity in traditional Fe3O4@SiO2 materials. The nitrogen-doped mesoporous carbon layer effectively prevents magnetic particle aggregation and enhances chemical stability. The magnesium-aluminum hydrotalcite layer provides high-density basic sites for loading urea-based functional groups. The outermost urea-functionalized oligomers can specifically recognize imazalil urea metabolites in urine through double hydrogen bonding, significantly improving adsorption capacity and selectivity. At the same time, the overall structure maintains excellent magnetic responsiveness, facilitating rapid separation and recovery.

[0009] Furthermore, the urea-functionalized oligomer layer is formed by a urea-silane coupling agent, which is prepared by reacting aminopropyltriethoxysilane with 2,4,6-trichlorophenylethyl isocyanate. This achieves directional, high-density covalent grafting of urea functional groups on the surface of the magnesium-aluminum hydrotalcite layer. This structure not only provides urea groups as hydrogen bond donors / acceptors but also introduces trichlorophenyl groups, which can additionally generate π-π stacking interactions, achieving efficient and specific capture of imazalil-deaminoimidazole and imazalil-deimidazole formamide groups.

[0010] A second aspect of the present invention provides a method for synthesizing the above-mentioned urea-functionalized ternary magnetic composite material, comprising the following steps: S1. Magnetic Fe3O4 microspheres were prepared by a solvothermal method; S2. The magnetic Fe3O4 microspheres obtained in step S1 are dispersed in a buffer solution, dopamine hydrochloride is added to carry out a polymerization reaction, and then carbonized at high temperature to obtain Fe3O4@N-MC composite material. S3. Disperse the Fe3O4@N-MC composite material obtained in step S2 in water, add magnesium salt, aluminum salt and urea, and obtain the Fe3O4@N-MC@MgAl-LDHs composite material through hydrothermal reaction. S4. The Fe3O4@N-MC@MgAl-LDHs composite material obtained in step S3 is reacted with a urea-based silane coupling agent under reflux in an organic solvent to obtain a urea-based functionalized ternary magnetic composite material.

[0011] The synthesis method of this invention achieves the controllable construction of ternary magnetic composite materials through a four-step reaction process involving solvothermal reaction, polymerization carbonization, hydrothermal growth, and surface functionalization. Each step involves mild reaction conditions and simple operation, ensuring uniform coating of the nitrogen-doped mesoporous carbon layer, directional growth of the magnesium-aluminum hydrotalcite layer, and stable grafting of urea functional groups. The resulting material exhibits good batch-to-batch reproducibility and is suitable for large-scale preparation.

[0012] Furthermore, in step S2, the high-temperature carbonization temperature is 500~600℃, and the time is 1~3 hours. These process conditions ensure that dopamine hydrochloride is fully carbonized on the Fe3O4 surface to form a nitrogen-doped mesoporous carbon structure, while avoiding excessively high temperatures that could lead to oxidation of the magnetic core or structural collapse.

[0013] Furthermore, in step S3, the hydrothermal reaction temperature is 100~140℃, and the time is 4~8 hours. Under these hydrothermal reaction conditions, the magnesium aluminum hydrotalcite layer can grow uniformly and densely on the nitrogen-doped mesoporous carbon surface, forming a stable layered structure.

[0014] Furthermore, in step S4, the reflux reaction temperature is 70~90℃, and the time is 8~16 hours. These process conditions can drive the urea-silane coupling agent to undergo a full condensation reaction with the hydroxyl groups on the surface of the magnesium-aluminum hydrotalcite layer, achieving efficient covalent grafting of urea functional groups.

[0015] A third aspect of the present invention provides a reagent kit comprising: The adsorbent is a urea-functionalized ternary magnetic composite material; The eluent is a methanol aqueous solution with a volume fraction of 2% to 20%; The eluent is methanol; The diluent is ultrapure water; The complex solution is an acetonitrile / water mixture; Centrifuge tubes.

[0016] The kit of this invention uses urea-functionalized ternary magnetic composite material as the core adsorbent and is equipped with diluent, eluent, elution solution, reconstitution solution, and centrifuge tubes, forming a complete integrated solution for urine pretreatment. This kit eliminates the need for expensive commercial solid-phase extraction columns, completing the operation through magnetic separation, significantly simplifying the pretreatment process and improving the efficiency and reliability of batch sample processing.

[0017] A fourth aspect of this invention provides an application of a urea-functionalized ternary magnetic composite material for detecting the urea metabolites imazalil-deaminoimidazole and imazalil-deimidazole formamide in urine. The application includes the following steps: diluting a urine sample, adding the urea-functionalized ternary magnetic composite material for vortex adsorption, magnetic separation followed by discarding the supernatant, adding eluent for vortex elution, magnetic separation followed by discarding the eluent, adding elution solution for vortex elution, magnetic separation followed by collecting the eluent, drying it under nitrogen, reconstituteing it, and then performing liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection.

[0018] This invention relates to a urea-functionalized ternary magnetic composite material designed for imazalil-deaminoimidazole and imazalil-deimidazole formamide groups in urine. It enables enzymatic analysis without overnight β-glucuronidase digestion, utilizing the specific adsorption capacity of urea-functionalized materials for free metabolites. This avoids the risk of target degradation caused by traditional enzymatic digestion steps, significantly shortens the pretreatment time, and improves the accuracy and stability of the detection results.

[0019] Furthermore, in the step of diluting the urine sample, the volume ratio of the urine sample to the diluent is 1:3~5. This dilution range can significantly reduce the ionic strength of high-concentration salts and urea in the urine, reducing competitive inhibition on the adsorption process, while avoiding excessive dilution that would cause the concentration of trace target substances to fall below the detection limit, thus ensuring the sensitivity and stability of the subsequent detection signal.

[0020] Furthermore, the amount of the urea-functionalized ternary magnetic composite material added to each 1 mL urine sample is 8–15 mg. This dosage range ensures sufficient adsorption sites to completely capture trace metabolites in the urine while avoiding waste or elution difficulties caused by excessive adsorbent, thus achieving an optimal balance between adsorption efficiency and cost control.

[0021] Furthermore, the vortex adsorption time is 4–8 minutes. This time range allows the urea-functionalized material to fully contact the target metabolite and form a stable double-hydrogen bond complex, ensuring that adsorption reaches saturation equilibrium while avoiding unnecessary waiting time.

[0022] Furthermore, the vortex elution time is 4-8 minutes. This time range is sufficient to break the hydrogen bonds between the urea groups and the target analyte, allowing the eluent to fully penetrate and displace the adsorbate, while avoiding excessively long elution times that could delay subsequent concentration and reconstitution steps, thus improving the overall detection throughput.

[0023] Compared with the prior art, the beneficial effects of the present invention include: (1) The urea-functionalized ternary magnetic composite material adopts a ternary hierarchical core-shell structure design. The nitrogen-doped mesoporous carbon layer effectively prevents particle agglomeration and provides a stable growth substrate for the outer layer. The magnesium-aluminum hydrotalcite layer serves as the core functional carrier, providing abundant basic sites to load high-density urea functional groups. The outermost urea-functionalized oligomer endows the material with specific molecular recognition ability. The overall structure is robust and has excellent magnetic responsiveness, laying a structural foundation for the rapid processing of large batches of samples.

[0024] (2) By introducing urea-containing trichlorobenzene ring urea functional sites, the urea-functionalized ternary magnetic composite material realizes a dual recognition mechanism for target metabolites. The urea functional group forms a specific double hydrogen bond with the urea bond structure of the target molecule. At the same time, the trichlorobenzene ring provides an additional π-π stacking effect. This synergistic effect enables the composite material to accurately capture the target from the complex urine matrix, effectively shielding the interference of salt, urea and endogenous impurities, and significantly reducing the matrix effect.

[0025] (3) An enzyme-free detection scheme for free metabolites was proposed, which completely eliminates the cumbersome and time-consuming overnight incubation enzymatic digestion step of β-glucuronidase in traditional methods, greatly shortens the sample pretreatment cycle, avoids detection errors caused by fluctuations in enzymatic digestion conditions or degradation of target substances, and significantly improves the stability and reproducibility of detection results.

[0026] (4) The operation mode based on magnetic separation technology enables rapid switching between adsorption, rinsing, and elution processes without relying on complex solid-phase extraction devices. Compared with traditional methods, the consumption of organic solvents is significantly reduced, and the material has excellent enrichment capabilities, which can meet the detection requirements of trace target substances in urine. The entire purification process is simple and quick, making it very suitable for operation by non-professionals and rapid on-site screening.

[0027] (5) The composite material structure of the present invention is stable and has excellent recyclability, which effectively reduces the cost of a single test and has good economic benefits. The detection technology constructed by the present invention is not only applicable to routine physical examinations of occupationally exposed populations, but can also be extended to environmental health screening and food safety risk assessment of the general population, and has extremely high promotion and application value and industrialization potential. Attached Figure Description

[0028] Figure 1 These are the chemical structural formulas of two prochlorazine urea metabolites.

[0029] Figure 2 MRM chromatograms of two prochlorazine urea metabolites in a urine spiked sample.

[0030] Figure 3 (a) TEM image and (b) SEM image of the urea-functionalized ternary magnetic composite material prepared in the embodiments of the present invention.

[0031] Figure 4 The VSM spectrum of Fe3O4 microspheres and urea-functionalized ternary magnetic composite material prepared in the embodiments of the present invention is shown.

[0032] Figure 5 XPS spectrum of urea-functionalized ternary magnetic composite material prepared in an embodiment of the present invention.

[0033] Figure 6 The figure shows the test results of the effect of urine sample dilution factor on the extraction efficiency of two prochlorazine urea metabolites.

[0034] Figure 7 The figure shows the test results of the effect of the amount of urea-functionalized ternary magnetic composite material on the extraction efficiency of two prochlorazine urea metabolites.

[0035] Figure 8 The figure shows the test results of the effect of extraction time on the extraction efficiency of two prochlorazine metabolites.

[0036] Figure 9 The figure shows the test results of the effect of elution time on the elution efficiency of two prochlorazine metabolites.

[0037] Figure 10 The figure shows the test results of the recycling performance of urea-functionalized ternary magnetic composite materials. Detailed Implementation

[0038] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0039] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0040] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0042] Example 1 This embodiment prepares a urea-functionalized ternary magnetic composite material, which is mainly used to extract and purify two urea metabolites of imazalil from urine: imazalil-deaminoimidazole (BTS 44595) and imazalil-deimidazole formamide (BTS 44596). Figure 1 The chemical structural formulas of the two prochlorazine urea metabolites mentioned above are shown, both of which contain urea bonds. Figure 2 The image shows the MRM chromatograms of two metabolites in a urine spiked sample (10.0 μg / L).

[0043] The preparation method of urea-functionalized ternary magnetic composite materials includes the following steps: S1. Preparation of magnetic Fe3O4 microspheres Using a solvothermal method, 0.8 g of sodium citrate and 1.5 g of FeCl3·6H2O were weighed into a 250 mL flask, and 80 mL of ethylene glycol was added. The mixture was mechanically stirred at room temperature for 30 min to dissolve, followed by the addition of 5 g of sodium acetate and stirring for another 30 min. The resulting solution was then transferred to a 100 mL high-pressure reactor and reacted at 200 °C for 10 h, followed by cooling to room temperature. The mixture was then washed three times with pure water and ethanol, and dried under vacuum at 60 °C overnight to obtain magnetic Fe3O4 microspheres.

[0044] S2. Preparation of Fe3O4@N-MC composite materials Weigh 1.0g of Fe3O4 microspheres obtained in step S1, sonicate them in 100mL of Tris-HCl buffer (pH=8.5), add 2.0g of dopamine hydrochloride, polymerize at room temperature for 24h, magnetically separate and dry, and then carbonize at 550℃ for 2h under nitrogen atmosphere to obtain Fe3O4@N-MC composite material. Wash with pure water and ethanol three times respectively, and vacuum dry at 60℃ overnight.

[0045] S3. Preparation of Fe3O4@N-MC@MgAl-LDHs composite materials Weigh 1.0g of the Fe3O4@N-MC composite material obtained in step S2, disperse it in 80mL of water, add 0.06mol Mg(NO3)2, 0.03mol Al(NO3)3 and 3.0g urea, stir mechanically for 0.5h to fully dissolve it, transfer it to a high-pressure reactor, and hydrothermally react at 120℃ for 6h. After magnetic separation and drying, the Fe3O4@N-MC@MgAl-LDHs composite material is obtained.

[0046] S4. Preparation of urea-functionalized ternary magnetic composite materials 0.1 mol of aminopropyltriethoxysilane and 0.1 mol of 2,4,6-trichlorophenylethyl isocyanate were mixed and added to a three-necked flask. 200 mL of anhydrous toluene was added, and the mixture was reacted at room temperature for 12 h with mechanical stirring at 500 rpm to prepare a urea-based silane coupling agent. 0.5 g of the Fe3O4@N-MC@MgAl-LDHs composite material obtained in step S3 was weighed and mixed with the above coupling agent. The mixture was refluxed in toluene at 80 °C for 12 h, washed, and dried to obtain a urea-functionalized ternary magnetic composite material.

[0047] Example 2 This embodiment provides a kit for extracting and purifying urea metabolites from urine, comprising: Adsorbent: Urea-functionalized ternary magnetic composite material prepared in Example 1; Eluent: A methanol aqueous solution with a volume fraction of 2%~20%; Eluent: Methanol; Diluent: Ultrapure water; Reconstituted solution: Acetonitrile / water mixture (1:1, v / v); 15mL polypropylene centrifuge tubes.

[0048] Example 3 This embodiment uses the kit from Example 2 to detect the concentration of imazalil metabolites (BTS 44595 and BTS 44596) in urine samples. The detection method includes the following steps: a. Transfer the urine sample into a polypropylene centrifuge tube, centrifuge, take the supernatant, and dilute and mix with ultrapure water.

[0049] b. Add urea-functionalized ternary magnetic composite material to the sample solution, vortex adsorption, magnetic separation, and discard the upper layer solution.

[0050] c. Add 20% methanol aqueous solution for rinsing, vortex rinsing, then place on a magnetic rack for magnetic separation, and pour off the supernatant.

[0051] d. Add methanol for elution, vortex elution, magnetic separation, and collect the supernatant.

[0052] e. Dry with nitrogen, reconstitute with acetonitrile / water solution (1:1, v / v), filter through a membrane, and then analyze by LC-MS / MS.

[0053] The LC-MS / MS instrument conditions are as follows: (1) Liquid phase conditions Chromatographic conditions: Column: ACQUITY™ PREMIER BEHC 18Column (1.7μm×100mm×2.1mm); Injection volume: 5.0μL; Mobile phase: Phase A is water (containing 0.1% formic acid-5mM ammonium formate, 2% methanol), Phase B is methanol (containing 0.1% formic acid-5mM ammonium formate, 2% water); Column temperature: 40℃; Flow rate: 0.3mL / min; Elution gradient: 0~2.0min, 10%B; 2.0~4.0min, 10%B~60%B; 4.0~6.0min, 60%B~90%B; 6.0~8.0min, 90%B; 8.0~8.01min, 90%B~10%B; 8.01~12.0min, 10%B.

[0054] (2) Mass spectrometry conditions Ion source: Electrospray ionization source (ESI source); Scanning mode: Positive ion scanning; Detection mode: Multiple reaction monitoring (MRM); Electrospray voltage (IS): 5500V; Nebulizer gas pressure (GS1): 50.0psi; Auxiliary gas flow rate (GS2): 50.0psi; Curtain gas pressure (CUR): 40.0psi; Collision gas (CAD): 6.0psi; Ion source temperature (TEM): 500℃; Collision chamber outlet voltage (CXP): 11.0V; Collision chamber inlet voltage (EP): 10.0V. The qualitative ion pairs, quantitative ion pairs, collision gas energy (CE), and declustering voltage (DP) of the target compound are shown in Table 1.

[0055] Table 1 Retention time, parent ion, fragment ion, declustering voltage, and collision energy of the target compound Example 1: Characterization of Urea-functionalized ternary magnetic composite materials The morphology of the urea-functionalized ternary magnetic composite material prepared in Example 1 was characterized using transmission electron microscopy (TEM) and scanning electron microscopy (SEM), such as... Figure 3 As shown. By Figure 3 (a) It can be seen that the synthesized composite material exhibits a significant core-shell structure, with the core being Fe3O4 and the shell layer of MgAl-LDHs uniformly distributed in a petal-like structure around the material. SEM results ( Figure 3 (b) further confirmed that the composite material is spherical with a petal-like structure on the surface, and the petal surface is modified with a thin deposit, which is a urea-functionalized oligomer modified on the surface of the MgAl-LDHs layer.

[0056] The magnetic properties of the Fe3O4 microspheres and urea-functionalized ternary magnetic composite material prepared in Example 1 were characterized using a vibrating sample magnetometer (VSM). The results are as follows: Figure 4As shown, both Fe3O4 microspheres and urea-functionalized ternary magnetic composites exhibit paramagnetism, with saturation magnetizations of 81.2 emu / g and 30.5 emu / g, respectively. The decrease in magnetism after functionalization modification indicates that the functional layers have been successfully coated, while the residual magnetization still meets the requirements for magnetic separation operations.

[0057] The elemental composition of the urea-functionalized ternary magnetic composite material prepared in Example 1 was analyzed by X-ray photoelectron spectroscopy (XPS), and the results are as follows: Figure 5 As shown. By Figure 5 It is known that the composite material is mainly composed of elements such as C, O, Fe, Mg, Al, N, and Cl. Specifically, Fe originates from the magnetic Fe3O4 core; C and N originate from the nitrogen-doped mesoporous carbon layer and urea-functionalized oligomers; Mg and Al originate from the magnesium-aluminum hydrotalcite layer; and Cl originates from the trichlorophenyl group in the urea-silane coupling agent. XPS analysis further confirms that all components of the composite material have been successfully synthesized, and the bonding between the functional layers is stable.

[0058] Experimental Example 2: Effect of urine sample dilution factor on extraction efficiency Because urine contains a large amount of inorganic salts, urea, uric acid, and other impurities, with high ionic strength and numerous organic interfering substances, the sample pretreatment process is quite challenging. This study selected 1 mL of low-concentration spiked urine sample (1.0 μg / L) as the investigation object. The sample was diluted with pure water at multiples of 1, 2, 5, 8, 10, and 20 times. The urea-functionalized ternary magnetic composite material prepared in Example 1 was used for purification according to the method in Example 3. The specific steps are as follows: 1.0 mL of urine sample was transferred to a 15 mL polypropylene centrifuge tube and centrifuged at 10000 r / min for 5 min. The supernatant was collected and diluted with 0, 1, 4, 7, 9, and 19 mL of ultrapure water, respectively. 10 mg of the urea-functionalized ternary magnetic composite material was added to the sample solution, vortexed for 5 min, magnetically separated for 10 s, and the supernatant was discarded. 5 mL of the supernatant was added... Elute with 20% methanol aqueous solution, vortex for 5 min, magnetically separate for 10 s, and decant the supernatant; add 5 mL of methanol for elution, vortex for 5 min, magnetically separate for 10 s, and aspirate the supernatant; dry with nitrogen, reconstitute with 1 mL of acetonitrile / water solution (1:1, v / v), filter through a membrane, and analyze by LC-MS / MS.

[0059] Analysis results as follows Figure 6As shown, when urine samples are purified directly without dilution, the recoveries and peak areas of the two metabolites BTS44595 and BTS44596 are low. With increasing dilution factor, the peak areas gradually increase. When the urine sample dilution factor increases to 5 times, the peak area reaches its maximum value, and further increases in dilution factor do not significantly change the peak areas of either metabolite. Considering economic efficiency and ease of operation, the preferred volume ratio of urine sample to diluent is 1:3 to 5. Subsequent experiments used a 5-fold dilution factor, i.e., a volume ratio of urine sample to diluent of 1:4.

[0060] Experimental Example 3: Effect of Urea-functionalized Ternary Magnetic Composite Material Dosage on Extraction Efficiency Add 1 mL of spiked urine sample (both prochloraz metabolites were spiked at a concentration of 100.0 μg / L) to each of six 10 mL polypropylene centrifuge tubes, and dilute with 4 mL of water. Accurately add 1–100 mg of the urea-functionalized ternary magnetic composite material prepared in Example 1 to the sample solution, and purify according to the method in Example 3. Analyze by LC-MS / MS. The results are as follows: Figure 7 As shown.

[0061] Depend on Figure 7 It can be seen that when the amount of adsorbent is small, the purification effect is relatively poor, and the peak areas of the two metabolites are relatively small. When the amount of adsorbent is 10 mg, the peak areas of both metabolites reach their maximum values, and further increasing the amount of adsorbent does not change the peak areas of both metabolites. Therefore, the preferred amount of urea-functionalized ternary magnetic composite material added to each 1 mL urine sample is 8-15 mg, and the subsequent experiments used an adsorbent amount of 10 mg.

[0062] Experiment Example 4: The Effect of Extraction Time on Extraction Efficiency 1 mL of spiked urine sample (both metabolites were spiked at a concentration of 100 μg / L) was pipetted into 15 mL polypropylene centrifuge tubes, and 4 mL of water was added for dilution and mixing. 10 mg of the urea-functionalized ternary magnetic composite material prepared in Example 1 was accurately added to the sample solution, and vortex extraction was performed for 1–60 min. Subsequent purification was carried out according to the method in Example 3. LC-MS / MS analysis was performed, and the results are as follows: Figure 8 As shown.

[0063] Depend on Figure 8 It was observed that for both metabolites, the peak area of ​​the target compound gradually increased with increasing extraction time. When the extraction time reached 5 minutes, the peak area no longer increased and reached a plateau. Therefore, considering both extraction efficiency and time saving, an extraction time of 4–8 minutes was optimal, and a 5-minute extraction time was used in subsequent experiments.

[0064] Effect of elution time on extraction efficiency in Experiment Example 5 1 mL of spiked urine sample (both metabolites were spiked at a concentration of 100 μg / L) was pipetted into 15 mL polypropylene centrifuge tubes and diluted with 4 mL of water. 10 mg of the urea-functionalized ternary magnetic composite material prepared in Example 1 was accurately added to the sample solution. The mixture was vortexed for 5 min, magnetically separated for 10 s, and the supernatant was discarded. Then, 5 mL of 20% methanol-water solution was added for rinsing, vortexed for 5 min, magnetically separated for 10 s, and the supernatant was decanted. 5 mL of methanol solution was added for elution, vortexed for 1–60 min, magnetically separated for 10 s, and the supernatant was collected, dried under nitrogen, and reconstituted with 1 mL of acetonitrile / water (1:1, v / v). The sample was analyzed by LC-MS / MS. The results are shown below. Figure 9 As shown.

[0065] Depend on Figure 9 It is evident that using methanol as the elution solvent can rapidly elute the two metabolites from the composite material, and a elution time of 5 minutes is sufficient to completely elute the target compound. Therefore, a elution time of 4–8 minutes is preferred, and a elution time of 5 minutes was used in subsequent experiments.

[0066] Experimental Example 6: Evaluation of Matrix Effect Accurately pipette 1 mL of urine blank sample into 15 mL polypropylene centrifuge tubes, add 4 mL of pure water and mix well. Then add 10 mg of the urea-functionalized ternary magnetic composite material prepared in Example 1, vortex extract for 5 min, then place on a magnetic rack for magnetic separation for 10 s, and decant the supernatant. Then add 5 mL of 20% methanol aqueous solution for washing (vortex for 5 min, magnetic separation for 10 s, decant the supernatant), add 5 mL of methanol for elution (vortex for 5 min, magnetic separation for 10 s), aspirate the supernatant, dry under nitrogen, and reconstitute with 1 mL of acetonitrile-water solution (1:1, v / v). Then add appropriate amounts of BTS 44595 and BTS 44596 standard stock solutions to prepare matrix-matched standard solutions of 0.1–200 μg / L using the blank urine purification solution. Simultaneously, compare the matrix-matched working curve purified by a commercial Waters HLB solid-phase extraction column with the solvent standard series solutions prepared with acetonitrile-water solution (1:1, v / v). The formula used is: [Matrix effect η = (Slope K of matrix matching standard curve)] a -Slope K of the solvent standard curve b Slope K of the solvent standard curve b The matrix effect was evaluated, and the results are shown in Table 2.

[0067] Table 2 Comparison of the matrix interference resistance of the kit of the present invention and the commercial Waters HLB solid phase extraction column. As shown in Table 2, after purification by the urea-functionalized ternary magnetic composite material of the present invention, the matrix effect of the two metabolites is... η The values ​​were -11.8% and -11.0%, respectively, indicating a weak matrix inhibition effect. Therefore, no matrix-matched working curve or isotopic internal standard was required during quantitative detection. However, for commercially available Waters HLB solid-phase extraction columns, the matrix effects of BTS 44595 and BTS 44596 were... η The values ​​of -35.3% and -38.1% were respectively, indicating a moderate matrix inhibition effect, requiring quantitative analysis using matrix-matched working curves or isotopic internal standards. Therefore, the kit of this invention can effectively remove interfering impurities in urine, effectively eliminate matrix interference effects during the detection process, and improve detection accuracy.

[0068] Experimental Example 7: Linear Equation, Correlation Coefficient, Accuracy, Precision, Limit of Detection and Limit of Quantitation The residual amounts of two prochlorazine urea metabolites in urine were simultaneously detected using the LC-MS / MS method described in Example 3. As shown in Table 3, BTS 44595 and BTS 44596 showed good linearity in the concentration range of 0.1–200.0 μg / L (correlation coefficient R). 2 >0.999). The method detection limit and quantitation limit are defined as signal-to-noise ratio (S / N) ≥3 and S / N ≥9, respectively. The method detection limits for BTS 44595 and BTS 44596 in urine are 0.01 μg / L and 0.03 μg / L, respectively, and the quantitation limits are 0.03 μg / L and 0.06 μg / L, respectively.

[0069] Table 3. Linear range, linear equation, correlation coefficient, limit of detection, and limit of quantitation of the detection method of the present invention. The accuracy and precision of the method of the present invention were investigated by controlling the spiked levels of the two metabolites in urine samples to be 0.5, 10.0, 50.0, and 100.0 μg / L, respectively, and detecting them using the method of Example 3. The results are shown in Table 4. Table 4 shows that the recoveries of BTS44595 and BTS44596 were 88.7%–102% and 89.1%–101%, respectively, with relative standard deviations (RSDs) of 1.5%–5.2%. This indicates that the method of the present invention has the advantages of being rapid, sensitive, and accurate.

[0070] Table 4. Spike recovery and precision of the detection method of the present invention (n=6) Experimental Example 8: Recycling Performance of Urea-functionalized Ternary Magnetic Composite Materials Following the detection method of Example 3, urine spiked samples were purified six times using 10 mg of the urea-functionalized ternary magnetic composite material prepared in Example 1. The results are as follows: Figure 10 As shown.

[0071] Depend on Figure 10 It can be seen that after six cycles of use, the peak areas of the two metabolites did not decrease significantly. Therefore, the urea-functionalized ternary magnetic composite material prepared in this invention exhibits good recyclability, which is beneficial for reducing detection costs and is more environmentally friendly.

[0072] While the present invention has been disclosed above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the scope of protection of this invention.

Claims

1. A urea-functionalized ternary magnetic composite material, characterized in that, The composite material is used to extract and purify prochloraz urea metabolites from urine, comprising: Magnetic Fe3O4 core; A nitrogen-doped mesoporous carbon layer is coated on the surface of the magnetic Fe3O4 core; A magnesium aluminum hydrotalcite layer is coated on the surface of the nitrogen-doped mesoporous carbon layer; A urea-functionalized oligomer layer is covalently grafted onto the surface of the magnesium aluminum hydrotalcite layer.

2. The urea-functionalized ternary magnetic composite material according to claim 1, characterized in that, The urea-functionalized oligomer layer is formed by a urea-silane coupling agent, which is prepared by reacting aminopropyltriethoxysilane with 2,4,6-trichlorophenylethyl isocyanate.

3. A method for synthesizing a urea-functionalized ternary magnetic composite material as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Magnetic Fe3O4 microspheres were prepared by a solvothermal method; S2. The magnetic Fe3O4 microspheres obtained in step S1 are dispersed in a buffer solution, dopamine hydrochloride is added to carry out a polymerization reaction, and then carbonized at high temperature to obtain Fe3O4@N-MC composite material. S3. Disperse the Fe3O4@N-MC composite material obtained in step S2 in water, add magnesium salt, aluminum salt and urea, and obtain the Fe3O4@N-MC@MgAl-LDHs composite material through hydrothermal reaction. S4. The Fe3O4@N-MC@MgAl-LDHs composite material obtained in step S3 is reacted with a urea-based silane coupling agent under reflux in an organic solvent to obtain a urea-based functionalized ternary magnetic composite material.

4. The synthesis method according to claim 3, characterized in that, In step S2, the high-temperature carbonization temperature is 500~600℃ and the time is 1~3 hours; In step S3, the hydrothermal reaction temperature is 100~140℃ and the time is 4~8 hours; In step S4, the reflux reaction temperature is 70~90℃ and the time is 8~16 hours.

5. A kit for extracting and purifying prochlorazine metabolites from urine, characterized in that, include: The adsorbent is the urea-functionalized ternary magnetic composite material according to claim 1 or 2; The eluent is a methanol aqueous solution with a volume fraction of 2% to 20%; The eluent is methanol; The diluent is ultrapure water; The complex solution is an acetonitrile / water mixture; Centrifuge tubes.

6. An application of a urea-functionalized ternary magnetic composite material as described in claim 1 or 2, characterized in that, The method for detecting the urea metabolites imazalil-deaminoimidazole and imazalil-deimidazole formamide in urine includes the following steps: diluting the urine sample, adding the urea-functionalized ternary magnetic composite material for vortex adsorption, discarding the supernatant after magnetic separation, adding eluent for vortex elution, discarding the eluent after magnetic separation, adding elution solution for vortex elution, collecting the eluent after magnetic separation, drying it with nitrogen, reconstituteing it, and then performing liquid chromatography-tandem mass spectrometry detection.

7. The application according to claim 6, characterized in that, In the step of diluting the urine sample, the volume ratio of the urine sample to the diluent is 1:3~5.

8. The application according to claim 6, characterized in that, The amount of the urea-functionalized ternary magnetic composite material added to each 1 mL urine sample is 8-15 mg.

9. The application according to claim 6, characterized in that, The vortex adsorption time is 4 to 8 minutes.

10. The application according to claim 6, characterized in that, The vortex elution time is 4 to 8 minutes.

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