A method for determining multiple estrogens in dairy products

Abstract

The present invention belongs to the field of analysis and detection, and provides a method for measuring multiple estrogens in dairy products, including: conjugated microporous polymer material (Fe3O4@TbDt), which has good magnetic responsiveness, high specific surface area, and abundant functional groups. It is an excellent magnetic solid phase extractor with good adsorption selectivity for estrogen compounds. The present invention is also combined with liquid chromatography-mass spectrometry (HPLC-MS / MS) technology to establish a method for simultaneously detecting 9 estrogen compounds (estriol, bisphenol F, bisphenol A, ethinylestradiol, α-estradiol, β-estradiol, estrone, hexane estradiol, dienestrol). The method has good linearity, low detection limit, and good repeatability. It can be used for the analysis of trace estrogens in dairy products such as milk and milk powder. Compared with other similar methods, the method is simple to operate, consumes less organic solvent, is highly sensitive, can achieve rapid detection of 9 estrogens in dairy products, and has the advantages of simplicity, efficiency, and environmental protection.

Description

Technical Field

[0001] The invention belongs to the field of analysis and detection, and particularly relates to a method for determining multiple estrogens in dairy products. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Estrogens are a class of environmental pollutants that have attracted considerable attention in recent years. They exhibit biotoxicity, including endocrine disruption, carcinogenicity, and teratogenicity, and are known to persist in the environment and bioaccumulate. Estrogens can be used as veterinary drugs or feed additives, but they are difficult to fully degrade in animals and are transported through the food chain, posing a direct threat to human health. Therefore, developing a rapid method for the analysis of trace estrogens, particularly in animal-derived foods such as dairy products, is crucial for early warning and monitoring of these pollutants, safeguarding ecosystems and human health.

[0004] Given the complex sample matrix of dairy products and the presence of trace levels of estrogen in these samples, sample pretreatment is essential before instrumental analysis to eliminate matrix effects and achieve enrichment of the target compound. Magnetic solid-phase extraction (MSPE) is a novel sample pretreatment technique. Its notable feature is the rapid separation of the adsorbent from the solution under the influence of an external magnetic field. Compared with traditional solid-phase extraction (SPE), MSPE eliminates the tedious steps of column packing and sample loading, avoiding the clogging of SPE columns and reducing organic solvent consumption. It is a simple, green, and efficient sample pretreatment technique. The core of MSPE is the adsorbent. Currently, a variety of novel nanomaterials have been applied in MSPE, such as carbon nanomaterials, graphene, metal-organic frameworks (MOFs), and covalent organic frameworks. However, their preparation and use inevitably encounter challenges. For example, some extraction and adsorption materials require harsh synthesis conditions, making them difficult to scale up, and some materials exhibit poor adsorption efficiency for highly polar compounds. Therefore, the development of materials that are easy to synthesize, highly selective, and have high extraction efficiency is crucial.

[0005] Conjugated microporous polymers (CMPs) are a class of porous organic framework materials that have attracted considerable attention in recent years. They can be synthesized through a single or several chemical reactions. Their large surface area, excellent physicochemical stability, and ease of functional group modification offer significant potential for application in MSPE. Currently, these materials have been used for the adsorption of gases such as H2 and CO2, but their application in MSPE is relatively limited. The industry has yet to identify a conjugated microporous polymer with sufficient adsorption for multiple estrogens in dairy products, and a method for detecting these compounds. Summary of the Invention

[0006] To address the above issues, the present invention provides a method for determining multiple estrogens in dairy products. Based on the Schiff base reaction principle, the present invention uses hydroxyl-rich trialdehyde phloroglucinol and 2-chloro-4,6-diamino-1,3,5-triazine as reactive monomers. A magnetic composite conjugated microporous polymer, Fe3O4@TbDt, with a core-shell structure is synthesized via a one-step reaction. This polymer is then used as an MSPE adsorbent. Combined with HPLC-MS / MS technology, an analytical method for the simultaneous detection of nine estrogens has been established. This method has been successfully applied to the detection of estrogens in dairy products such as milk and milk powder.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a conjugated microporous polymer material Fe3O4@TbDt, wherein the structural formula of the conjugated microporous polymer material Fe3O4@TbDt is as follows:

[0009]

[0010] The second aspect of the present invention provides a method for preparing a conjugated microporous polymer material Fe3O4@TbDt, comprising:

[0011] Disperse NH2-Fe3O4 in an organic solvent, add trialdehyde phloroglucinol and 2-chloro-4,6-diamino-1,3,5-triazine under stirring, and then add an organic solvent to dissolve the monomers. Under sealed conditions, perform nitrogen-vacuum exchange at least three times, raise the temperature to 120-130°C, react for 42-48 hours, collect the solid, wash, Soxhlet extraction for 24-32 hours, and dry to obtain the product.

[0012] The third aspect of the present invention provides a magnetic solid-phase extractant, the components of which include: the above-mentioned conjugated microporous polymer material Fe3O4@TbDt.

[0013] The fourth aspect of the present invention provides the use of the above-mentioned conjugated microporous polymer material Fe3O4@TbDt or the above-mentioned magnetic solid phase extraction agent in the HPLC-MS / MS determination of estrogen in dairy products.

[0014] A fifth aspect of the present invention provides a method for determining estrogen in dairy products by HPLC-MS / MS, comprising:

[0015] The above-mentioned conjugated microporous polymer material Fe3O4@TbDt was used as MSPE adsorbent and combined with HPLC-MS / MS method to detect 9 estrogens in dairy products.

[0016] The nine estrogens are estriol, α-estradiol, β-estradiol, hexaneestradiol, bisphenol estrogen, estrone, bisphenol A, bisphenol F, and ethinylestradiol.

[0017] Beneficial effects of the present invention

[0018] (1) This paper synthesized a core-shell magnetic conjugated microporous polymer, Fe3O4@TbDt, for the first time. This polymer was used as an adsorbent for magnetic solid-phase extraction of nine estrogens. By optimizing the MSPE conditions and combining it with HPLC-MS / MS, a method for detecting estrogen was established. The method has a wide linear range, a low detection limit (0.25 ng·L-1-5.1 ng·L-1), and good precision (RSD of 1.1-8.2%). The method was applied to the detection of milk, milk powder, and other dairy products. The spike recovery experiment achieved satisfactory recoveries of 82.1-101%, demonstrating the practical applicability of the method.

[0019] (2) Compared with other methods, the method established in the present invention is simple to operate, consumes less organic solvents, can simultaneously determine multiple estrogens in complex matrix samples such as dairy products, and has a lower detection limit, with the advantages of high efficiency and green environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0021] Figure 1 Schematic diagram of the synthesis of Fe3O4@TbDt in the present invention;

[0022] Figure 2 Characterization diagrams of the magnetic Fe3O4@TbDt of the present invention (a) solid-state NMR, (b) IR spectroscopy analysis, (c) hysteresis loop, (d) N2 adsorption-desorption isotherm, (e) transmission electron microscopy and scanning electron microscopy, (f) contact angle;

[0023] Figure 3 Optimization of MSPE parameters of the present invention: (a) adsorbent dosage; (b) sample solution pH; (c) MSPE time; (d) salt concentration; (e) desorption liquid type; (f) desorption liquid volume;

[0024] Figure 4 This is a chromatogram of nine estrogens of the present invention (1 estriol, 2 bisphenol F, 3 bisphenol A, 4 ethinylestradiol, 5β-estradiol, 6α-estradiol, 7 estrone, 8 dienestilbestrol, 9 hexaneestrol). DETAILED DESCRIPTION

[0025] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0026] A conjugated microporous polymer material Fe3O4@TbDt, wherein the structural formula of the conjugated microporous polymer material Fe3O4@TbDt is as follows:

[0027]

[0028] A method for preparing a conjugated microporous polymer material Fe3O4@TbDt, comprising:

[0029] Disperse NH2-Fe3O4 in an organic solvent, add trialdehyde phloroglucinol and 2-chloro-4,6-diamino-1,3,5-triazine under stirring, and then add an organic solvent to dissolve the monomers. Under sealed conditions, perform nitrogen-vacuum exchange at least three times, raise the temperature to 120-130°C, react for 42-48 hours, collect the solid, wash, Soxhlet extraction for 24-32 hours, and dry to obtain the product.

[0030] In some embodiments, the mass ratio of NH2-Fe3O4, trialdehyde phloroglucinol and 2-chloro-4,6-diamino-1,3,5-triazine is 5:4.2~4.5:2.91~3.

[0031] In some embodiments, the organic solvent is anhydrous DMF.

[0032] In some embodiments, the specific steps of dispersing NH2-Fe3O4 in an organic solvent include: adding NH2-Fe3O4 to anhydrous DMF, sealing, and ultrasonicating for 20 to 30 minutes.

[0033] In some embodiments, the specific step of the Soxhlet extraction includes: Soxhlet extraction at 95-96° C. using a mixture of methanol and tetrahydrofuran at a V / V ratio of 1:1.

[0034] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0035] In the following examples, trialdehyde phloroglucinol, 2-chloro-4,6-diamino-1,3,5-triazine, and 1,6-hexanediamine were purchased from Aladdin (Zhengzhou, China).

[0036] Example 1 Preparation of Estrogen Standard Stock Solution and Standard Working Solution

[0037] Estrogen standard stock solution: Accurately measure the concentration to 10 mg·L -11 mL each of the nine estrogen stock solutions of estriol, α-estradiol, β-estradiol, hexane estradiol, bisphenol estradiol, estrone, bisphenol A, bisphenol F, and ethinyl estradiol were placed in a 10 mL brown volumetric flask, and methanol was added to the volume to the mark to obtain a mixed standard solution with a concentration of 1 mg·L-1. The solution was then stored at 4°C.

[0038] Estrogen standard working solution: Dilute the standard stock solution with methanol step by step to prepare standard working solutions of different concentrations. The standard solutions must be prepared before use.

[0039] The structures and physicochemical properties of the nine estrogens are shown in Table 1.

[0040] Table 1 Structure and physicochemical properties of estrogen

[0041]

[0042]

[0043] Example 2 Synthesis of Fe3O4@TbDt Material

[0044] Synthesis of Fe3O4@TbDt: Weigh 500 mg of synthesized NH2-Fe3O4, add 20 mL of anhydrous DMF, seal the mixture, and sonicate for 20 minutes to achieve uniform dispersion. Pour the mixture into a three-necked flask equipped with a mechanical stirrer. While stirring, add two monomers: 420 mg of trialdehyde phloroglucinol (2,4,6-trihydroxybenzene-1,3,5-tricarbaldehyde, Tb) and 291 mg of 2-chloro-4,6-diamino-1,3,5-triazint (Dt). Then, add 10 mL of anhydrous DMF to dissolve the monomers in the reaction solution. A condenser was placed at the other end of the flask, connected to condensing water, and a double-row tube was connected to the top. The final end was sealed with a rubber stopper. Using the double-row tube, nitrogen was exchanged between the two openings for vacuum three times. The temperature was raised to 120°C and the reaction was allowed to proceed for 48 hours. After the reaction, a magnetic separation was used to obtain a brownish-red solid. The solid was washed twice with water, and then Soxhlet extracted with a mixture of methanol and tetrahydrofuran 120 mL (V / V = 1:1) at 95°C for 24 h, and then placed in a vacuum drying oven at 60°C to obtain a brownish-red powder. The synthesis route is as follows Figure 1 .

[0045] Example 3 MSPE experiment

[0046] 30 mg of the magnetic composite material Fe3O4@TbDt prepared in Example 2 was added to the glass bottle, followed by 60 mL of water sample with a pH of 10 and 0.6 mL of a 50 ppb estrogen mixed standard solution. The glass bottle was placed in an oscillator at 30°C and 240 rpm / min.-1 The sample was shaken at a speed of 100°C for 30 minutes. After extraction, the material was separated from the sample solution using a magnet. The material was left in a glass bottle, and 8 mL of methanol was added and shaken on a rotary mixer for 30 minutes for desorption. The Fe3O4@TbDt and the desorbent were again separated using an external magnet. The desorbent was collected and dried under a gentle nitrogen flow at 30°C. The sample was reconstituted with 0.3 mL of a mixture of methanol and water (V / V = 1:1), filtered through a 0.22 μm filter, and analyzed by LC-MS / MS.

[0047] Example 4 Chromatography Mass Spectrometry Instrument Conditions

[0048] The instrument model was SCIEX QTRAP 5500, and a C18 column (120 mm × 0.25 mm × 0.5 μm) was used for the separation of the nine estrogens. The liquid chromatography conditions were as follows: Phase A was methanol, Phase B was 10 mmol / L ammonium acetate solution; the mobile phase flow rate was 0.3 mL / min. -1 Gradient elution was performed. Phase A was 60% from 0 to 1 min, then gradually increased to 80% from 1 to 5 min, and then reduced to 60% from 5 to 8 min. Column temperature was 35°C, and autosampler temperature was 10°C. Mass spectrometry conditions included: ESI ionization mode, MRM scanning mode, and specific parameters as shown in Table 2. Curtain gas pressure was 275 kPa (40 psi), spray voltage was 5.5 kV, ion source temperature was 500°C, and nebulizer pressure was 345 kPa (50 psi). Collider and nebulizer were nitrogen.

[0049] Table 2 LC-MS / MS MRM data acquisition methods for nine estrogens

[0050]

[0051] Example 5 Collection and pretreatment of actual samples

[0052] Laboratory tap water (Jinan, Shandong), milk, and milk powder purchased from a local supermarket were used as actual samples. Water samples were filtered through a 0.45 μm filter membrane and stored in brown glass bottles at 4°C. The pH was adjusted to 10 before use.

[0053] Milk sample preparation: Weigh 1.00 g of milk and add 1.0 mL of estrogen standard working solution. Add 10 mL of acetonitrile to disrupt protein. Vortex and sonicate for 20 minutes to fully disrupt protein. Centrifuge and retain the supernatant. Add 5 mL of methanol to the lower precipitate to extract any residual estrogen. Centrifuge again and collect the supernatant. Combine the supernatants, air dry under nitrogen at 35°C, and reconstitute with 60 mL (3 × 20 mL) of pH 10 water before MSPE. Milk samples should be handled immediately upon use and should not be stored.

[0054] Milk powder sample treatment: Weigh 1.00 g of milk powder, add 3 mL of ultrapure water and shake to dissolve, add 1.0 mL of estrogen standard solution, add 10 mL of acetonitrile to destroy the protein, sonicate for 10 minutes, centrifuge to obtain the supernatant, add 5 mL of methanol to the lower white precipitate to extract possible residual estrogen, centrifuge and combine the supernatants, dry under a gentle nitrogen flow at 35°C, re-dissolve with 60 mL (3 × 20 mL) of pH = 10 water and perform MSPE.

[0055] Example 6 Methodological Investigation

[0056] A series of sample solutions with varying concentrations were prepared, subjected to MSPE, and then analyzed by LC-MS / MS. The linear range, limits of detection (LODs), and limits of quantification (LOQs) of the method were determined. LODs and LOQs were calculated using the baseline noise method at S / N ratios of 3 and 10, respectively. Precision was determined by the intra- and inter-day relative standard deviations (RSDs) of multiple replicate spike-in recovery experiments. The accuracy of the method was evaluated by the recoveries of real samples. For each real sample, three replicate spike-in recovery experiments were performed at low, medium, and high concentrations within the linear range. The average recovery and RSD of these three experiments were calculated.

[0057] In order to study the selectivity between Fe3O4@TbDt and estrogen, polycyclic aromatic hydrocarbons and glucocorticoids were added to the sample solution at the same concentration for selectivity exploration.

[0058] Experimental Example 1 Material Characterization

[0059] First, non-magnetic TbDt was synthesized according to the method of Example 2 and characterized by solid-state nuclear magnetic resonance. Figure 2 As shown in (a), the original C-Cl bond of monomer Dt is located at 162.38, and the C-N shift on the triazine ring is located at 158.80. The C-N shift between the benzene ring of monomer Tb and the hydroxyl group directly connected to the benzene ring is located at 150.68. The shift between the benzene ring and the C-OH bond directly connected to the benzene ring is 108.50. The C-N shift of the newly generated C-N bond formed by the reaction of the amino group and the aldehyde group is 56.59. Solid-state NMR results indicate the successful synthesis of the target product TbDt.

[0060] TbDt, NH2-Fe3O4 and the composite material Fe3O4@TbDt prepared in Example 2 were tested by infrared on the same instrument. The results are as follows: Figure 2 (b) The typical vibration of aldehyde and amino groups in the infrared spectrum of TbDt has disappeared, and the -1 There is a very broad stretching band at 1482 cm, which indicates that the product is connected through -C-NH- bond. -1 and 872cm -1There are two characteristic absorption peaks, indicating that the Fe3O4 surface has been functionalized by amino groups. -1 The absorption peak of the Fe3O4@TbDt composite material is consistent with the Fe-O absorption peak of the exposed amino-modified Fe3O4. Therefore, the infrared spectrum shows that the magnetic spheres are attached to TbDt and covalently linked with the two monomers to successfully synthesize the composite magnetic material Fe3O4@TbDt.

[0061] The hysteresis curves of bare NH2-Fe3O4 and the composite material Fe3O4@TbDt prepared in Example 2 are shown in Figure 2. Figure 2 As shown in (c), it can be seen that there is no hysteresis or remanence in the two magnetization curves, indicating that both NH2-Fe3O4 and Fe3O4@TbDt have good superparamagnetism. The saturation magnetization intensity value of Fe3O4@TbDt is 58.2emu·g -1 With NH2-Fe3O4(94.2emu·g -1 ), but still maintains a high magnetic induction intensity, can quickly perform magnetic separation, and meet the MSPE requirements for magnetic adsorbents.

[0062] Figure 2 (d) is the specific surface area and pore size diagram of the composite material Fe3O4@TbDt. The specific surface area is calculated to be 251.63 m2·g according to the BET algorithm. -1 Compared with naked ferroferric oxide, it has a larger specific surface area and can provide more adsorption sites for extraction, which is beneficial to improving the extraction efficiency.

[0063] TEM showed that the composite material was a core-shell structure, e.g. Figure 2 As shown in the upper part of (e), the material morphology was observed by SEM, and the results were as follows Figure 2 As shown in the lower part of (e), the smooth ferroferric oxide magnetic sphere has been covered with material and the surface has become rough. The contact angle characterization results of the composite material Fe3O4@TbDt are shown in Figure 2. Figure 2 In (f), they are 15.91° and 13.36°, respectively, indicating that the material is hydrophilic and is conducive to dispersion in water samples.

[0064] Experimental Example 2 Optimization of magnetic solid phase extraction parameters

[0065] To achieve the optimal extraction effect, the present invention investigated the magnetic solid-phase extraction parameters, taking the extraction efficiency of Fe3O4@TbDt for nine estrogens as the evaluation index. The main parameters investigated included adsorbent dosage, sample solution pH, extraction time, salt concentration, desorption liquid type, and eluent volume.

[0066] The amount of adsorbent is an important parameter that needs to be optimized. If the amount of adsorbent is too little, the adsorption is not thorough and the extraction and enrichment efficiency is low; if the amount of adsorbent is too large, it will cause material waste. The present invention uses 10-50mg (10, 20, 30, 40, 50) of different masses of adsorbent (Fe3O4@TbDt prepared in Example 2), and the results are as follows Figure 3 As shown in (a), when the adsorbent dosage gradually increases to 30 mg, the extraction efficiency shows a gradual upward trend. Further increasing the Fe3O4@TbDt dosage has no significant effect on improving the extraction efficiency. To achieve a good extraction recovery while using as little dosage as possible, 30 mg was selected for the experiment.

[0067] The pH of the sample solution is another important parameter. The present invention adjusts the pH of the sample solution and investigates the effect of pH at 4, 6, 8, 10, and 12 on the extraction of estrogen. Figure 3 Figure (b) shows that when the pH of the sample solution is 8, most compounds have good recovery rates, but the recovery rates of bisphenol A, bisphenol F, and dienestilbestrol have not yet reached 80%. When the pH value is increased to 10, all nine estrogens can achieve high extraction efficiency. When the alkalinity of the solution is further increased to 12, the recovery rate does not increase significantly. This is mainly because in an alkaline environment, estrogens are more likely to lose hydrogen protons on the hydroxyl group and exist as negatively charged ions. The lone pair of electrons on the N atom on the triazine ring in the material structure is shielded by the formed π bond. In addition, the N=N double bond is conjugated, and the electron-absorbing property of the chlorine atom itself makes the material overall lack of electricity, and there is electrostatic attraction between it and the target compound.

[0068] In the MSPE process, one factor that affects the extraction and enrichment efficiency is the extraction time. The effect of extraction time on the recovery rate was investigated in the range of 10-50 min (i.e. 10, 20, 30, 40, 50 min). Figure 3 In (c), within 10-30 min, as the extraction time increases, the recovery rate shows an upward trend, but there is no significant improvement in the recovery rate when the time is further extended. In order to improve the pretreatment efficiency, 30 min is selected as the extraction time.

[0069] The presence of NaCl mainly affects the ionic strength of the sample solution, and thus affects the diffusion rate of the target in the sample. The present invention examines the effect of different ionic strengths on the extraction efficiency. The NaCl mass fractions of the water sample were adjusted to 0, 0.25, 0.5, 0.75, and 1% wt. Figure 3 As shown in (d), when no NaCl is added, the recovery rate of MSPE is the highest. This is mainly because the addition of NaCl makes it difficult for estrogen to move to the adsorbent. In the same time, the adsorbed estrogen decreases, which leads to a decrease in the recovery rate. Therefore, the ionic strength is selected as 0.

[0070] The desorption liquid determines whether the analyte can be smoothly eluted from the adsorbent and has a significant impact on the extraction efficiency. The present invention selects methanol, 0.5% formic acid methanol, 0.5% ammonia methanol, acetonitrile, and ethyl acetate as the eluent. Figure 3 (e) shows the elution effects of the five solvents. It is obvious that methanol has the best desorption effect on the nine estrogens. This result is because the estrogen has a higher solubility in methanol and is therefore more easily eluted by methanol.

[0071] The amount of desorption liquid is also one of the important factors affecting the extraction efficiency. If the volume used is insufficient, the desorption will be incomplete, the analyte will remain on the material, affecting the reuse of the adsorbent, and at the same time leading to low extraction efficiency. Using too much desorption liquid will cause waste and contamination of organic reagents, and the subsequent nitrogen blowdown will also take more time. In this experiment, the eluent volume was tested to be 4-12mL (4, 6, 8, 10, 12mL), and the results are as follows Figure 3 As shown in (f), when the eluent volume increased from 4 mL to 8 mL, the extraction efficiency continued to improve, and further increasing the eluent volume had no obvious effect on the recovery rate. 8 mL of eluent was used.

[0072] The optimal experimental conditions for the extraction of nine estrogen compounds by Fe3O4@TbDt were as follows: Fe3O4@TbDt material dosage was 30 mg, sample solution pH = 10, extraction time was 30 min, salt concentration was 0, methanol was used as the desorption solution, and the desorption solution volume was 8 mL.

[0073] Experimental Example 3 Methodological Investigation

[0074] Table 3 lists the methodological data of the established method. The results show that the method has good linearity (R 2 ≥0.996), wide linear range, low LODs (0.25ng·L -1 -5.1ng·L -1 ), the limit of quantification is (1.7 ng·L -1 -8.2ng·L -1 ), good precision, intra-day (n=6) RSD was 1.1-4.2%, and inter-day (n=6) RSD was 1.7-8.2%.

[0075] Table 3 Methodology validation

[0076]

[0077]

[0078] Experimental Example 4: Actual Sample Analysis

[0079] The present invention establishes a method for rapid detection of nine estrogens based on Fe3O4@TbDt MSPE coupled with HPLC-MS / MS technology. This method can be used to analyze estrogens in milk samples such as milk and milk powder, and spike recovery experiments at low, medium, and high concentrations have been carried out. Figure 4 At low concentrations (1 ng·L of dienestilbestrol and hexaneestrol -1 , estrone is 5 ng·L -1 , estriol, bisphenol A, and bisphenol F concentrations were 10 ng·L -1 The concentrations of ethinylestradiol, α-estradiol, and β-estradiol were 20 ng·L -1 ), 500, 1000ng·L -1 Spike recovery experiments were performed at concentrations of 100 mg / mL and the recovery was calculated using peak area. The results are shown in Table 4. The recovery range was 82.1-101%, with an RSD of less than 8.2%. This demonstrates the applicability and reliability of this method for the analysis of trace estrogens in complex matrices such as milk samples.

[0080] Table 4 Actual sample spiked

[0081]

[0082]

[0083] Low concentration: dienestilbestrol, hexestrol concentration 1ng·L -1 , estrone concentration 5 ng·L -1 , estriol, bisphenol A, and bisphenol F concentrations 10 ng·L -1 , ethinylestradiol, α-estradiol, β-estradiol concentration 20ng·L -1

[0084] Experimental Example 5 Method Comparison

[0085] The method developed by the present invention is compared with the previous method of LC-MS / MS detection of estrogen using the same MSPE pre-treatment method. Table 5 summarizes the linear range, number of types of estrogen detected, sample form, recovery rate and LODs of these methods. The method developed by the present invention has the advantage of being able to detect more types of estrogen simultaneously and can be used for the detection of food samples such as milk and milk powder other than water. Compared with the method that can detect multiple types of estrogen at the same time, the LODs of the method of the present invention are lower and the linear range is wider. Compared with the method similar to LODs, the present method can take into account the detection of multiple types of estrogen at the same time and maintain a higher recovery rate.

[0086] Table 5 Method comparison

[0087]

[0088]

[0089] Experimental Example 6 Discussion on adsorption mechanism

[0090] This study investigates the mechanism of Fe3O4@TbDt's action on estrogen. First, the Fe3O4@TbDt unit possesses a large conjugated system, which creates π-π interactions with estrogen, enhancing adsorption affinity. Second, Fe3O4@TbDt contains multiple amino and hydroxyl groups, which can form multiple hydrogen bonds with the target compound. Third, electrostatic attraction exists between Fe3O4@TbDt and the target compound. Estrogen's pKa indicates that at a pH of 10, estrogen exists as a negative ion. The material contains abundant N=N double bonds, which can be considered charge-withdrawing groups. This results in an overall charge-deficient state, leading to electrostatic attraction between the material and the target compound. Furthermore, the material's large specific surface area provides abundant adsorption sites for estrogen. Therefore, π-π interactions, multiple hydrogen bonds, electrostatic interactions, and a large specific surface area all play a role in the adsorption process.

[0091] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A conjugated microporous polymer material Fe3O4@TbDt, characterized in that: The structural formula of the conjugated microporous polymer material Fe3O4@TbDt is as follows: Formula I.

2. A method for preparing a conjugated microporous polymer material Fe3O4@TbDt, characterized in that: include: NH2-Fe3O4 is dispersed in an organic solvent, and trialdehyde phloroglucinol and 2-chloro-4,6-diamino-1,3,5-triazine are added under stirring. Then, an organic solvent is added to dissolve the monomers. Under sealed conditions, nitrogen-vacuum exchange is performed at least three times, the temperature is raised to 120-130°C, and the reaction is carried out for 42-48 hours. The solid is collected, washed, and Soxhlet extracted for 24-32 hours, and dried to obtain the product. The mass ratio of NH2-Fe3O4, trialdehyde phloroglucinol and 2-chloro-4,6-diamino-1,3,5-triazine is 5:4.2-4.5:2.91-3.

3. The method for preparing the conjugated microporous polymer material Fe3O4@TbDt according to claim 2, characterized in that: The organic solvent is anhydrous DMF.

4. The method for preparing the conjugated microporous polymer material Fe3O4@TbDt according to claim 2, characterized in that: The specific steps of dispersing NH2-Fe3O4 in an organic solvent include: adding NH2-Fe3O4 into anhydrous DMF, sealing and then ultrasonicating for 20 to 30 minutes.

5. The method for preparing the conjugated microporous polymer material Fe3O4@TbDt according to claim 2, characterized in that: The specific steps of the Soxhlet extraction include: using a mixture of methanol and tetrahydrofuran with a V / V ratio of 1:1 to perform Soxhlet extraction at 95-96°C.

6. A magnetic solid phase extractant, characterized in that The components of the magnetic solid phase extractor include: the conjugated microporous polymer material Fe3O4@TbDt according to claim 1.

7. Use of the magnetic solid phase extraction agent according to claim 6 in the determination of estrogen in dairy products by HPLC-MS / MS.

8. A method for determining estrogen in dairy products by HPLC-MS / MS, characterized in that: include: The conjugated microporous polymer material Fe3O4@TbDt according to claim 1 is used as an MSPE adsorbent, and 9 estrogens in dairy products are detected by HPLC-MS / MS method. The nine estrogens are estriol, α-estradiol, β-estradiol, hexaneestradiol, bisphenol estrogen, estrone, bisphenol A, bisphenol F, and ethinylestradiol.

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