An estrogen-like substance-enriched material and a method for analyzing the same

Abstract

The present application belongs to the technical field of analysis and detection, and particularly relates to an enrichment material of estrogen substances and an analysis method thereof. The enrichment material of estrogen substances (Fe3O4@SiO2@CMP-SO3Na) comprises a magnetic carrier and an ionic conjugated microporous polymer loaded on the surface of the magnetic carrier. The composite material exhibits high adsorption capacity, fast adsorption kinetics and excellent reusability by virtue of the synergistic effect of the conjugated system-ion group-porous structure. On this basis, the present application first establishes an MSPE-HPLC-MS / MS combined analysis method based on Fe3O4@SiO2@CMP-SO3Na, and successfully applies the method to efficient enrichment and accurate detection of trace EEs in food samples (such as honey, dairy products) and environmental water (such as surface water, wastewater).

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to an enrichment material for estrogen-like substances and its analytical method. Background Technology

[0002] Estrogenous compounds (EEs), typical endocrine disruptors, mainly include natural steroidal estrogens (such as estrone (E1), 17β-estradiol (E2), and estriol (E3)) and synthetic estrogen derivatives (such as diethylstilbestrol (DES) and ethinylestradiol (EE)). These substances play important physiological functions in organisms, participating in the regulation of endocrine signaling pathways, hormone synthesis, and metabolism. However, with the widespread use in livestock farming and the pharmaceutical industry, EEs continuously enter the environment through wastewater discharge and medical waste, and are frequently detected in surface water, groundwater, and various agricultural and livestock products. Studies have found that EEs can produce significant biological effects at trace levels (ng / L-μg / L). By interfering with the normal function of the endocrine system, they may lead to various health problems such as metabolic disorders, cardiovascular disease, and diabetes, and increase the risk of cancer. In aquatic ecosystems, long-term exposure to EEs can lead to serious ecological consequences such as reproductive abnormalities and population decline, threatening biodiversity and ecosystem stability.

[0003] Currently, detection techniques for EEs mainly include enzyme-linked immunosorbent assay (ELISA), immunochromatography, electrochemical analysis, and chromatography-mass spectrometry (GC-MS). Among these, high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) has become the standard analytical method for EE detection due to its excellent separation capabilities and high sensitivity. However, the concentration of EEs in real-world environmental samples is extremely low (typically in the ng / L-μg / L range), and the complex matrix results in severe matrix interference, making direct injection analysis difficult to meet detection requirements. Therefore, developing efficient and reliable sample pretreatment techniques to achieve effective enrichment and purification of target analytes has become a key step in improving the accuracy and sensitivity of EE detection.

[0004] Magnetic solid-phase extraction (MSPE) is the core of solid-phase extraction technology. It uses magnetic materials as adsorbents and achieves rapid separation with the help of an external magnetic field, avoiding the cumbersome steps of centrifugation and filtration in traditional solid-phase extraction techniques. It offers advantages such as simple operation, high enrichment efficiency, low organic solvent consumption, and minimal clogging, showing promising application prospects in trace pollutant analysis. Magnetic nanomaterials are key to improving their selectivity and adsorption capacity for target analytes. Ionic conjugated microporous polymers (i-CMPs) are a class of multifunctional materials with permanent porosity, extended π-conjugated frameworks, and ionic sites. Their unique structural characteristics, such as tunable pore size distribution, abundant π-electron systems, and ionic functional groups in the framework, enable them to bind to target analytes through various interactions, including electrostatic interactions, π-π stacking, and ion-dipole interactions, giving them potential advantages in adsorption separation. However, current adsorption materials used for EE enrichment still face many bottlenecks and challenges in practical applications: First, the poor mass transfer kinetics of the materials prolong the extraction equilibrium time, severely limiting the overall efficiency of the detection process; second, it is difficult to achieve synergistic optimization of the material's structural stability and regeneration performance, leading to structural collapse and loss of active sites after adsorption-desorption cycles, resulting in a significant decrease in adsorption capacity. Furthermore, existing research has not yet provided an in-depth and systematic analysis of the adsorption mechanism between the materials and EEs, directly causing a significant deviation between the actual extraction effect and theoretical expectations, severely restricting their practical application and promotion in trace EE analysis. Summary of the Invention

[0005] The purpose of this invention is to provide an enrichment material for estrogen-like substances and its analytical method, thereby overcoming the shortcomings of the prior art. For the first time, an MSPE-HPLC-MS / MS coupled analytical method based on Fe3O4@SiO2@CMP-SO3Na has been established and successfully applied to the efficient enrichment and accurate detection of trace EEs in food samples (such as honey and dairy products) and environmental water bodies (such as surface water and wastewater).

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides an estrogen-like substance enrichment material, comprising a magnetic carrier and an ionic conjugated microporous polymer loaded on its surface.

[0008] The magnetic carrier has a core-shell structure, including an Fe3O4 core and a SiO2 shell covering the surface of the Fe3O4 core;

[0009] Ionic conjugated microporous polymers have the following repeating structural units:

[0010] .

[0011] In some other embodiments, the estrogen-rich material has a three-dimensionally interconnected network porous structure with a specific surface area >158.0 m². 2 ·g -1 The average pore size is 3.5-4.0 nm.

[0012] In some other embodiments, the method for preparing estrogen-rich materials includes the following steps:

[0013] The magnetic support is added to a solvent and dispersed evenly. Then, 2,4,6-tris(4-ethynylphenyl)-1,3,5-triazine, 1,4-dibromo-2,5-bis(3-sulfopropoxy)benzene and a catalyst are added, and a coupling reaction is carried out under a protective atmosphere to obtain the final product.

[0014] In some other embodiments, the magnetic carrier is prepared as follows: ferric chloride is added to ethylene glycol and mixed, then sodium acetate and citric acid are added and mixed, followed by hydrothermal reaction. After washing and drying, Fe3O4 microspheres are obtained.

[0015] Fe3O4 microspheres were added to a mixed solvent of ethanol, deionized water and ammonia, and tetraethyl orthosilicate was added dropwise for stirring and reaction. After magnetic separation, washing and drying, Fe3O4@SiO2 magnetic carrier was obtained.

[0016] Specifically, in the preparation method of Fe3O4 microspheres, the mass ratio of ferric chloride, sodium acetate and citric acid is (3-3.5):1:6; the hydrothermal reaction temperature is 190-210℃ and the time is 20-30 h; the solvent used for washing is ethanol and deionized water; and the drying is carried out under vacuum at 55-65℃ for 5-7 h.

[0017] In the preparation method of Fe3O4@SiO2 magnetic carrier, the volume ratio of ethanol, deionized water and ammonia is (28-22):3:1; the volume concentration of ammonia is 20-30%; 4-6 L of mixed solvent is added per gram of Fe3O4 microspheres; the volume ratio of mixed solvent to tetraethyl orthosilicate is (400-600):1; the stirring reaction temperature is room temperature and the time is 5-7 h; the solvent used for washing is ethanol and deionized water; drying is carried out under vacuum at 55-65℃ for 5-7 h.

[0018] In some other embodiments, the solvent is a mixture of toluene and triethylamine, wherein the volume ratio of toluene to triethylamine is 1:(2-2.2);

[0019] The mass ratio of 2,4,6-tris(4-ethynylphenyl)-1,3,5-triazine, 1,4-dibromo-2,5-bis(3-sulfopropoxy)benzene to the catalyst is (4-4.5):(14-18):1.

[0020] Specifically, the volume ratio of toluene to triethylamine is 1:2, 1:2.1, or 1:2.2;

[0021] The mass ratio of 2,4,6-tris(4-ethynylphenyl)-1,3,5-triazine, 1,4-dibromo-2,5-bis(3-sulfopropoxy)benzene to the catalyst is 4:14:1, 4:18:1, 5:14:1, or 5:18:1.

[0022] In some other embodiments, the catalyst is a mixture of dichlorobis(triphenylphosphine)palladium and cuprous iodide, wherein the mass ratio of dichlorobis(triphenylphosphine)palladium to cuprous iodide is (3.5-3.6):1;

[0023] The coupling reaction is carried out at a temperature of 80-100℃ for 20-30 hours.

[0024] Specifically, the mass ratio of dichlorobis(triphenylphosphine)palladium and cuprous iodide is 3.5:1, 3.55:1, or 3.6:1;

[0025] The coupling reaction is carried out at a temperature of 80, 90 or 100 °C for 20, 24 or 30 h.

[0026] Secondly, the present invention provides an analytical method for estrogen-like substances, comprising the following steps:

[0027] The estrogen-like substance enrichment material described in the first aspect is used as an adsorbent, dispersed in the sample to be tested for adsorption, the adsorbent is separated by an external magnetic field, desorption solvent is added for desorption, the eluent is collected and dried with nitrogen to obtain the residue, which is then reconstituted, filtered and analyzed by liquid chromatography-tandem mass spectrometry.

[0028] In some other embodiments, the estrogenic compound is one or more of estrone, 17β-estradiol, estriol, ethinylestradiol, and hexanestilbene.

[0029] Materials for enriching estrogen-like substances possess unique characteristics, including an extended π-conjugated system, a permanent microporous structure, and embedded ionic groups within the framework. This structural design enables the construction of charged transport channels within a nanoscale confined space, which not only improves ion transport efficiency but also significantly enhances the electrostatic affinity for polar analytes through ion-ion, ion-dipole, and dipole-dipole interactions, thus facilitating the efficient enrichment of EEs.

[0030] In some other implementations, the sample to be tested is water, honey, or milk powder;

[0031] The pH of the sample should be 2-10; 5-25 L of sample should be added per gram of adsorbent; the adsorption time should be 0.5-3 min.

[0032] The desorption solvent is one of methanol, acetonitrile, and dichloromethane; the volume ratio of the desorption solvent to the sample is (1-4):50; the desorption time is 2-5 min.

[0033] In some other embodiments, the sample to be tested needs to be pretreated before magnetic solid-phase extraction. Specifically, the pretreatment method is as follows:

[0034] When the sample to be tested is water, the water is filtered and then refrigerated.

[0035] When the sample to be tested is honey, simply dissolve the honey in water and filter it.

[0036] When the sample to be tested is milk powder, the milk powder is dissolved in water, acetonitrile is added, and the supernatant and residue are collected by extraction and centrifugation. Methanol is added to the residue, and the supernatant is collected by extraction and centrifugation. The two supernatants are combined, dried by nitrogen blowing, and then reconstituted with water to obtain the final product.

[0037] More specifically, when the sample to be tested is water, the filter membrane used for filtration has a pore size of 0.2-0.3 μm, and the refrigerated storage temperature is 3-5℃;

[0038] When the sample to be tested is honey, add 8-12 mL of water per gram of honey, vortex for 4-6 min, sonicate for 8-12 min, and then filter with a filter membrane with a pore size of 0.2-0.3 μm.

[0039] When the sample to be tested is milk powder, add 5-7 mL of water per gram of milk powder, vortex for 4-6 min, add 8-12 mL of acetonitrile to precipitate the protein, vortex for 4-6 min, then sonicate for 8-12 min, then centrifuge at 10,000-15,000 rpm for 8-12 min, collect the supernatant, add 3-6 mL of methanol to the residue, vortex for 1-3 min, then sonicate for 4-6 min a second time, centrifuge, combine the two supernatants, blow dry with nitrogen under a 30-40℃ water bath, and finally redissolve the residue with 40-60 mL of water.

[0040] In some other embodiments, the liquid chromatography column is a Waters XBridge BEH C18 column, mobile phase A is an aqueous solution of ammonium acetate, and mobile phase B is methanol. The elution program is as follows: 0–1.0 min, methanol volume ratio linearly increases from 0% to 60%; 1.0–5.0 min, methanol volume ratio linearly increases from 60% to 80%; 5.1–8.0 min, isocratic elution is performed while maintaining a methanol volume ratio of 60%, and the flow rate is 0.2–0.4 mL / min. -1 The column temperature is 35-37℃, and the injection volume is 4-6 μL;

[0041] The ion source for mass spectrometry analysis is electrospray ionization in negative ion mode; the scanning mode is multiple reaction monitoring mode, and the ion source temperature is 450-550℃.

[0042] Specifically, the concentration of mobile phase A (ammonium acetate aqueous solution) in the liquid chromatography is 8-12 mmol·L⁻¹. -1 The flow rate is 0.2, 0.3, or 0.4 mL / min. -1 The column temperature is 35℃, and the injection volume is 4, 5, or 6 μL.

[0043] The ion source temperature for mass spectrometry analysis was 450, 550, or 550 °C. The estrogenic compounds included estrone, 17β-estradiol, estriol, ethinylestradiol, and hexanestilbene. Specific mass spectrometry parameters are shown in Table 1.

[0044] The beneficial effects of this invention are:

[0045] Based on the polar phenolic hydroxyl functional groups in the molecular structure of EEs, it is hypothesized that i-CMPs can form a strong adsorption affinity with them through electrostatic interactions. This invention successfully prepared a sodium sulfonate-functionalized magnetic conjugated microporous polymer composite material (Fe3O4@SiO2@CMP-SO3Na). This composite material exhibits high adsorption capacity, rapid adsorption kinetics, and excellent reusability through the synergistic effect of the conjugated system, ionic groups, and porous structure. Building upon this, this study established for the first time an MSPE-HPLC-MS / MS method based on Fe3O4@SiO2@CMP-SO3Na, and successfully applied it to the efficient enrichment and accurate detection of trace EEs in food samples (such as honey and dairy products) and environmental water bodies (such as surface water and wastewater). Attached Figure Description

[0046] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0047] Figure 1 This is a schematic diagram of the preparation of Fe3O4@SiO2@CMP-SO3Na in Example 1 of the present invention;

[0048] Figure 2 The images shown are systematic characterization images of the morphological features of the synthesized materials using scanning electron microscopy and transmission electron microscopy in Example 1 of this invention. Among them, (a) is the SEM image of Fe3O4, (b) is the SEM image of Fe3O4@SiO2, (c) is the SEM image of Fe3O4@SiO2@CMP-SO3Na, (d) is the TEM image of Fe3O4, (e) is the TEM image of Fe3O4@SiO2, and (f) is the TEM image of Fe3O4@SiO2@CMP-SO3Na.

[0049] Figure 3 The images show the Fourier transform infrared spectrum, N2 adsorption-desorption isotherm and pore size distribution, XRD pattern and hysteresis loop diagram of the material prepared in Example 1 of this invention, wherein (a) is the Fourier transform infrared spectrum, (b) is the N2 adsorption-desorption isotherm and pore size distribution diagram, (c) is the XRD pattern and (d) is the hysteresis loop diagram.

[0050] Figure 4 Thermogravimetric analysis curves of Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2@CMP-SO3Na in Example 1 of this invention;

[0051] Figure 5 The adsorption performance diagrams of Fe3O4@SiO2@CMP-SO3Na and Fe3O4@SiO2@CMP in Example 1 of this invention are shown.

[0052] Figure 6 The graphs show the parameters affecting the adsorption performance of Fe3O4@SiO2@CMP-SO3Na in Example 1 of the present invention, where (a) is the pH value graph, (b) is the adsorbent dosage graph, (c) is the adsorption time graph, and (d) is the ionic strength graph.

[0053] Figure 7 The graph shows the parameters affecting the desorption performance of Fe3O4@SiO2@CMP-SO3Na in Example 1 of the present invention, where (a) is a graph of the types of desorption solvents, (b) is a graph of desorption volume, and (c) is a graph of desorption time.

[0054] Figure 8 The adsorption performance diagrams of Fe3O4@SiO2 and Fe3O4@SiO2@CMP-SO3Na in Example 1 of this invention are shown.

[0055] Figure 9 This is a visualization of non-covalent interactions in the complex (i-CMP@E3) after the ionic conjugated microporous polymer adsorbs estriol in Example 1 of the present invention; wherein, (a) is a visualization of ion-dipole interactions and hydrogen bonding in i-CMP@E3, (b) is a visualization of π-π stacking interactions in i-CMP@E3, and (c) is a visualization of the classification of non-covalent interactions in i-CMP@E3;

[0056] Figure 10 The electrostatic potential surface diagram calculated in Example 1 of the present invention is shown, wherein (a) is an ionic conjugated microporous polymer (i-CMP) and (b) is estriol (E3).

[0057] Figure 11The above are XPS analysis results from Example 1 of this invention, where (a) is a full scan spectrum, (b) is a high-resolution C1s spectrum of Fe3O4@SiO2@CMP-SO3Na before E3 adsorption (Fe3O4@SiO2@CMP-SO3Na), (c) is a high-resolution C1s spectrum of Fe3O4@SiO2@CMP-SO3Na+E3 after E3 adsorption (Fe3O4@SiO2@CMP-SO3Na+E3), (d) is a high-resolution O1s spectrum of Fe3O4@SiO2@CMP-SO3Na before E3 adsorption (Fe3O4@SiO2@CMP-SO3Na+E3), and (e) is a high-resolution O1s spectrum of Fe3O4@SiO2@CMP-SO3Na+E3 after E3 adsorption.

[0058] Figure 12 This is a graph showing the effect of urea on the adsorption performance of Fe3O4@SiO2@CMP and Fe3O4@SiO2@CMP-SO3Na in Example 1 of the present invention;

[0059] Figure 13 This is a batch-to-batch stability diagram of Fe3O4@SiO2@CMP-SO3Na in Example 1 of the present invention;

[0060] Figure 14 This is a diagram illustrating the reusability of Fe3O4@SiO2@CMP-SO3Na in Example 1 of this invention.

[0061] in, Figures 5-14 In the formula, E1 represents estrone, E2 represents 17β-estradiol, E3 represents estriol, EE represents ethinylestradiol, and HE represents hexanestilbene. Detailed Implementation

[0062] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Specific conditions not specified in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Components whose manufacturers are not specified are all commercially available conventional products.

[0063] Example 1

[0064] 1. Experimental Section

[0065] (1) Material preparation

[0066] The method for preparing Fe3O4 microspheres using the solvothermal method is as follows: Figure 1 As shown, the specific process is as follows:

[0067] First, accurately weigh 0.65 g of ferric chloride hexahydrate (FeCl3·6H2O) and dissolve it in 60 mL of ethylene glycol, stirring continuously until completely dissolved. Then, add 0.2 g of sodium acetate and 1.2 g of citric acid to the above solution, stirring thoroughly to ensure homogeneous mixing. Finally, transfer the completely dissolved and homogenized mixture to a stainless steel reactor lined with polytetrafluoroethylene (PTFE), seal it, and react at 200°C for 24 h. After the reaction, allow the reactor to cool naturally to room temperature. Wash the resulting black precipitate repeatedly with excess ethanol and deionized water until the supernatant is clear, then dry it in a vacuum drying oven at 60°C for 6 h to obtain Fe3O4 microspheres.

[0068] Using St The specific process for preparing Fe3O4@SiO2 microspheres by the Ber method is as follows:

[0069] First, 20 mg of Fe3O4 microspheres were dispersed in 100 mL of a mixture of anhydrous ethanol, deionized water, and 25% ammonia in a volume ratio of 20:3:1, and stirred continuously for 30 min. Then, 0.2 mL of tetraethyl orthosilicate (TEOS) was slowly added dropwise to the solution while stirring. Afterward, the reaction mixture was mechanically stirred at room temperature for 6 h. Once the reaction was complete, the product was magnetically separated, thoroughly washed with excess ethanol and deionized water to remove unreacted substances, and finally dried in a vacuum drying oven at 60°C for 6 h to obtain Fe3O4@SiO2 microspheres.

[0070] The specific process for preparing Fe3O4@SiO2@CMP-SO3Na via the Sonogashira-Hagihara coupling reaction is as follows:

[0071] First, 100 mg of Fe3O4@SiO2 was dispersed in a mixed solution of 6.6 mL toluene and 13.3 mL triethylamine, and sonicated for 30 min to achieve uniform dispersion. Then, 2,4,6-tris(4-ethynylphenyl)-1,3,5-triazine (TEPT, 25.4 mg) and 1,4-dibromo-2,5-bis(3-sulfonylpropoxy)benzene (DBSB, 74 mg), along with catalysts dichlorobis(triphenylphosphine)palladium (Pd(PPh3)2Cl2, 4.6 mg) and cuprous iodide (CuI, 1.3 mg), were added to the dispersion. After purging, the reaction system was stirred at 90°C for 24 h under a nitrogen atmosphere.

[0072] The preparation method of Fe3O4@SiO2@CMP differs from that of Fe3O4@SiO2@CMP-SO3Na in that 1,4-dibromo-2,5-bis(3-sulfonylpropoxy)benzene is replaced with an equal amount of 1,4-dibromo-2,5-di(butoxy)benzene, while the other preparation methods are the same.

[0073] (2) Sample preparation

[0074] Environmental water samples: Surface water samples were collected from the Xiaoqing River in Jinan City, and wastewater samples were taken from the inlet of the city's main sewage treatment plant. All water samples were filtered through a 0.22 μm polyethersulfone (PES) membrane and then stored at 4°C for analysis.

[0075] Honey sample: Purchased from Jinan Agricultural Trade Market, Shandong Province. Accurately weigh 0.5 g of honey sample, dilute with 50 mL of pure water, vortex for 5 min, and sonicate for 10 min until completely dissolved. Filter through a 0.22 μm PES membrane for later use.

[0076] Milk powder sample: The sample was purchased from a local supermarket in Jinan. 0.5 g of sample was accurately weighed and placed in a 50 mL polypropylene centrifuge tube. 3.0 mL of ultrapure water was added, and the mixture was vortexed for 3 min to dissolve completely. 10.0 mL of acetonitrile was slowly added to precipitate the protein. After vortexing for 5 min, the mixture was ultrasonically extracted for 10 min, followed by centrifugation at 12,000 rpm for 10 min. The supernatant was collected. 5.0 mL of methanol was added to the residue, and the mixture was vortexed for 2 min, followed by a second ultrasonic extraction for 5 min. The supernatants from both extractions were combined and transferred to a 15 mL centrifuge tube. The mixture was then dried under nitrogen in a 35℃ water bath. Finally, the residue was reconstituted with 50 mL of water for subsequent magnetic solid-phase extraction.

[0077] (3) The magnetic solid-phase extraction process is as follows:

[0078] An appropriate amount of Fe3O4@SiO2@CMP-SO3Na was dispersed in 50 mL of sample solution and shaken at 240 rpm for 3 min on a shaker. The adsorbent was then separated using an external magnetic field, and 4 mL of methanol was added for vortex elution. The eluent was collected and dried under a gentle nitrogen stream. The residue was treated with 400 μL of a methanol-water mixture (V... 甲醇 / V 水 The solution was reconstituted at a ratio of 1:1, filtered through a filter membrane, and then analyzed by HPLC-MS / MS.

[0079] (4) Calculation method

[0080] The adsorption behavior of the complex (i-CMP@E3) after adsorption of estriol by CMP-SO3Na was studied using the Gaussian09 software package and Multiwfn (version 3.8). Since the support could not be included in the theoretical calculations, adsorption experiments were conducted to demonstrate that the adsorption effect mainly originated from i-CMP. Therefore, i-CMP@E3 was used for subsequent theoretical calculations. The specific calculation method is as follows:

[0081] First, density functional theory (DFT) calculations were performed at the B3LYP / 6-31G(d,p) basis set level to optimize the most stable conformations of CMP-SO3Na (i-CMP), estriol (E3) monomers, and the i-CMP@E3 complex. To further explore the molecular-level interaction mechanism, independent gradient model (IGMH) analysis based on Hirshfeld segmentation revealed that the IGMH isosurfaces visualized by VMD (version 1.9.3) clearly showed the spatial region and relative strength of the interaction between i-CMP and E3. Furthermore, the adsorption energy (Ei) was calculated using formulas. adsorption To quantitatively assess adsorption strength:

[0082] E adsorption = E i-CMP@E3 - ( E i-CMP + E E3 )

[0083] Where E i-CMP@E3 E i-CMP and E E3 These represent the total energy of the i-CMP@E3 complex, free i-CMP, and free E3, respectively. A larger negative adsorption energy indicates a stronger adsorption interaction.

[0084] (5) Liquid chromatography-tandem mass spectrometry conditions

[0085] Instrument model: Ultimate 3000 high performance liquid chromatograph in tandem with QTRAP 5500 triple quadrupole-linear ion trap tandem mass spectrometer.

[0086] Liquid chromatography separation conditions: Separation was performed using a Waters XBridge BEH C18 column (100 mm × 2.1 mm, particle size 2.6 μm), with a binary mobile phase of 10 mmol·L⁻¹. -1 Ammonium acetate aqueous solution (phase A) and methanol (phase B). The gradient elution program was optimized as follows: 0–1.0 min: methanol concentration linearly increased from 0% to 60% (v / v); 1.0–5.0 min: methanol concentration linearly increased from 60% to 80% (v / v); 5.1–8.0 min: methanol concentration maintained at 60% (v / v), isocratic elution. Flow rate: 0.3 mL / L -1 Column temperature: 35 ℃, injection volume: 5 μL.

[0087] Mass spectrometry detection conditions: Ion source: electrospray ionization, negative ion mode; Scanning mode: multiple reaction monitoring (MRM); Ion source temperature: 500℃; Other parameters are shown in Table 1.

[0088] Table 1 shows the structure, physicochemical properties, and MS parameters of the five EEs.

[0089]

[0090] 2. Results and Analysis

[0091] (1) Structural analysis of Fe3O4@SiO2@CMP-SO3Na

[0092] First, the morphological characteristics of the synthesized material were systematically characterized using scanning electron microscopy and transmission electron microscopy. The results... Figure 2 As shown.

[0093] like Figure 2 (a) and Figure 2 As shown in (d), the original Fe3O4 nanoparticles exhibit a well-dispersed spherical morphology with no significant agglomeration and good particle size uniformity. After SiO2 coating modification, the particle size of the Fe3O4@SiO2 composite material slightly increases. Figure 2 (b) in the image, its TEM characterization image ( Figure 2 (e) clearly shows a core-shell bilayer structure, confirming that the amorphous silicon layer has been successfully coated on the Fe3O4 core surface. Compared with the precursor material, the morphology of the final product Fe3O4@SiO2@CMP-SO3Na composite material has changed significantly, forming a rough surface and a three-dimensional interconnected network porous structure. Figure 2 (c) and Figure 2 (f)). This unique morphology not only provides a larger specific surface area but also creates abundant adsorption active sites, providing a favorable structural basis for the rapid mass transfer and efficient adsorption of estrogen-like compounds.

[0094] The surface functional groups of the composite material were further characterized using Fourier transform infrared spectroscopy (FTIR). Figure 3 (a)). All magnetic composite materials (Fe3O4, Fe3O4@SiO2 and Fe3O4@SiO2@CMP-SO3Na) are at 578 cm⁻¹. -1 The characteristic peak at 1183 cm⁻¹ is attributed to the Fe-O stretching vibration of the Fe₃O₄ core, demonstrating its structural stability throughout the synthesis process. For Fe₃O₄@SiO₂ and Fe₃O₄@SiO₂@CMP-SO₃Na, the characteristic peak at 1183 cm⁻¹ is also observed. -1 The newly emerging characteristic peak can be attributed to Si-O stretching vibrations, confirming successful SiO2 shell coating. Furthermore, although the raw materials TEPT and DBSB show peaks at 3293 cm⁻¹... -1 (–C≡CH) and 548cm -1These peaks show distinct characteristic peaks at (–C–Br), but they completely disappear in the Fe3O4@SiO2@CMP-SO3Na spectrum, replaced by peaks at 2170 cm⁻¹. -1 A new peak at the point was observed, attributed to the C≡C stretching vibration. This series of spectral changes provides direct evidence for the successful synthesis of the Fe3O4@SiO2@CMP-SO3Na composite material.

[0095] The specific surface area and pore size distribution of Fe3O4@SiO2@CMP-SO3Na were characterized using an N2 adsorption-desorption isotherm. Figure 3 As shown in (b), the specific surface area of ​​the material, determined by the Brunauer-Emmett-Teller (BET) method, is 158.69 m². 2 ·g -1 Pore ​​size distribution analysis further revealed that Fe3O4@SiO2@CMP-SO3Na possesses a mesoporous structure with an average pore size of 3.8 nm.

[0096] The crystal structures of Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2@CMP-SO3Na were characterized by X-ray diffraction (XRD), and the results are as follows: Figure 3 As shown in (c), the XRD pattern of pure Fe3O4 shows obvious characteristic diffraction peaks at 2θ = 30.1°, 35.5°, 43.2°, 57.1°, and 62.7°, corresponding to the (220), (311), (400), (440), and (511) crystal planes in the Fe3O4 standard card (e.g., JCPDS No. 19-0629), indicating that the prepared pure Fe3O4 has a good crystal structure. It is noteworthy that the above characteristic diffraction peaks are clearly present in the XRD patterns of both Fe3O4@SiO2 and Fe3O4@SiO2@CMP-SO3Na, indicating that the crystal structure of the magnetic Fe3O4 core was not destroyed during the SiO2 coating and subsequent Sonogashira-Hagihara coupling reaction. The complete preservation of the magnetic core crystal structure ensures that the composite material possesses good magnetic response performance, which is of crucial significance for its practical application in the separation field.

[0097] The magnetic properties of the three materials were tested at room temperature using a vibrating sample magnetometer (VSM), and the results are as follows: Figure 3 As shown in (d) above. All tested samples exhibited negligible coercivity and remanence, indicating typical superparamagnetic properties. Saturation magnetization (M s The test results show that the M of the original Fe3O4 s The value is 54 emu·g -¹, After being coated with SiO2, the M of Fe3O4@SiO2 s The value drops to 21 emu·g -1 After further coating with i-CMP, the M of Fe3O4@SiO2@CMP-SO3Na s The value further decreased to 16 emu·g -1 The decreasing magnetization is mainly attributed to the introduction of the non-magnetic SiO2 shell and i-CMP coating. The presence of these non-magnetic components dilutes the proportion of the magnetic phase in the material. It is noteworthy that although the magnetization of the composite material is ultimately reduced, it still maintains sufficient magnetic response capability, enabling rapid separation from the solution under an applied magnetic field, thus laying the foundation for its practical application in the field of magnetic separation.

[0098] Thermogravimetric analysis (TGA) was used to evaluate the thermal stability of the material. For example... Figure 4 As shown, the three materials Fe3O4, Fe3O4@SiO2, and Fe3O4@SiO2@CMP-SO3Na all exhibited a mass loss of approximately 4 wt% in the temperature range of 25–200°C. This phenomenon can be attributed to the desorption and evaporation of physically adsorbed moisture on the material surface. In the temperature range of 200–500°C, all samples further showed a mass loss of approximately 10 wt%, mainly due to the thermal decomposition of residual organic components in the materials. Upon reaching the high-temperature stage (550–800°C), Fe3O4@SiO2 and Fe3O4@SiO2@CMP-SO3Na showed an additional mass loss of approximately 10 wt%, primarily caused by the thermally induced collapse of the material's framework structure. In summary, Fe3O4@SiO2@CMP-SO3Na exhibits excellent thermal stability below 500°C.

[0099] (2) Selection of adsorption materials

[0100] This study synthesized two types of CMPs: a sulfonate-modified magnetic conjugated microporous polymer material (Fe3O4@SiO2@CMP-SO3Na) and a non-functionalized magnetic conjugated microporous polymer material (Fe3O4@SiO2@CMP). Under the same magnetic solid-phase extraction conditions, 10 mg of each adsorbent was dispersed in 50 mL of EEs solution, and their extraction efficiency for EEs was evaluated. Figure 5 As shown, Fe3O4@SiO2@CMP-SO3Na exhibits significantly superior adsorption performance. This performance improvement is attributed to its inherent ionic framework structure, which enhances both electrostatic and polar interactions with the target EEs. Based on these results, Fe3O4@SiO2@CMP-SO3Na was selected as the optimal adsorbent for all subsequent magnetic solid-phase extraction experiments.

[0101] (3) Optimization of extraction conditions for magnetic solid phase extraction

[0102] This study used Fe3O4@SiO2@CMP-SO3Na as the adsorbent and systematically investigated the effects of key experimental parameters such as sample pH, adsorbent dosage, adsorption time, ionic strength, type of desorption solvent, volume of desorption solvent, and desorption time. The recovery rate of five target EEs was used as the evaluation index of magnetic solid phase extraction efficiency.

[0103] Sample pH is crucial for the MSPE process because it simultaneously affects the ionization state of EEs and the surface charge properties of Fe3O4@SiO2@CMP-SO3Na. To investigate its influence, extraction experiments were conducted at pH values ​​of 2, 4, 6, 10, and 12. Figure 6 As shown in (a), when pH < 10, all five target EEs achieved high recoveries; however, when pH > 10, the extraction efficiency of all EEs decreased significantly. This trend is closely related to the dissociation constants of the EEs (pKa = 9.8~10.77): when pH < 10 (below pKa), the acidic functional groups (such as phenolic hydroxyl groups) in the EE molecules are mainly in a protonated state, and the overall molecule is neutral, which facilitates binding with Fe3O4@SiO2@CMP-SO3Na through hydrogen bonding and π-π stacking interactions; when pH > 10 (above pKa), the extraction efficiency of all EEs decreases significantly. a When the value is (), the acidic functional groups undergo deprotonation, causing the EEs molecules to carry a negative charge, which ultimately leads to a decrease in extraction efficiency.

[0104] The effect of adsorbent dosage on the extraction efficiency of five target EEs was investigated by examining five different dosages of Fe3O4@SiO2@CMP-SO3Na: 2, 4, 6, 8, and 10 mg. Figure 6 As shown in (b), the extraction efficiency of all five EEs gradually increased when the adsorbent dosage was increased to 8 mg; beyond this dosage, further increases to 10 mg did not significantly improve the extraction efficiency, indicating that 8 mg of adsorbent was sufficient for quantitative adsorption of the target analyte. Therefore, 8 mg was selected as the optimal adsorbent dosage for all subsequent experiments.

[0105] The effect of adsorption time was also investigated. For example... Figure 6 As shown in (c), the recoveries of all five EEs steadily increased when the adsorption time was extended from 0.5 min to 3 min; further extension of the adsorption time did not significantly improve the extraction efficiency. This result confirms that an adsorption time of 3 min is sufficient to reach adsorption equilibrium.

[0106] By adding concentrations of 0, 0.1, 0.2, 0.4, and 0.8 mol·L⁻¹ to the sample solution -¹NaCl was used to investigate the effect of ionic strength on extraction efficiency. For example... Figure 6 As shown in (d), the extraction efficiency of the five target compounds was basically unaffected within the test concentration range, indicating that no adjustment of ion strength was required in subsequent experiments.

[0107] The desorption solvent directly affects the desorption efficiency of the target analyte. This study investigated the desorption efficiencies of three solvents—methanol (MeOH), acetonitrile (ACN), and dichloromethane (DCM)—to screen for the optimal desorption solvent. Figure 7 As shown in (a), methanol has the highest desorption efficiency for all five target EEs, and therefore was selected as the desorption solvent for subsequent experiments.

[0108] The effect of solvent absorption volume was investigated within the range of 1.0–5.0 mL. Figure 7 As shown in (b), when the desorption solvent volume increased to 3.0 mL, the recovery rates of the five EEs gradually increased and reached a plateau; further increases in volume did not significantly improve the recovery rates. Therefore, 4 mL was selected as the optimal desorption solvent volume.

[0109] Furthermore, to determine the time required for complete desorption of EEs, this study investigated the effect of desorption time. For example... Figure 7 As shown in (c), within the range of 2–5 min, the recovery rate of the target analyte increased with increasing desorption time; however, when the desorption time exceeded 5 min, the recovery rate did not show a significant increase. Therefore, 5 min was ultimately selected as the optimal desorption time.

[0110] (4) Adsorption mechanism

[0111] To elucidate the adsorption mechanism of EEs by the Fe3O4@SiO2@CMP-SO3Na composite material, a comparative magnetic solid-phase extraction experiment was conducted using Fe3O4@SiO2 as a reference material. Figure 8 As shown, the extraction efficiency of Fe3O4@SiO2@CMP-SO3Na for EEs is significantly better than that of the pure support Fe3O4@SiO2, clearly confirming the key role of the i-CMP layer in the adsorption process. Therefore, a density functional theory (DFT) optimized i-CMP model was constructed to conduct mechanistic studies.

[0112] Based on the density functional theory (DFT) optimized structure, E3 was selected as a representative EE to simulate its adsorption behavior on i-CMP. Simulation results show that three main interactions exist between the two (e.g., ...). Figure 9 As shown in (c) in i-CMP: (i) The –OH in E3 acts as a proton donor, forming a -OH···O- hydrogen bond with the oxygen atom (proton acceptor) of -SO3Na in i-CMP; (ii) Na in i-CMP +There is an ion-dipole interaction between the oxygen atom in the –OH group of E3 (e.g. Figure 9 (a) shows that the E3 molecules are oriented parallel to the i-CMP benzene ring and achieve stable bonding mainly through misaligned face-to-face π-π stacking (as shown in (a)). Figure 9 (as shown in (b)).

[0113] To further elucidate the driving force of the adsorption process, the electrostatic potential distribution of E3 and i-CMP was analyzed. Figure 10 As shown, in the electrostatic potential equipotential surface, the red area corresponds to the positively charged sites in the system, while the blue area represents the negatively charged regions. The results show that Na in i-CMP... + Positively charged, while the oxygen atom in i-CMP-SO3Na (such as...) Figure 10 (as shown in (a)) and the oxygen atom of –OH in E3 as shown in (a) Figure 10 (b) shown in the diagram all carry a negative charge. This complementary charge distribution creates an ideal electrostatic environment: Na + E3 binds to the negatively charged oxygen atom in the E3-OH group via ion-dipole interactions, while the -OH group of E3 forms hydrogen bonds with the oxygen atom of the -SO3Na group in the i-CMP. The synergistic effect of hydrogen bonds and ion-dipole interactions forms a multidimensional non-covalent network, effectively stabilizing the adsorption configuration of E3 on the i-CMP.

[0114] X-ray photoelectron spectroscopy (XPS) was used to analyze the changes in elemental electronic states and surface composition of Fe3O4@SiO2@CMP-SO3Na before and after E3 adsorption. Figure 11 As shown in (a), the O 1s peak intensity is significantly enhanced after adsorption, indicating that the chemical environment of oxygen atoms has changed. Figure 11 Images (b) and (c) in section 11 are high-resolution C 1s spectra before and after E3 adsorption. It can be seen that the binding energy of the C=C peak shifted from 284.78 eV to 284.63 eV, and its relative content increased from 44.12% to 50.45%. These changes strongly suggest the existence of π-π interactions between the E3 aromatic ring and the CMP framework. Furthermore, the O 1s spectrum (… Figure 11 As shown in (d) of 11 and (e) of 11, the -OH binding energy shifted from 535.22 eV to a lower binding energy direction to 533.94 eV, while the relative content increased from 9.23% to 10.86%. This phenomenon indicates that hydrogen bonds were formed between the E3 molecule and the oxygen atom of the -SO3Na group in i-CMP.

[0115] Using urea (at concentrations of 0 and 6 mol·L⁻¹), a known hydrogen bonding competitor, -1By comparing the adsorption effects of Fe3O4@SiO2@CMP and Fe3O4@SiO2@CMP-SO3Na on EEs, the role of hydrogen bonding in the adsorption process was further explored. Figure 12 As shown, urea significantly inhibited the adsorption performance of Fe3O4@SiO2@CMP-SO3Na, while having little effect on the adsorption performance of Fe3O4@SiO2@CMP. This difference confirms that sulfonate groups are crucial for the formation of hydrogen bonds in EEs during adsorption.

[0116] (5) Reusability and reproducibility of Fe3O4@SiO2@CMP-SO3Na

[0117] This study systematically investigated the reproducibility of the Fe3O4@SiO2@CMP-SO3Na material prepared in five different batches (n=5, i.e., batches 1 to 5) by evaluating its adsorption performance. Experimental results showed that all batches achieved satisfactory recoveries, and the relative standard deviations (RSDs) were all less than 5%. Figure 13 This confirmed that Fe3O4@SiO2@iCMP exhibits good reproducibility in synthesis. Based on this, 10 mg of Fe3O4@SiO2@CMP-SO3Na was used in 11 cyclic experiments. After 10 consecutive adsorption-desorption cycles, the recovery rate of EEs remained above 80% (e.g., ...). Figure 14 ,in Figure 14 The numbers 1-11 in the table represent the number of adsorption-desorption cycles. This result fully demonstrates that Fe3O4@SiO2@CMP-SO3Na exhibits excellent cycling stability.

[0118] In summary, the excellent batch-to-batch reproducibility and reusability of Fe3O4@SiO2@CMP-SO3Na provide a key guarantee for its practical application.

[0119] (6) Analytical performance of the developed method

[0120] The matrix effect (ME) was evaluated by comparing the matrix-matched calibration curves with the solvent calibration curves. The analytical performance of the MSPE-HPLC-MS / MS method based on Fe3O4@SiO2@CMP-SO3Na was assessed by linearity, correlation coefficient (r), limit of detection (LOD, S / N=3), limit of quantitation (LOQ, S / N=10), and precision (shown in Tables 2 and 3).

[0121] Table 2 Analytical results of food samples

[0122]

[0123] Table 3. Analytical results of water samples

[0124]

[0125] The results in Tables 2 and 3 show that the food samples ranged from 1 to 800 ng·g -1 Concentration range, water sample 1~800 ng·L -1 A good linear relationship was observed within the concentration range, with a correlation coefficient r ≥ 0.9935. The LOD and LOQ of the food samples were 0.12–5.22 ng·g, respectively. -1 and 0.43~17.85 ng·g -1 The LOD and LOQ of surface water were 0.14–4.00 ng·L⁻¹. -1 and 5.77~7.26 ng·L -1 The LOD and LOQ of the wastewater were 0.11~3.73 ng·L⁻¹. -1 and 0.39~12.42 ng·L -1 The relative standard deviations (RSDs) of intra-day precision (n=6) and inter-day precision (n=6) were less than 7.45% and 9.78%, respectively. These results demonstrate that the established method possesses high sensitivity and reproducibility, exhibiting significant application potential in the quality control of complex sample analysis.

[0126] (7) Analysis of actual samples

[0127] To evaluate the practical application value of the established method, it was used to detect EEs in food samples, surface water, and wastewater samples. The results showed that no EE residues were detected in any of the analyzed samples. To examine the accuracy of the method, a spiked recovery experiment was conducted, and the results are shown in Table 4.

[0128] Table 4. Measurement results of EEs in actual samples

[0129]

[0130] As shown in Table 4, the spiked recoveries of food samples ranged from 81.9% to 111.1%, and the spiked recoveries of water samples ranged from 82.0% to 117.4%, both achieving satisfactory recovery results and confirming the reliability of this method for the detection of EEs in complex matrices.

[0131] In summary, to achieve accurate quantification of EEs in food and environmental samples, this study prepared a magnetic ionic conjugated microporous polymer (Fe3O4@SiO2@CMP-SO3Na) via a Sonogashira-Hagihara coupling reaction, using Fe3O4@SiO2 as the magnetic support and 1,4-dibromo-2,5-bis(3-sulfopropyl)benzene (DBSB) and 2,4,6-tris(4-ethynylphenyl)-1,3,5-triazine (TEPT) as building blocks. Using this material as the adsorbent, an MSPE-HPLC-MS / MS analytical method was established and applied to the detection of EEs in honey, milk powder samples, and environmental water. Fe3O4@SiO2@CMP-SO3Na possesses a conjugated structure, ionic properties, and a porous structure; its excellent adsorption performance stems from the synergistic effect of hydrogen bonding, ion-dipole interactions, and π-π stacking interactions. Under optimized experimental conditions, the established method achieved rapid analysis of EEs with satisfactory sensitivity and accuracy. This study not only provides a stable and reliable analytical method for the detection of trace EEs, but also offers new insights into the rational design of functionalized ionic conjugated microporous polymers for efficient adsorption and removal of organic pollutants from complex samples.

[0132] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A material for enriching estrogen-like substances, characterized in that, Including magnetic carriers and ionic conjugated microporous polymers coated on their surfaces; The magnetic carrier has a core-shell structure, including an Fe3O4 core and a SiO2 shell covering the surface of the Fe3O4 core; The ionic conjugated microporous polymer has the following repeating structural units: 。 2. The estrogen-enriching material according to claim 1, characterized in that, The ionic conjugated microporous polymer has a three-dimensional interconnected network porous structure with a specific surface area >158.0 m². 2 ·g -1 The average pore size is 3.5-4.0 nm.

3. The estrogen-enriching material according to claim 1, characterized in that, A method for preparing estrogen-rich materials includes the following steps: The magnetic support is added to a solvent and dispersed evenly. Then, 2,4,6-tris(4-ethynylphenyl)-1,3,5-triazine, 1,4-dibromo-2,5-bis(3-sulfopropoxy)benzene and a catalyst are added, and a coupling reaction is carried out under a protective atmosphere to obtain the final product.

4. The estrogen-enriching material according to claim 3, characterized in that, The solvent is a mixture of toluene and triethylamine, wherein the volume ratio of toluene to triethylamine is 1:(2-2.2).

5. The estrogen-like substance enrichment material according to claim 3, characterized in that, The mass ratio of 2,4,6-tris(4-ethynylphenyl)-1,3,5-triazine, 1,4-dibromo-2,5-bis(3-sulfopropoxy)benzene to the catalyst is (4-4.5):(14-18):

1.

6. The estrogen-enriching material according to claim 3, characterized in that, The catalyst is a mixture of dichlorobis(triphenylphosphine)palladium and cuprous iodide, wherein the mass ratio of dichlorobis(triphenylphosphine)palladium and cuprous iodide is (3.5-3.6):1; The coupling reaction is carried out at a temperature of 80-100℃ for 20-30 h.

7. A method for analyzing estrogen-like substances, characterized in that, Includes the following steps: The estrogen-like substance enrichment material according to any one of claims 1-6 is used as an adsorbent, dispersed in the sample to be tested for adsorption, the adsorbent is separated by an external magnetic field, desorption solvent is added for desorption, the eluent is collected and dried with nitrogen to obtain the residue, which is then reconstituted, filtered and analyzed by liquid chromatography-tandem mass spectrometry.

8. The analytical method for estrogen-like substances according to claim 7, characterized in that, The estrogenic compound is one or more of estrone, 17β-estradiol, estriol, ethinylestradiol, and hexanestilbene.

9. The analytical method for estrogen-like substances according to claim 7, characterized in that, The sample to be tested is water, honey, or milk powder; The pH of the sample to be tested is 2-10; 5-25 L of sample to be tested is added per gram of adsorbent; the adsorption time is 0.5-3 min; The desorption solvent is one of methanol, acetonitrile, and dichloromethane; the volume ratio of the desorption solvent to the sample is (1-4):50; the desorption time is 2-5 min.

10. The analytical method for estrogen-like substances according to claim 7, characterized in that, The liquid chromatography column was a Waters XBridge BEH C18 column. Mobile phase A was ammonium acetate aqueous solution, and mobile phase B was methanol. The elution program was as follows: 0–1.0 min, methanol volume ratio linearly increased from 0% to 60%; 1.0–5.0 min, methanol volume ratio linearly increased from 60% to 80%; 5.1–8.0 min, isocratic elution was performed while maintaining a methanol volume ratio of 60%, with a flow rate of 0.2–0.4 mL / min. -1 The column temperature is 35-37℃, and the injection volume is 4-6 μL; The ion source for mass spectrometry analysis is electrospray ionization in negative ion mode; the scanning mode is multiple reaction monitoring mode, and the ion source temperature is 450-550℃.

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