Preparation and application method of selective magnetic solid-phase extraction material for purine compounds
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTHEAST UNIV
- Filing Date
- 2026-04-15
- Publication Date
- 2026-05-29
Smart Images
Figure CN122098521A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a magnetic solid-phase extraction method based on functionalized nanocomposite materials, and particularly to a nanocomposite material for selective magnetic solid-phase extraction of pterin compounds, its preparation method, and a method for selective magnetic solid-phase extraction of pterin compounds in complex samples using the nanocomposite material. Background Technology
[0002] Pterin compounds are nitrogen-containing heterocyclic aromatic compounds formed by the fusion of pyrimidine and pyrazine rings. They are key metabolic derivatives of guanosine triphosphate (GTP) and are widely involved in physiological processes in the body, such as enzymatic reactions in nitric oxide synthesis, the synthesis of neurotransmitters like dopamine and serotonin, and the activation of immune cells. In the human body, pterin compounds mainly exist in oxidized forms such as neopterin and biopterin, and reduced forms such as 7,8-dihydrobiopterin and 5,6,7,8-tetrahydrobiopterin. Their content and redox balance directly reflect the pathological state of the body. For example, neopterin is synthesized by monocytes / macrophages under interferon-γ stimulation, and its serum level can serve as a marker of inflammatory activity in viral infections such as COVID-19 and autoimmune diseases such as systemic lupus erythematosus. 5,6,7,8-tetrahydrobiopterin, as an essential cofactor for phenylalanine hydroxylase, can lead to phenylketonuria (PKU) due to deficiency, and its abnormal levels are closely related to vascular endothelial dysfunction, Parkinson's disease, and other neurodegenerative diseases.
[0003] Research on pteridine derivatives as drugs covers two core areas: cancer treatment and autoimmune disease treatment. Among them, methotrexate, pemetrexed, pralatrexate, leflunomide, and sulfasalazine are widely used in clinical practice and each has its own advantages. In the field of cancer treatment, methotrexate inhibits folate metabolism by potently inhibiting dihydrofolate reductase, thereby blocking the synthesis of nucleic acids in tumor cells and inhibiting their proliferation. It is a basic drug for various cancers such as acute lymphoblastic leukemia and breast cancer. Pemetrexed, with its multi-target inhibitory properties, acts simultaneously on key enzymes such as thymidylate synthase, and has shown significant efficacy in the treatment of advanced non-small cell lung cancer and malignant pleural mesothelioma. Pralatrexate, with its high affinity for folate transport proteins, is more easily taken up by tumor cells and has become an important therapeutic drug for relapsed and refractory peripheral T-cell lymphoma. In the treatment of autoimmune diseases, low-dose methotrexate exerts its immunosuppressive effect by inhibiting lymphocyte proliferation and is a first-line drug for rheumatoid arthritis; leflunomide metabolites inhibit dihydroorotate dehydrogenase, blocking pyrimidine synthesis to relieve inflammation, and are suitable for rheumatoid arthritis, systemic lupus erythematosus, etc.; sulfasalazine is broken down into active pterin products in the intestine, combining immunosuppression and intestinal anti-inflammatory effects, and is effective for ulcerative colitis and rheumatoid arthritis complicated with intestinal lesions.
[0004] In clinical treatment, monitoring the blood concentration of these pterin drugs allows for individualized dosage adjustments: in cancer treatment, it can enhance the efficacy of combined chemotherapy with methotrexate and pemetrexed, especially optimizing the leucovorin rescue regimen after high-dose methotrexate chemotherapy; in the treatment of autoimmune diseases, it can precisely control drug exposure and reduce the risk of side effects from long-term use. However, pterin drugs generally have a narrow therapeutic window and significant individual metabolic variability. Overdose can easily lead to serious adverse reactions. For example, methotrexate and pemetrexed may cause bone marrow suppression and liver and kidney damage, leflunomide may cause skin toxicity, and sulfasalazine is prone to causing gastrointestinal reactions. Furthermore, pharmacological studies have confirmed that methotrexate and pemetrexed are converted into polyglutamylated metabolites after entering cells. These metabolites have a higher affinity for the target enzyme and a longer retention time, which is key to their long-lasting pharmacological effects.
[0005] Pterin compounds in biological samples are present in trace amounts and exhibit complex redox states, making them highly susceptible to environmental interference and thus challenging to detect. In recent years, high-performance liquid chromatography (HPLC) or liquid chromatography-tandem mass spectrometry (LC-MS / MS) has been the most commonly used methods for pterin analysis in clinical practice. However, these methods still face numerous challenges in practical applications. Traditional HPLC-FLD analysis often employs potassium iodide-iodine solution and manganese oxide for oxidation pretreatment to enhance pterin stability and obtain higher fluorescence response, but this is an indirect detection method, unable to distinguish pterin isomer structures, sensitive to reaction conditions, introduces additional errors, requires large amounts of chemical reagents, and lacks environmental friendliness. Although LC-MS / MS can achieve direct detection, the complexity of the human body fluid matrix often leads to severe matrix effects and ion inhibition, typically requiring high-level dilution to reduce sensitivity. Therefore, there is an urgent need to develop an efficient, sustainable sample pretreatment technique suitable for complex biological samples to directly detect analytes, effectively removing matrix interferences and enriching the target analyte.
[0006] Based on the dinitrogen ring core structure of pterin, its 4-position amino group, 5-position heterocyclic N atom, and side-chain carboxyl and carbonyl groups provide clear targets for coordination and binding with metal ions. To address the need for trace analysis of pterin in complex biological samples, there is an urgent need to develop an enrichment material that combines high selectivity, rapid separation capability, and pH responsiveness. The iron(III) oxide@polypyrrole / palladium and other noble metal ion composite system perfectly meets this requirement: the superparamagnetism of the iron(III) oxide nanoparticles ensures high efficiency of magnetically responsive separation; the polypyrrole layer achieves pre-enrichment of the target analyte through π-π stacking and hydrogen bonding; and noble metal ions such as palladium can form a highly stable chelate structure with the 4-NH2 and 5-position heterocyclic N atoms of the pterin molecule, significantly improving adsorption selectivity; simultaneously, the pH sensitivity of this chelate structure enables efficient desorption of the target analyte. Therefore, the iron(II) oxide@polypyrrole / noble metal ion functionalized nanocomposite nanomaterial constructed in this invention can effectively solve the technical problems of insufficient selectivity, cumbersome separation, and low desorption efficiency in existing enrichment methods, and provide a reliable pretreatment solution for the accurate detection of pterin-like substances in complex samples. Summary of the Invention
[0007] Objective of the Invention: To address the problems existing in the prior art, this invention provides a functionalized nanocomposite material for selective magnetic solid-phase extraction of pterin compounds in complex samples, and provides a method for preparing the nanocomposite material. This nanocomposite material has high extraction efficiency and can achieve rapid magnetic solid-phase extraction of pterin compounds in complex samples. In addition, this invention also provides a method for selective magnetic solid-phase extraction of pterin compounds in complex samples using the nanocomposite material as an adsorbent.
[0008] Technical solution: The present invention discloses a selective magnetic solid-phase extraction material for pterin compounds, comprising, from the center outwards, a core layer of iron oxide nanoparticles, a modification layer I, and a modification layer II; wherein, the core layer of iron oxide nanoparticles is iron oxide nanoparticles; the modification layer I is obtained by in-situ polymerization of pyrrole monomers onto the surface of the iron oxide nanoparticle core layer under the action of an initiator; the modification layer II is obtained by dissolving a metal compound in solvent A to obtain a modification solution, and coating the surface of modification layer I with the metal compound in the modification solution.
[0009] The core layer of the iron oxide nanoparticles has a particle size of 2-50 nm and a crystallinity of 70%-95%.
[0010] The raw materials for preparing the modified layer I include pyrrole monomer, initiator and solvent; the initiator is ferric chloride solution; the mass ratio of pyrrole monomer to iron oxide nanoparticle core layer is 1:5-10, and the molar ratio of initiator to pyrrole monomer is 2-3:1.
[0011] The metal compound of the modified layer II is a chloride, a metal complex acid, or a metal nitrate; the metal chloride is one or a mixture of palladium chloride and rhodium chloride; the metal complex acid is one or a mixture of chloroauric acid, chloroplatinic acid, and chloropalladium acid; the metal nitrate is silver nitrate; the solvent A is a 0.1 M hydrochloric acid solution or pure water, and the concentration of the metal compound in the solvent is 10-500 mg / L.
[0012] A method for preparing the pterin-based selective magnetic solid-phase extraction material includes the following steps: Step 1, Preparation of the core layer of iron oxide nanoparticles: The core layer of iron oxide nanoparticles is prepared by chemical co-precipitation of ferrous salt and ferric salt, or by solvothermal method of ferrous salt, ferric salt and organic solvent, or by thermal decomposition method of organic iron precursor, or by microemulsion method of ferrous salt, ferric salt and surfactant. Step 2, Preparation of Modification Layer I: Modification layer I is obtained by in-situ polymerization of pyrrole monomers on the surface of the core layer of iron oxide nanoparticles obtained in Step 1 under the action of an initiator, thus obtaining iron oxide@polypyrrole intermediate; Step 3, Preparation of Modification Layer II: The metal compound is dissolved in solvent A to obtain a modification solution. The metal compound in the modification solution is coated on the surface of modification layer I to obtain modification layer II, thus obtaining a surface-functionalized nanocomposite material.
[0013] The application of the pterin-based selective magnetic solid-phase extraction material in the selective extraction of pterin-based compounds from complex samples.
[0014] The application includes the following steps: Step 1: After obtaining the sample, quickly freeze or process the sample to obtain the test solution or the processing solution; Step 2: Take the pterin-based compound selective magnetic solid-phase extraction material and activate it sequentially with the first activation solution and the second activation solution to obtain the activated magnetic nanocomposite material. Step 3: Add the activated magnetic nanocomposite material to the test solution or treatment solution obtained in Step 1, and stir at room temperature to allow the pterin compounds in the sample to be fully adsorbed onto the surface of the nanocomposite material. Step 4: Place the adsorption system in an external magnetic field and let it stand to allow the pterin selective magnetic solid phase extraction material containing pterin compounds to quickly separate from the sample matrix, and discard the supernatant. Step 5: Add eluent to the separated pterin compounds selective magnetic solid-phase extraction material, stir and elute at room temperature, then place it in an external magnetic field again to achieve solid-liquid separation, collect the eluent, and complete the selective magnetic solid-phase extraction of pterin compounds.
[0015] The specific activation method for the selective magnetic solid-phase extraction material of pterin compounds in step 2 is as follows: Place the nanocomposite material in a centrifuge tube, add the first activation solution, mix, and then separate it in an external magnetic field, discarding the activation waste liquid; then add the second activation solution to the centrifuge tube, perform mixing, magnetic response separation, and discard the waste liquid to complete the activation; the liquid-to-solid ratio of the first activation solution to the nanocomposite material is 5-10 mL:2-10 mg, and the liquid-to-solid ratio of the second activation solution to the nanocomposite material is the same as that of the first activation solution; the stirring rate in step 3 is 100-300 r / min; the eluent in step 5 is a mixed solution of acetonitrile and formic acid with a volume ratio of 70:30, and the liquid-to-solid ratio of the eluent to the nanocomposite material is 0.2-5 mL:2-10 mg; the magnetic field strength of the external magnetic field is 0.1-0.5 T.
[0016] The first activating solution is selected from one or more of methanol, ethanol, formic acid, and acetic acid; the second activating solution is selected from one or more of methanol, ethanol, and water.
[0017] The eluent consists of an acidic or alkaline solution and an organic solvent. The acidic solution is selected from sulfuric acid, hydrochloric acid, formic acid, and acetic acid; the alkaline solution is selected from ammonia water and sodium hydroxide solution; the organic solvent is selected from methanol, ethanol, and acetonitrile, or any combination of two or more of them. The mass fraction of the acidic solution is 1% to 10%, the mass fraction of the alkaline solution is 1% to 10%, the volume ratio of the acidic solution to the organic solvent is 1:10 to 1:1, the volume ratio of the alkaline solution to the organic solvent is 1:10 to 1:1, the pH range of the acidic solution is 1 to 6, and the pH range of the alkaline solution is 7 to 11. Beneficial effects: Compared with the prior art, the advantages of the present invention are: (1) The functionalized nanocomposite material of the present invention uses iron oxide as the magnetic response substrate, is coated with polypyrrole and loaded with noble metal ions, has a large specific surface area, and has many sites on the material surface that interact with the target molecules with high selectivity. Only a small amount of material is needed to achieve the same extraction effect, and the adsorption extraction efficiency is greatly improved; (2) When the functionalized nanocomposite material of the present invention is used for selective magnetic solid phase extraction of pterin compounds in complex samples, solid-liquid separation is achieved by means of an external magnetic field. There is no need for cumbersome operations such as centrifugation and filtration. The operation process is simple and fast. This feature is especially suitable for the rapid extraction of unstable pterin compounds that are easily oxidized and destroyed; (3) In the process of selective magnetic solid phase extraction of pterin compounds in complex samples using the functionalized nanocomposite material of the present invention, the target substances enriched on the surface of the functionalized nanocomposite material can be desorbed with a small volume of acidic or weak base and organic mixture solution. This reduces or eliminates the energy consumption of the operation of evaporating solvent to concentrate the target substances in conventional solid phase extraction, and also reduces the risk of pterin compounds being destroyed; (4) The functionalized nanocomposite material of the present invention can be rapidly separated and recovered by magnetic field, and can be reused 5-8 times after activation, which greatly reduces the extraction cost and has good application prospects. Attached Figure Description
[0018] Figure 1 A transmission electron microscope image of a material used in this patent; Figure 2 Hysteresis loop diagram of Fe3O4@ppy / Pd (Ⅱ) functionalized nanocomposite material prepared in this patent; Figure 3 This is a chromatogram of the eluent after selective magnetic solid phase extraction of a real urine sample with methotrexate standard solution using a material in Example 1. Figure 4 This is a chromatogram of the eluent after a real urine sample was magnetically selectively separated using a material in Example 2.
[0019] in: Figure 1 In the figure, (a) shows Fe3O4@ppy / Pd (II) functionalized nanocomposite material prepared in this patent; (b) shows Fe3O4@ppy / Pd (II) functionalized nanocomposite material prepared in this patent after modification with modification layer I; (c) shows Fe3O4@ppy / Pd (II) functionalized nanocomposite material prepared in this patent after modification with modification layer I and modification layer II; (d) shows the transmission electron microscopy energy spectrum of Fe3O4@ppy / Pd (II) functionalized nanocomposite material prepared in this patent. Figure 3In the figures, (a) is the chromatogram of a 10 μg / ml methotrexate standard solution; (b) is the chromatogram of a real urine sample with a 500 ng / ml methotrexate standard solution; and (c) is the chromatogram of the eluent after magnetic solid-phase extraction of a real urine sample with a 500 ng / ml methotrexate standard solution using the Fe3O4@ppy / Pd (Ⅱ) functionalized nanocomposite material prepared in this patent as an adsorbent. Figure 4 In the figures, (a) is a chromatogram of a mixed standard solution of neopterin, isoxopterin, biopterin and pterin at 500 ng / ml; (b) is a chromatogram of a real urine sample; and (c) is a chromatogram of the eluent after magnetic solid-phase extraction of a real urine sample using the Fe3O4@ppy / Pd (Ⅱ) functionalized nanocomposite material prepared in this patent as an adsorbent. Detailed Implementation The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.
[0020] This invention discloses a functionalized nanocomposite material for selective magnetic solid-phase extraction of pterin compounds from complex samples. The functionalized nanocomposite material comprises a magnetite nanoparticle core layer, a modification layer I, and a modification layer II. The magnetite nanoparticle core layer is prepared by chemical co-precipitation using ferrous and ferric salts as raw materials, or by a solvothermal method using ferrous and ferric salts with an organic solvent, or by a thermal decomposition method using an organic iron precursor, or by a microemulsion method using ferrous and ferric salts with a surfactant. Modification layer I is obtained by in-situ polymerization of pyrrole monomers on the surface of the magnetite nanoparticle core layer under the action of an initiator. A modification solution II is obtained by dissolving a metal chloride, metal complex acid, or metal nitrate in solvent A. The metal compound in modification solution II is then coated onto the surface of modification layer I through an immersion coating treatment to obtain modification layer II. In the raw materials for preparing the iron(III) oxide nanoparticle core layer, the molar ratio of ferrous salt to ferric salt is 1:1.8-1:2.0; the polymer concentration in modification solution I containing modification layer I is 0.5-0.83 wt%; and the concentration of the metal compound in modification solution II containing modification layer II is 10-500 mg / L. The iron(III) oxide nanoparticle core layer is dispersed in pure water, and pyrrole monomer and initiator are added to prepare modification solution I. The concentration of pyrrole monomer in modification solution I is 0.03-0.05 M by solvent volume.
[0021] Preferably, the ferrous salt used in the preparation of the iron(III) oxide nanoparticle core layer includes one or more of ferrous chloride, ferrous sulfate, and ferrous acetate; the iron salt includes one or more of ferric chloride, ferric sulfate, and ferric nitrate; the organic iron precursor includes one or more of ferric oleate, ferric acetylacetone, and ferric stearate; the organic solvent used in the solvothermal method includes one or more of ethylene glycol, diethylene glycol, and 1-octadecene; and the surfactant used in the microemulsion method includes one or more of trisodium citrate, polyethylene glycol, sodium dodecyl sulfate, Triton X-100, and oleic acid.
[0022] The metal chloride of the modification layer II includes one or a mixture of two of palladium chloride and rhodium chloride; the metal complex acid includes one or a mixture of several of chloroauric acid, chloroplatinic acid, and chloropalladium acid; and the metal nitrate includes silver nitrate.
[0023] Solvent A contains 0.1 M hydrochloric acid solution or pure water.
[0024] The initiator used in the preparation of modified layer I contains ferric chloride, and the molar ratio of the initiator to the pyrrole monomer is 2:1-3:1.
[0025] The present invention discloses a method for preparing functionalized nanocomposites for selective magnetic solid-phase extraction of pterin compounds from complex samples, comprising the following steps: The ferrous salt (one or a mixture of ferrous chloride, ferrous sulfate, or ferrous acetate) and the ferric salt (one or a mixture of ferric chloride, ferric sulfate, or ferric nitrate) were added to deionized water at a molar ratio of 1:1.8 to 1:2.0. After purging the system with N2 gas for 30 min to remove oxygen, the mixture was placed in a constant temperature environment of 65℃ and magnetically stirred at a rate of 550 r / min. The pH was adjusted to 9.5 by rapidly adding 1.8 mol / L sodium hydroxide solution, and the mixture was kept at this temperature for 1.2 h. After the reaction was completed, the mixture was centrifuged and washed alternately with deionized water and anhydrous ethanol until the filtrate was neutral. The filtrate was then vacuum dried at 55℃ for 5 h to obtain the core layer of iron(III) oxide nanoparticles.
[0026] Step 2: Take 50 mg of the iron oxide nanoparticle core layer obtained in Step 1, add 200 mL of pure water to prepare a suspension, cool the suspension to 5℃, add 0.675 mL of pyrrole monomer, and stir at 500 r / min for 30 min; then slowly add 25 mL of 0.5 mol / L ferric chloride solution, and continue stirring for 24 h to carry out in-situ polymerization reaction; after the reaction is completed, filter, wash repeatedly with deionized water until the washing liquid is colorless, and dry at 50℃ for 24 h to allow polypyrrole to complex onto the surface of the iron oxide nanoparticle core layer to form modification layer I, and obtain nanoparticle material coated with modification layer I.
[0027] Step 3: Dissolve the metal compound (one or a mixture of palladium chloride and rhodium chloride, or one or a mixture of chloroauric acid and chloroplatinic acid, or silver nitrate) in 0.1 M hydrochloric acid solution or pure water, and sonicate for 20 min. Then adjust the concentration of the metal compound to 10-500 mg / L to obtain modification solution II. Take 50 mg of the nanoparticle material coated with modification layer I obtained in step 2, soak it in 2 mL of modification solution II, and let it stand at room temperature for 2-4 h, stirring at 300 r / min for 10 min every 1 h. Then place the system in an external magnetic field and let it stand for 5-10 min to achieve solid-liquid separation. Discard the supernatant.
[0028] Step 4: Place the separated nanoparticles in a 50°C oven to dry and remove moisture, and coat the surface of the metal compound onto the surface of the modification layer I to form the modification layer II, thus obtaining the surface-functionalized nanocomposite material.
[0029] Preferably, in step 1 above, the N2 gas introduction rate is 45-55 mL / min; the sodium hydroxide solution dropping rate is 0.4-0.6 mL / s; and the temperature control accuracy for the heat preservation and curing process is ±1℃.
[0030] In step 2 above, the dropping rate of the ferric chloride solution is 0.2-0.4 mL / s, and the stirring rate of the in-situ polymerization process is stable at 100-550 r / min; in step 3, the power of the ultrasonic dispersion is 250-350 W, and the magnetic field strength of the external magnetic field is 0.1-0.5 T.
[0031] The present invention discloses a method for selective magnetic solid-phase extraction of pterin compounds from complex samples using functionalized nanocomposite materials, comprising the following steps: Step 1: After obtaining the sample, it needs to be frozen or processed as soon as possible to obtain the test solution or the processing solution.
[0032] Step 2: Weigh 5-20 mg of functionalized nanocomposite material and place it in a centrifuge tube. Add 0.5-5 mL of the first activation solution and 1-5 mL of the second activation solution in sequence. Vortex mix for 2-3 min each. Then place the centrifuge tube in an external magnetic field with a magnetic field strength of 0.1-0.5 T and let it stand for 5-10 min to achieve solid-liquid separation. Discard the activation waste liquid to complete the material activation.
[0033] Step 3: Add 0.5-1 mL of the test solution or treatment solution to the activated functionalized nanocomposite material, and stir at 100-300 r / min for 10-30 min at room temperature to allow pterin compounds to be fully adsorbed onto the material surface; then place the centrifuge tube in an external magnetic field and let it stand for 5-10 min. After solid-liquid separation, discard the supernatant; then add 0.2-5 mL of eluent to the centrifuge tube, vortex mix for 5-15 min, and place it in an external magnetic field again for 5-10 min. After solid-liquid separation, collect the eluent to complete the separation of pterin compounds.
[0034] Before adsorbing pterin compounds from the sample, the functionalized nanocomposite material is first activated. The specific activation method is as follows: weigh the functionalized nanocomposite material and place it in a centrifuge tube, add the first activation solution, vortex mix for 2-3 minutes, and then place it in an external magnetic field for separation, discarding the activation waste liquid; then add the second activation solution to the centrifuge tube, and repeat the operation of vortex mixing, magnetic response separation, and discarding the waste liquid, so that the nanocomposite material can reach a state in which it can efficiently adsorb pterin compounds.
[0035] In step 3, the method for adsorbing pterin compounds in complex samples using functionalized nanocomposite materials includes the following steps: adding the test solution or treatment solution to a centrifuge tube containing activated functionalized nanocomposite materials, stirring at a rate of 100-300 r / min for 10-30 min at room temperature, and adsorbing pterin compounds in the sample or its treatment solution onto the functionalized nanocomposite materials by utilizing the chelation effect between noble metal ions on the material surface and the -NH2 at the 4-position and the N atom of the heterocyclic ring at the 5-position of the pterin molecule.
[0036] The first activating solution is selected from one or more of methanol, ethanol, formic acid, and acetic acid; the second activating solution is selected from one or more of methanol, ethanol, and water.
[0037] The eluent consists of an acidic solution and an organic solvent. The acidic solution is selected from compounds that can form acidic solutions, such as sulfuric acid, hydrochloric acid, formic acid, and acetic acid. The organic solvent is selected from one or more combinations of methanol, ethanol, and acetonitrile.
[0038] The mass fraction of the acidic solution is 1%-10%, the volume ratio of the acidic solution to the organic solvent is 1:10-1:1, and the pH range of the acidic solution is 1-6.
[0039] The iron(II,III) oxide@polypyrrole / noble metal ion functionalized nanocomposite material of this invention can be used as a magnetic solid-phase extraction (MSPE) adsorbent to achieve rapid and selective MSPE extraction of pterin compounds in complex samples. The process of selective MSPE extraction of pterin compounds using this functionalized nanocomposite material includes three core steps: activation, adsorption, and elution. The entire operation requires no complex equipment and is rapid and efficient. This characteristic is particularly suitable for the rapid extraction of easily oxidized and degraded pterin compounds and their polymers, maximizing the preservation of the structural integrity of the target analytes.
[0040] Before adsorbing pterin compounds from a sample, the functionalized nanocomposite material needs to be activated to remove impurities remaining from the material preparation and storage process, while simultaneously wetting the material surface and exposing the coordination sites of noble metal ions to ensure adsorption efficiency. The specific activation method is as follows: Weigh 10-20 mg of the functionalized nanocomposite material into a centrifuge tube, add 0.5-5 mL of the first activation solution, vortex mix for 2-3 min, then place the centrifuge tube in an external magnetic field with a strength of 0.1-0.5 T and let it stand for 5-10 min to achieve solid-liquid separation, discarding the activation waste liquid; subsequently, add 0.5-5 mL of the second activation solution to the centrifuge tube, repeating the vortex mixing, magnetic response separation, and waste liquid discarding process until the functionalized nanocomposite material reaches a state capable of efficiently adsorbing pterin compounds. The first activation solution is selected from one or more combinations of methanol, ethanol, formic acid, and acetic acid, and the second activation solution is selected from one or more combinations of methanol, ethanol, and water. The purpose of activation is to remove impurities and debris remaining during the preparation and storage of nanocomposite materials to avoid contaminating the target sample; to fully wet the polypyrrole modification layer and metal ion active sites on the surface of the nanocomposite materials to form the interfacial environment required for adsorption; and to activate the surface-specific adsorption groups by sequential rinsing with methanol and water to enhance the adsorption capacity and selectivity for pterin compounds.
[0041] Adsorption involves the selective binding of pterin compounds in complex samples or their treatment solutions using functionalized nanocomposites. The specific steps are as follows: 0.5-1 mL of the sample or its treatment solution is added to the activated functionalized nanocomposites, and the mixture is stirred at 100-300 r / min for 10-30 min at room temperature. Utilizing the π-π stacking, hydrogen bonding, and selective chelation between noble metal ions and the -NH2 at the 4-position and the N atom of the heterocyclic ring at the 5-position of the pterin molecules on the surface of the polypyrrole layer of the material, the pterin compounds in the sample or its treatment solution are fully adsorbed onto the surface of the functionalized nanocomposites. After adsorption, the centrifuge tube is placed in an external magnetic field and allowed to stand for 5-10 min to achieve solid-liquid separation. The supernatant is then discarded, completing the enrichment of the target analyte.
[0042] The eluent consists of an acidic solution and an organic solvent. The acidic solution is selected from compounds that can form acidic solutions, such as sulfuric acid, hydrochloric acid, formic acid, and acetic acid. The organic solvent is selected from one or more combinations of methanol, ethanol, and acetonitrile. The mass fraction of the acidic solution is 1%-10%, the volume ratio of the acidic solution to the organic solvent is 1:10-1:1, and the pH range of the acidic solution is 1-6. Elution is the efficient desorption of pterin compounds adsorbed on the functionalized nanocomposite material. Specifically, through vortex soaking, shaking rinsing, etc., the coordination equilibrium is controlled by the acidic environment, causing the chelate between noble metal ions and pterin molecules to dissociate, allowing the target analyte to quickly detach from the material surface and enter the eluent. After desorption, the centrifuge tube is placed in an external magnetic field and allowed to stand for 5-10 minutes to achieve solid-liquid separation and collect the eluent, which can then be used for subsequent detection and analysis.
[0043] The functionalized nanocomposite material of this invention selectively separates and enriches pterinary compounds in complex samples. The amount used depends on the content of pterinary compounds in the sample; if the content is high, the amount of material should be increased appropriately. In complex samples to be detected (such as biological samples like blood and urine), pterinary compounds are usually present at trace levels (i.e., parts per million or even lower). For such trace samples or their processing solutions, generally 5 mg of functionalized nanocomposite material is weighed and adsorbed through the above-mentioned magnetic solid-phase extraction process, followed by elution with 0.2-5 mL of eluent to ensure a high recovery rate of the target analyte. For analytical applications involving complex biological samples, to achieve quantitative recovery of the target analyte, the adsorption operation is usually performed at a ratio of 0.1-0.5 mL of biological sample or its processing solution to 5 mg of functionalized nanocomposite material.
[0044] As can be seen, when the functionalized nanocomposite material of the present invention is used as an adsorbent, its dosage is extremely small. This is because the material is based on nano-sized iron oxide, and the polypyrrole layer coated on the surface and the noble metal ions loaded form abundant active sites. The specific surface area is significantly higher than that of traditional particulate adsorbents. Generally, only 1 / 10 to 1 / 100 of the dosage of existing particulate adsorbents (the volume of the stationary phase is significantly reduced) is used to achieve the same or even better extraction effect, and the adsorption and extraction efficiency is greatly improved.
[0045] In addition, the amount of eluent used in the above process is extremely small, only 0.5-5 mL is needed to achieve efficient desorption of the target analyte. There is no need to perform the solvent evaporation and concentration operation of the eluent in conventional solid phase extraction. This not only reduces energy consumption, but also reduces the risk of oxidation and damage of the target analyte during the concentration process, further ensuring the accuracy of the detection results.
[0046] Example 1: Selective magnetic solid-phase extraction and detection of methotrexate in real urine Methotrexate, a commonly used pterin chemotherapy drug in clinical practice, is of great significance for the trace detection of in biological samples for therapeutic drug monitoring. Real urine has a complex matrix, containing interfering components such as proteins and metabolic waste. Traditional separation methods suffer from problems such as cumbersome operation, low separation efficiency, and high target analyte loss. This embodiment uses the Fe3O4@ppy / Pd(II) functionalized nanocomposite material prepared in this patent as an adsorbent. Its superparamagnetism enables rapid solid-liquid separation. Combined with the selective chelation of Pd(II) with the -NH2 at the 4-position and the heterocyclic N atom at the 5-position of the methotrexate molecule, highly efficient separation of spiked methotrexate in real urine is achieved. Quantitative analysis is performed using HPLC-UV detection. The specific steps are as follows: Step 1: Preparation of Fe3O4@ppy / Pd(Ⅱ) functionalized nanocomposites (1) Preparation of the core layer of iron(III) oxide nanoparticles: 0.4 g of ferrous chloride (FeCl2·4H2O) and 1.1 g of ferric chloride (FeCl3·6H2O) were weighed and placed in a 250 mL three-necked flask at a ferrous salt to ferric salt molar ratio of 1:1.8. 100 mL of deionized water was added, and nitrogen (N2) gas was introduced for 30 min to remove oxygen from the system. The flask was placed in a 65℃ constant temperature water bath and magnetically stirred at a rate of 550 r / min. 1.8 mol / L sodium hydroxide solution was rapidly added dropwise to adjust the pH of the system to 9.5, and the mixture was kept at this temperature for 1.2 h. After the reaction was completed, the reaction solution was placed in an external magnetic field for static separation. The precipitate was washed alternately with deionized water and anhydrous ethanol until the filtrate was neutral. The precipitate was then vacuum dried at 55℃ for 5 h to obtain the core layer of iron(III) oxide nanoparticles (e.g., ferric oxide nanoparticles). Figure 1 (a) is shown.
[0047] (2) Coating with Modification Layer I: 50 mg of the above-mentioned iron oxide nanoparticle core layer was added to 200 mL of pure water and ultrasonically dispersed for 15 min to prepare a uniform suspension; after cooling the suspension to 5℃, 0.675 mL of pyrrole monomer was added, and the mixture was stirred at 100 r / min for 30 min to allow the monomer to be fully adsorbed; then 25 mL of 0.5 mol / L ferric chloride solution (initiator) was slowly added dropwise, with the dropping rate controlled at 0.3 mL / s, and stirring was continued for 24 h to carry out the in-situ polymerization reaction; after the reaction, magnetic response separation was performed, and the mixture was repeatedly washed with deionized water until the washing liquid was colorless, and dried at 50℃ for 24 h to obtain iron oxide@polypyrrole composite material (modified only with modification layer I, such as...). Figure 1 (b) is shown.
[0048] (3) Loading of Modification Layer II: A suitable amount of palladium chloride (PdCl2) was dissolved in 0.1 mol / L hydrochloric acid solution and ultrasonically dispersed for 20 min. The palladium chloride concentration was adjusted to 200 mg / L to prepare Modification Solution II. 50 mg of the above-mentioned Fe3O4@ppy / Pd(II) functionalized nanocomposite material was immersed in 2 mL of Modification Solution II and stirred at 150 r / min for 60 min. Subsequently, the supernatant was discarded after magnetic response separation. The material was dried in a 50℃ oven to remove moisture, yielding Fe3O4@ppy / Pd(II) functionalized nanocomposite material (Modification Layer I + Modification Layer II, as shown in the image). Figure 1 (c) and Figure 1 (d) is shown.
[0049] Step 2: Preparation of real urine spiked samples Take 5 mL of real urine from healthy volunteers, place it in a centrifuge tube, centrifuge at 3000 r / min for 5 min, discard the precipitate to remove insoluble impurities, and use the supernatant as the urine matrix; accurately transfer an appropriate amount of methotrexate standard stock solution (100 μg / mL) to the urine matrix to prepare a real urine spiked sample with a methotrexate concentration of 500 ng / mL, and use it immediately after preparation.
[0050] Step 3: Selective magnetic solid phase extraction operation (1) Material activation: Weigh 5 mg of Fe3O4@ppy / Pd(Ⅱ) functionalized nanocomposite material and place it in a 5 mL centrifuge tube. Add 0.5 mL of methanol (first activation solution), vortex mix for 2 min, and then place the centrifuge tube in an external magnetic field with a magnetic field strength of 0.3 T for 8 min to achieve solid-liquid separation. Discard the activation waste liquid. Then add 0.5 mL of deionized water (second activation solution), repeat the vortex mixing and magnetic response separation operation, discard the waste liquid, and complete the material activation. Figure 2 The material has been shown to have good magnetic responsiveness, enabling rapid solid-liquid separation.
[0051] (2) Adsorption: Add 0.5 mL of real urine spiked sample to the activated material and stir at 100 r / min for 60 min at room temperature to form a stable chelate structure between methotrexate molecules and Pd(II) on the surface of the material. After stirring, place it in an external magnetic field and let it stand for 8 min. After separation, discard the supernatant and retain the composite material adsorbed with the target substance.
[0052] (3) Elution: Add 0.2 mL of elution buffer (acetonitrile-pH1 formic acid solution, volume ratio 3:7) to the centrifuge tube, vortex mix for 3 min, and use the acidic environment and organic molecules to destroy the chelation structure to desorb methotrexate; place it in an external magnetic field again and let it stand for 8 min, collect the elution buffer after separation, filter it through a 0.22 μm organic phase filter membrane, and use it for later use.
[0053] Step 4: High Performance Liquid Chromatography-Ultraviolet Detection High-performance liquid chromatography (HPLC) with a UV detector was used for detection; the chromatographic column was a C18 column (250 mm × 4.6 mm, 5 μm); the mobile phase was methanol-acetonitrile-0.01 mol / L phosphate buffer (pH 7.4), with a volume ratio of 2:7:91; the flow rate was 0.8 mL / min; the detection wavelength was 313 nm; the column temperature was 30℃; and the injection volume was 20 μL. Separately, 10 μg / mL methotrexate standard solution (e.g.,...) was used for detection. Figure 3 (a) Real urine sample mixed with methotrexate standard solution (e.g.) Figure 3 (b)) and the above-mentioned elution solution (e.g.) Figure 3 (c) Perform sample injection and testing.
[0054] Results Analysis: Figure 3 It can be seen that, Figure 3 (c) The target peak (methotrexate) is sharp and clear, the baseline is stable, and there are no obvious matrix interference peaks, which proves that the Fe3O4@ppy / Pd(Ⅱ) functionalized nanocomposite material prepared in this patent can selectively extract methotrexate from real urine using magnetic solid phase extraction.
[0055] Example 2: Selective magnetic solid-phase extraction and detection of neopterin, isoxopterin, biopterin, and pterin in real urine. Neopterin, isoxopterin, biopterin, and pterin are endogenous pterin compounds, and their levels in urine are closely related to the body's immune function and disease state. The content of these endogenous pterins in real urine is extremely low, and matrix interference is complex, requiring efficient extraction and separation for accurate detection. This embodiment uses the Fe3O4@ppy / Ag(Ⅰ) functionalized nanocomposite material of this invention for magnetic solid-phase extraction, combined with the high sensitivity of high-performance liquid chromatography-fluorescence detection, to achieve the separation, extraction, and detection of three endogenous pterins in real urine. The specific steps are as follows: Step 1: Preparation of Fe3O4@ppy / Ag(Ⅰ) functionalized nanocomposites The preparation method is the same as step 1 in Example 1, wherein (3) Loading of Modification Layer II: Weigh an appropriate amount of silver nitrate (AgNO3) and dissolve it in pure water. Adjust the AgNO3 concentration to 200 mg / L to prepare modification solution II. Take 50 mg of the above iron(III) oxide@polypyrrole composite material and soak it in 2 mL of modification solution II. Stir at 150 r / min for 60 min. Then, magnetic response separation is performed to remove the supernatant. Place the material in a 50℃ oven to dry and remove moisture to obtain Fe3O4@ppy / Ag(I) functionalized nanocomposite material.
[0056] Step 2: Preparation of real urine samples Take 10 mL of real urine from healthy volunteers, centrifuge at 3000 r / min for 5 min, and discard the precipitate; take the supernatant and filter it with a 0.45 μm aqueous filter membrane to remove soluble macromolecular impurities, and obtain real urine treatment solution.
[0057] Step 3: Selective magnetic solid phase extraction operation (1) Material activation: Weigh 10 mg of Fe3O4@ppy / Ag(Ⅰ) functionalized nanocomposite material and place it in a 5 mL centrifuge tube. Add 0.5 mL of methanol (first activation solution), vortex mix for 3 min, and then separate by magnetic response. Discard the waste liquid. Add 0.5 mL of deionized water (second activation solution), repeat vortex mixing and magnetic response separation, and discard the waste liquid to complete the activation.
[0058] (2) Adsorption: Add 0.5 mL of real urine treatment solution to the activated material and stir at 100 r / min for 25 min at room temperature to allow neopterin, isoxopterin, biopterin and pterin in the urine to specifically chelate with Ag(Ⅰ) on the surface of the material; discard the supernatant after magnetic response separation.
[0059] (3) Elution: Add 0.2 mL of eluent (acetonitrile-pH1 formic acid solution, volume ratio 3:7), vortex mix for 5 min, collect the eluent after magnetic response separation, filter through a 0.22 μm organic phase filter membrane, and set aside for later use.
[0060] Step 4: High Performance Liquid Chromatography-Fluorescence Detection High-performance liquid chromatography (HPLC) with a fluorescence detector was used for detection; the column was Inertsil ODS-3 (250 mm × 4.6 mm, 5 μm); the mobile phase was methanol-0.015 mol / L phosphate buffer (pH 7.2), v / v ratio 99.5:0.5; the flow rate was 1 mL / min; the fluorescence detection parameters were Ex / Em = 350 / 450 nm; the column temperature was 30℃; and the injection volume was 10 μL. 500 ng / mL neopterin, isoxopterin, biopterin, and a mixed pterin standard solution (e.g.,...) were used for detection. Figure 4 (a)), Real urine (e.g.) Figure 4 (b)) and the above-mentioned elution solution (e.g.) Figure 4 (c) Perform sample injection and testing.
[0061] Results Analysis: Figure 4 It can be seen that, Figure 4 (c) can be clearly detected as Figure 4(a) The four target peaks (neopterin, isoxopterin, biopterin, and pterin) corresponding to the mixed standard solution have symmetrical peak shapes, few interfering peaks, and good separation, proving that the Fe3O4@ppy / Ag(Ⅰ) functionalized nanocomposite material prepared in this patent can effectively separate and enrich trace amounts of endogenous pterin compounds in real urine. Combined with high performance liquid chromatography-fluorescence detection, it can achieve accurate quantification, providing a reliable pretreatment solution for the diagnosis and monitoring of clinically related diseases.
[0062] Example 3: Highly Selective Magnetic Solid Phase Extraction and Detection of Methotrexate in Real Urine In this embodiment, rhodium chloride is used as the metal compound of the modification layer II, and 0.1 M hydrochloric acid is used as solvent A to prepare Fe3O4@ppy / Rh (III) nanocomposite material for highly selective magnetic solid phase extraction of methotrexate in real urine. The reaction conditions are adjusted to reflect the flexibility of the process.
[0063] Step 1: Preparation of Fe3O4@ppy / Rh (Ⅲ) nanocomposite material. (1) Preparation of Fe3O4 nanoparticle core layer: Same as in Example 1. (2) Coating with modification layer I: Same as in Example 1, to obtain Fe3O4@ppy / Rh (Ⅲ) nanocomposite material. (3) Loading with modification layer II: Weigh an appropriate amount of rhodium chloride and dissolve it in 0.1 M hydrochloric acid solution (solvent A), ultrasonically disperse for 20 min, adjust the concentration of metal compound to 200 mg / L, and prepare modification solution II; take 50 mg of Fe3O4@ppy / Rh (Ⅲ) material and soak it in 2 mL of modification solution II, stir at room temperature for 50 min; after separation by external magnetic field, dry at 45 ℃ to obtain Fe3O4@ppy / Rh (Ⅲ) nanocomposite material.
[0064] Step 2: Preparation of real urine spiked samples is the same as in Example 1, preparing urine samples spiked with methotrexate at a concentration of 500 ng / mL.
[0065] Step 3: The activation conditions for the high-selectivity magnetic solid phase extraction operation material are the same as in Example 1; the adsorption conditions are adjusted to: stirring at 100 r / min for 50 min at room temperature; the elution conditions are the same as in Example 1.
[0066] Step 4: HPLC-UV detection. Detection conditions are the same as in Example 1.
[0067] The results showed that the Fe3O4@ppy / Rh(Ⅲ) nanocomposite material achieved a relative recovery of 93.2% for methotrexate in urine spiked (500 ng / mL), with a relative standard deviation (RSD) of 3.5% for three parallel determinations. The calculated saturated adsorption capacity of the material was 31.8 mg / g. High-performance liquid chromatography-ultraviolet (HPLC-UV) chromatography revealed that the methotrexate target peak was symmetrical, free from interference from impurity peaks, and the matrix effect was negligible. These data demonstrate that the Fe3O4@ppy / Rh(Ⅲ) nanocomposite material remains highly effective for the highly selective magnetic solid-phase extraction of pterin compounds, reflecting the flexibility of the process described in this invention.
[0068] Example 4: Highly selective magnetic solid-phase extraction and detection of neopterin, isoxopterin, biopterin, and pterin in real urine. In this embodiment, chloroauric acid is used as the metal compound of the modification layer II, and pure water is used as solvent A to prepare Fe3O4@ppy / Au (Ⅲ) nanocomposite material for highly selective magnetic solid phase extraction of endogenous pterin compounds in real urine. The reaction conditions are fine-tuned to reflect the process adaptability.
[0069] Step 1: Preparation of Fe3O4@ppy / Au (Ⅲ) functionalized nanocomposite material. (1) Preparation of Fe3O4 nanoparticle core layer: Same as in Example 1. (2) Coating with modification layer I: Same as in Example 1, to obtain Fe3O4@polypyrrole composite material. (3) Loading with modification layer II: Weigh an appropriate amount of chloroauric acid and dissolve it in pure water (solvent A), ultrasonically disperse for 20 min, adjust the concentration to 200 mg / L to prepare modification solution II; take 50 mg of Fe3O4@polypyrrole material and soak it in 2 mL of modification solution II, stir at room temperature for 50 min; after separation by an external magnetic field, dry at 45℃ to obtain Fe3O4@ppy / Au (Ⅲ) functionalized nanocomposite material.
[0070] Step 2: Preparation of real urine samples is the same as in Example 2.
[0071] Step 3: The activation conditions for the high-selectivity magnetic solid phase extraction operation material are the same as in Example 2; the adsorption conditions are adjusted to: stirring at 100 r / min for 30 min at room temperature; the elution conditions are the same as in Example 2.
[0072] Step 4: HPLC-fluorescence detection. Detection conditions are the same as in Example 2.
[0073] The results showed that the recoveries of the four target compounds by the Fe3O4@ppy / Au(Ⅲ) material were: neopterin 91.5%, isoxopterin 88.3%, biopterin 94.1%, and pterin 89.7%, with RSDs of less than 4.2% for three repeated determinations. The four pterins showed good separation in HPLC-fluorescence detection (R>1.5). These data demonstrate that the Fe3O4@ppy / Au(Ⅲ) nanocomposite material still exhibits excellent selective extraction performance for pterin compounds, further validating the variability and adaptability of the process of this invention.
Claims
1. A selective magnetic solid-phase extraction material for pterin compounds, characterized in that, From the center outwards, it includes a core layer of iron oxide nanoparticles, a modification layer I, and a modification layer II. The core layer of iron oxide nanoparticles consists of iron oxide nanoparticles. The modification layer I is obtained by in-situ polymerization of pyrrole monomers onto the surface of the iron oxide nanoparticle core layer under the action of an initiator. The modification layer II is obtained by dissolving a metal compound in solvent A to obtain a modification liquid, and coating the surface of modification layer I with the metal compound in the modification liquid.
2. The selective magnetic solid-phase extraction material for pterin compounds according to claim 1, characterized in that, The core layer of the iron oxide nanoparticles has a particle size of 2-50 nm and a crystallinity of 70%-95%.
3. The selective magnetic solid-phase extraction material for pterin compounds according to claim 1, characterized in that, The raw materials for preparing the modified layer I include pyrrole monomer, initiator and solvent; the initiator is ferric chloride solution; the mass ratio of pyrrole monomer to iron oxide nanoparticle core layer is 1:5-10, and the molar ratio of initiator to pyrrole monomer is 2-3:
1.
4. The selective magnetic solid-phase extraction material for pterin compounds according to claim 1, characterized in that, The metal compound of the modified layer II is a chloride, a metal complex acid, or a metal nitrate; the metal chloride is one or a mixture of palladium chloride and rhodium chloride; the metal complex acid is one or a mixture of chloroauric acid, chloroplatinic acid, and chloropalladium acid; the metal nitrate is silver nitrate; the solvent A is a 0.1 M hydrochloric acid solution or pure water, and the concentration of the metal compound in the solvent is 10-500 mg / L.
5. A method for preparing a selective magnetic solid-phase extraction material for pterin compounds according to any one of claims 1 to 4, characterized in that, Includes the following steps: Step 1, Preparation of Modification Layer I: Modification layer I is obtained by in-situ polymerization of pyrrole monomers on the surface of the core layer of iron oxide nanoparticles under the action of an initiator, thus obtaining iron oxide@polypyrrole intermediate; Step 2, Preparation of Modification Layer II: The metal compound is dissolved in solvent A to obtain a modification solution. The metal compound in the modification solution is coated on the surface of modification layer I to obtain modification layer II, thus obtaining a surface-functionalized nanocomposite material.
6. The application of the selective magnetic solid-phase extraction material for pterin compounds according to any one of claims 1-4 in the selective extraction of pterin compounds from complex samples.
7. The application according to claim 6, characterized in that, Includes the following steps: Step 1: After obtaining the sample, quickly freeze or process the sample to obtain the test solution or the processing solution; Step 2: Take the pterin-based compound selective magnetic solid-phase extraction material and activate it sequentially with the first activation solution and the second activation solution to obtain the activated magnetic nanocomposite material. Step 3: Add the activated magnetic nanocomposite material to the test solution or treatment solution obtained in Step 1, and stir at room temperature to allow the pterin compounds in the sample to be fully adsorbed onto the surface of the nanocomposite material. Step 4: Place the adsorption system in an external magnetic field and let it stand to allow the pterin selective magnetic solid phase extraction material containing pterin compounds to quickly separate from the sample matrix, and discard the supernatant. Step 5: Add eluent to the separated pterin compounds selective magnetic solid-phase extraction material, stir and elute at room temperature, then place it in an external magnetic field again to achieve solid-liquid separation, collect the eluent, and complete the selective magnetic solid-phase extraction of pterin compounds.
8. The application according to claim 7, characterized in that, The specific activation method for the selective magnetic solid-phase extraction material of pterin compounds in step 2 is as follows: Place the nanocomposite material in a centrifuge tube, add the first activation solution, mix, and then separate it in an external magnetic field, discarding the activation waste liquid; then add the second activation solution to the centrifuge tube, perform mixing, magnetic response separation, and discard the waste liquid to complete the activation; the liquid-to-solid ratio of the first activation solution to the nanocomposite material is 5-10 mL:2-10 mg, and the liquid-to-solid ratio of the second activation solution to the nanocomposite material is the same as that of the first activation solution; the stirring rate in step 3 is 100-300 r / min; the eluent in step 5 is a mixed solution of acetonitrile and formic acid with a volume ratio of 70:30, and the liquid-to-solid ratio of the eluent to the nanocomposite material is 0.2-5 mL:2-10 mg; the magnetic field strength of the external magnetic field is 0.1-0.5 T.
9. The application according to claim 7, characterized in that, The first activating solution is selected from one or more of methanol, ethanol, formic acid, and acetic acid; the second activating solution is selected from one or more of methanol, ethanol, and water.
10. The application according to claim 7, characterized in that, The eluent is composed of an acidic or alkaline solution and an organic solvent. The acidic solution is selected from sulfuric acid, hydrochloric acid, formic acid, and acetic acid; the alkaline solution is selected from ammonia water and sodium hydroxide solution; the organic solvent is selected from methanol, ethanol, and acetonitrile, or any combination of two or more of them. The mass fraction of the acidic solution is 1% to 10%, the mass fraction of the alkaline solution is 1% to 10%, the volume ratio of the acidic solution to the organic solvent is 1:10 to 1:1, the volume ratio of the alkaline solution to the organic solvent is 1:10 to 1:1, the pH range of the acidic solution is 1 to 6, and the pH range of the alkaline solution is 7 to 11.