7-ketolithocholic acid molecularly imprinted polymer microspheres and their application in solid phase extraction

Abstract

The application discloses a 7-ketolithocholic acid molecularly imprinted polymer microsphere and application thereof in solid phase extraction, and belongs to the field of functional polymer materials and separation and analysis technologies. The microsphere is a hollow structure formed by dissolving a silica core, the overall particle size is 3-5 mu m, the particle size distribution index PDI is 0.05-0.08, the molecularly imprinted polymer shell is formed by taking lithocholic acid as a template molecule and through a thermal initiation free radical copolymerization reaction, and is copolymerized by the template molecule, a functional monomer 4-vinylphenylboronic acid and acrylamide, and a crosslinking agent ethylene glycol dimethacrylate in a molar ratio of 1:1:2:20. The application further provides a preparation method of the microsphere and a solid phase extraction column containing the microsphere. The molecularly imprinted polymer microsphere is used as a solid phase extraction adsorbent, can selectively enrich and purify 7-ketolithocholic acid from serum or bile samples, has high selectivity and high recovery rate, and completely avoids the template leakage problem.

Description

Technical Field

[0001] This invention relates to the fields of functional polymer materials and separation and analysis technology, specifically to 7-ketolithocholic acid molecularly imprinted polymer microspheres and their application in solid-phase extraction. Background Technology

[0002] 7-Ketolithocholic acid is an important secondary bile acid produced by the metabolism of primary bile acids by human intestinal microorganisms. Recent studies have shown that its concentration changes in serum and bile are closely related to various hepatobiliary diseases, intestinal inflammation, and metabolic syndrome. Therefore, accurate detection of 7-ketolithocholic acid in biological samples, such as serum and bile, is of great significance for the diagnosis, prognostic assessment, and pathological mechanism research of related diseases.

[0003] Because biological samples have complex matrices and the concentration of target analytes is usually low, solid-phase extraction (SPE) is typically used to pretreat the samples before instrumental analysis to enrich the target analytes and purify the matrix. Currently, commercially available SPE adsorbents are mainly represented by C18 bonded silica gel. However, these traditional adsorbents rely on non-specific hydrophobic interactions, resulting in poor selectivity when faced with coexisting bile acid homologues with similar structures. This leads to unstable recovery rates of target analytes and severe matrix interference, affecting the accuracy and sensitivity of subsequent detection. Molecularly imprinted polymers (MIPs) are synthetic materials with pre-defined recognition capabilities, often referred to as plastic antibodies. The principle behind them is that in the presence of a template molecule, functional monomers and crosslinking agents copolymerize to form a three-dimensional network structure. After the template is eluted, specific cavities that are complementary to the template molecule in shape, size, and functional group arrangement are left in the polymer, thereby achieving specific recognition of the target molecule. However, applying traditional molecularly imprinted polymers directly to solid-phase extraction, especially for biomolecules such as 7-ketolithocholic acid, faces two prominent technical challenges: First, there is the issue of template molecule leakage. Traditional self-molding strategies directly use the target analyte 7-ketolithocholic acid itself as a template. However, due to the large number of firmly bound sites generated during the imprinting process, a small number of template molecules are always deeply embedded and difficult to completely remove during the subsequent template elution step. In subsequent solid-phase extraction applications, these residual template molecules are slowly released during the elution stage, causing so-called template leakage. This can lead to false positive signals when analyzing blank samples, seriously interfering with the accurate quantification of low concentrations of 7-ketolithocholic acid in actual samples. Second, there are issues with mass transfer efficiency and site accessibility. Traditional bulk imprinted polymers, after being pulverized, produce particles with irregular shapes and wide particle size distributions, which can easily lead to excessively high column pressure and uneven flow rates when packed into SPE columns. More importantly, a large number of imprinted sites are buried deep within the polymer network, requiring target molecules to diffuse over long distances to reach the binding sites, resulting in slow adsorption / desorption kinetics and low extraction efficiency. Although some studies have improved the morphology by preparing microspheres, achieving both high mass transfer efficiency and high site accessibility simultaneously remains a challenge. What's more challenging is that the two technical problems mentioned above are interconnected and form a vicious cycle. In order to completely elute the template and solve the leakage problem, stronger elution conditions are often required, such as stronger acids and longer elution times. However, this will exacerbate the destruction of the polymer network and the embedding of imprinted sites, further reducing mass transfer efficiency. Conversely, if the degree of crosslinking is reduced or the pore structure is optimized in order to pursue high mass transfer efficiency, it may lead to a decrease in the stability of imprinted holes, making it impossible to effectively fix template molecules, which will instead increase the difficulty of elution and the risk of leakage. Therefore, there is an urgent need in the field to develop a solid-phase extraction material that can fundamentally avoid template leakage, while possessing high mass transfer efficiency and high selectivity for 7-ketolithocholic acid, in order to meet the stringent requirements for accuracy, sensitivity and reproducibility in the analysis of complex biological samples. Summary of the Invention

[0004] This invention provides 7-ketolithocholic acid molecularly imprinted polymer microspheres and their application in solid-phase extraction, in order to solve the problem of the lack of solid-phase extraction materials with high mass transfer efficiency and high selectivity for 7-ketolithocholic acid in the prior art. 7-Ketolithocholic acid molecularly imprinted polymer microspheres for solid-phase extraction have a hollow structure with a particle size of 3-5 μm. The hollow structure is formed by the dissolution of a silica core. The molecularly imprinted polymer shell is formed by thermally initiated free radical copolymerization using lithocholic acid as a template molecule. The hollow structure works synergistically with the template molecules, enabling the microspheres to achieve both template molecule leakage-free operation and rapid adsorption and elution of 7-ketolithocholic acid when used for solid-phase extraction.

[0005] Preferably, the molecularly imprinted polymer shell is copolymerized from template molecules, functional monomers, and crosslinking agents; The functional monomers are 4-vinylphenylboronic acid and acrylamide; The crosslinking agent is ethylene glycol dimethacrylate.

[0006] Preferably, the molar ratio of template molecule, 4-vinylphenylboronic acid, acrylamide and crosslinking agent is 1:1:2:20.

[0007] Preferably, the particle size distribution index (PDI) of the microspheres is 0.05-0.08.

[0008] Preferably, a method for preparing 7-ketolithocholic acid molecularly imprinted polymer microspheres includes the following steps: S1, prepare monodisperse silica microspheres as the core; S2, on the surface of the silica core, the template molecules, functional monomers and crosslinking agents undergo a thermally initiated free radical copolymerization reaction in the presence of an initiator to form a molecularly imprinted polymer shell, resulting in core-shell structured composite microspheres; S3, the silica core in the composite microspheres obtained in step S2 is dissolved by dilute hydrofluoric acid solution or strong alkali solution, and the template molecules are simultaneously eluted to obtain hollow 7-ketolithocholic acid molecularly imprinted polymer microspheres.

[0009] Preferably, in step S2, the initiator for the thermally initiated free radical copolymerization reaction is azobisisobutyronitrile, and the reaction temperature is 60-70℃.

[0010] Preferably, a solid-phase extraction column includes a column tube, sieve plates located at both ends of the column tube, and packing material filled inside the column tube. The packing material is 7-ketolithocholic acid molecularly imprinted polymer microspheres. The solid-phase extraction column can specifically enrich and purify 7-ketolithocholic acid from serum or bile samples, and there is no template molecule leakage during operation.

[0011] Preferably, the application of 7-ketolithocholic acid molecularly imprinted polymer microspheres as a solid-phase extraction adsorbent for the enrichment and purification of 7-ketolithocholic acid from serum or bile samples can avoid detection interference caused by template molecule leakage.

[0012] Preferably, the above application includes the following steps: Activation and equilibration: a solid-phase extraction device containing molecularly imprinted polymer microspheres was balanced with methanol and water. Sample loading: The serum or bile sample solution containing 7-ketolithocholic acid is loaded onto the equilibrated solid-phase extraction device. Rinsing was performed using an acetonitrile-water solution with a volume ratio of 9:1. Elution was performed using a methanol solution containing 0.5-1% acetic acid as the eluent to elute and collect the specifically adsorbed 7-ketolithocholic acid.

[0013] The beneficial effects of this invention are as follows: Compared with the prior art, the present invention has the following beneficial effects: To fundamentally and collaboratively solve the problems of template leakage and mass transfer, this invention adopts a collaborative strategy that combines virtual templates with hollow structures. The virtual template (lithocholic acid) cuts off the source of the leaked substance (7-ketolithocholic acid) at the molecular level, and even if there are trace amounts of residue, it will not interfere with the detection. The hollow structure provides an ideal mass transfer channel for the complete removal of the virtual template, making it easy to remove the virtual template that might otherwise be buried, thus laying the foundation for high-fidelity regeneration of the hole. The two work together to break through the traditional dilemma of site destruction caused by strong elution, and achieve the acquisition of leakage-free imprinted materials with high mass transfer efficiency under mild conditions; Enhanced selectivity is achieved by using a mixed functional monomer system of 4-vinylphenylboronic acid and acrylamide, which can form multiple interactions with the ketone, carboxyl, and hydroxyl groups on the 7-ketolithocholic acid molecule to construct a highly selective recognition hole that can effectively distinguish bile acid homologues with similar structures. Solid-phase extraction columns have high efficiency. The microspheres have a regular spherical shape of 3-5μm and excellent monodispersity, making them easy to pack into uniform and stable solid-phase extraction columns, exhibiting advantages such as low column pressure and good reproducibility. It exhibits excellent application results. When used for solid-phase extraction pretreatment of complex biological samples such as serum and bile, it can effectively remove matrix interference and achieve efficient enrichment and purification of 7-ketolithocholic acid. Attached Figure Description

[0014] Figure 1 This is a comparison of the adsorption isotherms of the 7-ketolithocholic acid molecularly imprinted polymer microspheres prepared in Example 1 of the present invention and the non-imprinted polymer microspheres prepared in Comparative Example 1. Figure 2 The adsorption kinetics curve of the 7-ketolithocholic acid molecularly imprinted polymer microspheres prepared in Example 1 of this invention; Figure 3 This is a bar chart showing the competitive adsorption of MIP-1 and NIP-1 on 7-ketolithocholic acid and its structural analogs in Example 2 of the present invention. Figure 4 This is a comparison diagram of template leakage in solid-phase extraction between the molecularly imprinted polymer microspheres using 7-ketolithocholic acid as the real template in Comparative Example 2 of the present invention and the microspheres in Example 1 of the present invention. Detailed Implementation

[0015] To further illustrate the technical means and effects adopted by the present invention to achieve its intended purpose, exemplary embodiments will be described in detail below, examples of which are illustrated in the accompanying drawings. In the following description relating to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. Rather, they are merely examples of methods and systems consistent with some aspects of this application as detailed in the appended claims.

[0016] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The singular forms “a” and “the” as used in this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0017] To better illustrate the purpose, technical solutions, and advantages of this application, the following description, in conjunction with specific embodiments and comparative examples, aims to provide a detailed understanding of the content of this application, rather than limiting it. All other embodiments obtained by those skilled in the art without inventive effort are within the protection scope of this application. Unless otherwise specified, the experimental reagents and instruments involved in the implementation of this application are commonly used reagents and instruments. In this application, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0018] The following detailed description of the specific implementation methods, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided in detail.

[0019] Example 1 This embodiment provides a 7-ketolithocholic acid molecularly imprinted polymer microsphere, the preparation method of which is as follows: Monodisperse silica microspheres were prepared using the Stöber method as the core. Add 50 mL of anhydrous ethanol, 5 mL of ultrapure water, and 28 wt% of concentrated ammonia to a 250 mL round-bottom flask and stir magnetically at 300 rpm for 10 minutes at 25 °C to form a homogeneous solution. Slowly add 3 mL of tetraethyl orthosilicate (TEOS) and continue the reaction for 6 hours to obtain a white suspension. The silica microspheres were collected by centrifugation at 8000 rpm for 10 minutes, washed three times each with anhydrous ethanol and ultrapure water, and then dried under vacuum at 60°C for 12 hours. The average particle size of the obtained silica microspheres was measured by a laser particle size analyzer to be 3.0 μm, with good monodispersity and a particle size distribution index (PDI) of 0.05. A thermally initiated free radical copolymerization reaction is carried out on the surface of the silica core to form a molecularly imprinted polymer shell: 0.1 mmol of lithocholic acid (the template molecule), 0.1 mmol of 4-vinylphenylboronic acid, and 0.2 mmol of acrylamide (the functional monomer) were dissolved in 50 mL of a 1:1 mixture of acetonitrile and toluene and sonicated for 30 minutes to allow the template and functional monomer to be fully pre-assembled. Add 2.0 mmol of ethylene glycol dimethacrylate (EGDMA), i.e., crosslinking agent, and 10 mg of azobisisobutyronitrile (AIBN), i.e., initiator, to the above solution. Stir magnetically for 30 minutes until homogeneous. Add 200 mg of silica microspheres and purge with nitrogen gas to remove oxygen for 20 minutes. The reaction was carried out at 65℃ for 12 hours. After the reaction, the core-shell structured microspheres were collected by vacuum filtration and washed three times with acetonitrile. The molar ratio of template molecule, 4-vinylphenylboronic acid, acrylamide and crosslinking agent is strictly controlled at 1:1:2:20; Template elution and core dissolution: The composite microspheres were extracted with methanol / acetic acid 9:1, v / v, by Soxhlet extraction for 24 hours, and then dispersed in 20 mL of 0.5 mol / L HF and shaken for 4 hours to dissolve the core. Washed with ultrapure water until neutral, and vacuum dried at 60°C for 12 hours to obtain 7-ketolithocholic acid molecularly imprinted polymer microspheres, denoted as hollow MIP-1 microspheres; Please refer to Figure 2 The adsorption kinetics curves show that MIP-1 reaches adsorption equilibrium within 30 minutes, far exceeding traditional bulk imprinted polymers, which typically take several hours. This is attributed to the hollow structure, which enriches the imprinted sites in the shell and shortens the diffusion path. The final product was measured by a laser particle size analyzer, and the average particle size was 4.2 μm, with a PDI of 0.08. To verify the material properties, adsorption kinetics and static adsorption capacity tests were conducted: Prepare 50 mL of 50 mg / L 7-ketolithocholic acid ethanol solution and add 20 mg of MIP-1 microspheres; The sample was taken at different time points (5, 10, 20, 30, 60, and 120 min) after constant temperature oscillation at 25℃, and the concentration of the supernatant was determined after centrifugation. Please refer to Figure 1 The adsorption capacity was calculated and kinetic curves were plotted. The results showed that MIP-1 reached adsorption equilibrium within 30 minutes, with an equilibrium adsorption capacity of 17.8 mg / g, demonstrating its rapid adsorption capacity. Prepare a series of 7-ketolithocholic acid ethanol solutions with concentrations of 10-100 mg / L, and add 20 mg of MIP-1 or NIP-1 to each solution. NIP-1 is prepared as described in Comparative Example 1 below. After oscillating at a constant temperature to equilibrium, the concentration is measured and the equilibrium adsorption capacity is calculated. Please refer to Figure 1 The maximum adsorption capacity of MIP-1 for 7-ketolithocholic acid was 18.3 mg / g, while that of NIP-1 was only 6.1 mg / g, proving the successful construction of imprinted holes.

[0020] Comparative Example 1 This comparative example illustrates the preparation process of non-imprinted polymer microspheres, denoted as NIP-1. This comparative example serves as a blank control to verify the molecular imprinting effect. The preparation process is exactly the same as in Example 1, except that the template molecule lithocholic acid is not added. Monodisperse silica microspheres were prepared using the same Stöber method as in Example 1 as the core. The average particle size of the obtained silica microspheres was 3.0 μm and the PDI was 0.06, as determined by a laser particle size analyzer. A thermally initiated free radical copolymerization reaction is carried out on the surface of a silica core to form a non-imprinted polymer shell: Dissolve 0.1 mmol of 4-vinylphenylboronic acid and 0.2 mmol of acrylamide, the functional monomer, in 50 mL of a 1:1 volume ratio of acetonitrile / toluene mixture and sonicate for 30 minutes. No template molecule is added in this step. Add 2.0 mmol of ethylene glycol dimethacrylate (EGDMA), a crosslinking agent, and 10 mg of azobisisobutyronitrile (AIBN) and an initiator to the above solution. Stir magnetically for 30 minutes until homogeneous. Add 200 mg of the above silica microspheres and purge with nitrogen to remove oxygen for 20 minutes. The reaction was carried out in a constant temperature water bath at 65°C for 12 hours, maintaining the same reaction temperature and time as in Example 1. The molar ratio of 4-vinylphenylboronic acid, acrylamide, and crosslinking agent is maintained at 1:2:20; The same post-processing steps as in Example 1 were used; After the reaction was completed, the composite microspheres were collected by vacuum filtration and washed three times with acetonitrile to remove unreacted monomers. The microspheres were dispersed in 20 mL of 0.5 mol / L hydrofluoric acid solution and shaken at room temperature for 4 hours to completely dissolve the silica core. Washed with ultrapure water until neutral, pH≈7, and vacuum dried at 60℃ for 12 hours to obtain hollow non-imprinted polymer microspheres NIP-1; The average particle size of NIP-1 microspheres was 4.3 μm and the particle size distribution index (PDI) was 0.08, as determined by a laser particle size analyzer. They have similar physical morphology to MIP-1. Static adsorption capacity was determined using the same test method as in Example 1. Please refer to Figure 1 The maximum adsorption capacity of NIP-1 for 7-ketolithocholic acid was only 6.1 mg / g, which was lower than that of MIP-1 (18.3 mg / g), demonstrating that non-imprinted materials lacking imprinted holes have a weaker adsorption capacity for target molecules.

[0021] Comparative Example 2 This embodiment provides a molecularly imprinted polymer microsphere, denoted as MIP-2, using 7-ketolithocholic acid as a template, and its preparation method is as follows: The preparation process is basically the same as in Example 1, except that 0.1 mmol of 7-ketolithocholic acid is used instead of lithocholic acid as the template molecule. Dissolve 0.1 mmol of 7-ketolithocholic acid (template molecule), 0.1 mmol of 4-vinylphenylboronic acid and 0.2 mmol of acrylamide in 50 mL of acetonitrile / toluene mixed solvent at a volume ratio of 1:1; Add 2.0 mmol EGDMA and 10 mg AIBN; Add 200 mg of silica microspheres (3.0 μm, PDI = 0.06); The reaction was carried out at 65°C for 12 hours, followed by Soxhlet extraction with methanol / acetic acid = 9:1 and HF etching. The molar ratio of template molecule, functional monomer and crosslinking agent in all samples was maintained at 1:1:2:20; Accurately weigh 50.0 mg of each sample and pack it into an SPE column. Equilibrate with 5 mL of methanol and 5 mL of ultrapure water sequentially. Perform blank elution with 3 mL of methanol solution containing 1% acetic acid. Concentrate the eluent to 0.5 mL with nitrogen. Detect by LC-MS / MS using an electrospray ionization source in negative ion mode and monitor with MRM. Table 1. Template leakage test results (LC-MS / MS analysis) ; As can be seen from the data in Table 1, all samples using 7-ketolithocholic acid as the real template showed significant template leakage, ranging from 12.1 to 13.2 ng / mL. Even after 48 hours of Soxhlet extraction, the template molecules could not be completely removed, and the leakage level would seriously interfere with the actual detection, which was usually within the range of 5-100 ng / mL.

[0022] Example 2 To verify the specific recognition ability of the 7-ketolithocholic acid molecularly imprinted polymer microspheres MIP-1 prepared in Example 1 to the template molecule, the molecular recognition selectivity was evaluated by calculating its selectivity coefficient for 7-ketolithocholic acid relative to two structural analogs, such as lithocholic acid and chenodeoxycholic acid. The adsorbent was 7-ketolithocholic acid molecularly imprinted polymer microspheres MIP-1 prepared in Example 1; Non-imprinted polymer microspheres NIP-1 prepared in Comparative Example 1; Target and interfering substances: 7-ketolithocholic acid (7-KLCA), purity ≥98%; Lithocholic acid (LCA), purity ≥97%; Chedeoxycholic acid (CDCA), purity ≥98%; Solvent: Ethanol, analytical grade; Accurately weigh 10.0 mg each of 7-ketolithocholic acid, lithocholic acid and chenodeoxycholic acid, place them in the same 100 mL volumetric flask, dissolve them in ethanol and dilute to the mark, shake well, and prepare a single stock solution with a concentration of 100 mg / L for each of the three cholic acids. Before use, dilute the stock solution with ethanol to obtain a ternary mixed competitive adsorption solution with a concentration of 10.0 mg / L for each of the three cholic acids. Accurately weigh 20.0 mg each of MIP-1 prepared in Example 1 and NIP-1 prepared in Comparative Example 1, accurate to 0.1 mg, and place them in 50 mL stoppered conical flasks. Add 20.0 mL of the above ternary mixed competitive adsorption solution to each conical flask, i.e., the initial amount of each cholic acid is 0.20 mg. After sealing the conical flasks, place them in a constant temperature shaker and shake at 150 rpm at 25°C for 4 hours to ensure that adsorption equilibrium is reached. Meanwhile, a blank control group without any adsorbent was set up to determine the initial concentration of the target analyte; After adsorption equilibrium, the supernatant was filtered through a 0.22 μm organic microporous membrane, and the equilibrium concentration Ce of the three bile acids in the filtrate was determined by HPLC. The HPLC analysis conditions were as follows: mobile phase: acetonitrile: 20 mL potassium dihydrogen phosphate buffer, pH 4.5 = 60:40 (v / v), flow rate 1.0 mL / min, detection wavelength 205 nm, column temperature 30 °C, and injection volume 10 μL. Calculation of equilibrium adsorption capacity Qe: The equilibrium adsorption capacities of MIP-1 and NIP-1 for each bile acid were calculated using the formula:

[0023] Where C0 is the initial concentration of the target analyte, which is 10.0 mg / L in this experiment; Ce is the equilibrium concentration of the target analyte; V is the solution volume, which is 0.020 L in this experiment; and m is the adsorbent mass, which is 0.0200 g in this experiment. Allocation coefficient Kd = Qe / Ce; The selectivity coefficient α was calculated relative to lithocholic acid LCA and chenodeoxycholic acid CDCA using 7-ketolithocholic acid 7-KLCA as the template molecule.

[0024] Where X represents LCA or CDCA; The relative selectivity coefficient β is used to eliminate the influence of nonspecific adsorption, and the relative selectivity coefficient is further calculated.

[0025] Table 2. Competitive adsorption experimental data and results of selectivity coefficient calculation. ; Please refer to Figure 3 According to Table 2, in a competitive environment where structural analogs coexist, MIP-1 adsorbed 17.10 mg / g of the template molecule 7-ketolithocholic acid, which was higher than the adsorption amounts of lithocholic acid (3.20 mg / g) and chenodeoxycholic acid (2.38 mg / g). MIP-1 exhibits excellent selectivity for 7-ketolithocholic acid, with selectivity coefficients α as high as 14.8 and 20.6 relative to lithocholic acid and chenodeoxycholic acid, respectively. The relative selectivity coefficients β calculated by comparison with NIP-1 were 3.5, relative to lithocholic acid and 4.1, relative to chenodeoxycholic acid. The imprinted holes formed by the template molecule (lithocholic acid) and functional monomers (4-vinylphenylboronic acid and acrylamide) in a molar ratio of 1:1:2:20 have a precise and specific recognition ability for 7-ketolithocholic acid, rather than simple physical adsorption.

[0026] Example 3 In this embodiment, the 7-ketolithocholic acid molecularly imprinted polymer microspheres MIP-1 prepared in Example 1 were applied to the solid-phase extraction process of actual biological samples, and their performance in practical applications was verified through spike recovery experiments and template leakage investigation. 50 mg of the MIP-1 microspheres prepared in Example 1 were accurately weighed and used as packing material. They were then packed into a 3 mL solid phase extraction empty column using a dry method. A sieve plate with a 20 μm pore size was added to each end to prepare a MIP-SPE column. The MIP-1 microspheres used had an average particle size of 4.2 μm and a PDI of 0.07. Commercially available healthy human blank mixed serum was used as the experimental matrix. Each package contained 5 mL, and a total of 3 vials were used, for a total of 15 mL. Prepare a stock solution of 7-ketolithocholic acid standard with methanol, and dilute it with blank serum to accurately prepare spiked samples at low, medium, and high concentrations: Low concentration: 50 ng / mL; Medium concentration: 200 ng / mL High concentration: 500 ng / mL Five samples were prepared in parallel for each concentration level (n=5); Activation and equilibration were performed by sequentially passing 5 mL of methanol and 5 mL of ultrapure water through a MIP-SPE column at a flow rate of approximately 1 mL / min. For sample loading, accurately take 5 mL of spiked serum sample and load it onto the equilibrated solid-phase extraction device at a flow rate of 0.5 mL / min. Rinsing was performed using 3 mL of acetonitrile-water solution at a volume ratio of 9:1 to remove non-specific adsorbed impurities from the serum matrix. Elution was performed using 3 mL of methanol solution containing 1% (v / v) acetic acid as the eluent, and the specifically adsorbed 7-ketolithocholic acid was eluted and collected at a flow rate of 0.5 mL / min. The collected eluent was purged with nitrogen at 40°C until nearly dry, then reconstituted with 100 μL of methanol and detected by LC-MS / MS. Chromatographic conditions: C18 column, 2.1×100mm, 1.8μm, mobile phase: acetonitrile-5 mM ammonium acetate aqueous solution, gradient elution, electrospray ionization source in negative ion mode, multiple reaction monitoring (MRM); Recovery rate (%) = (detected concentration / spiked concentration) × 100%; Table 3. Results of spiked recovery experiments (n=5) ; The solid-phase extraction column packed with MIP-1 microspheres prepared in this invention has excellent enrichment ability for 7-ketolithocholic acid in serum, with a recovery rate that is stable between 92.6% and 97.8%. To verify the advantages of this invention in solving the template leakage problem, a comparative experiment was conducted: Experimental group: SPE columns filled with MIP-1 microspheres prepared in Example 1 and using lithocholic acid as a virtual template; Control group: SPE columns filled with MIP-2B microspheres prepared in Comparative Example 2, using 7-ketolithocholic acid as the real template; The eluent was collected using the same solid-phase extraction process, concentrated, and then detected by high-sensitivity LC-MS / MS. Table 4 Template Leakage Detection Results ; Please refer to Figure 4 As shown in Table 4, the MIP-1 microspheres prepared using lithocholic acid as a virtual template avoid template leakage in subsequent solid-phase extraction applications. This excellent characteristic stems not only from the correct selection of the virtual template but also from the unique hollow structure of the microspheres. This structure enables the template molecules to be efficiently eluted during the preparation process through the core dissolution and Soxhlet extraction steps, fully exposing the imprinted sites on the shell surface. This solves the core technical problem of incomplete template elution caused by the deep embedding of imprinted sites in traditional bulk imprinted polymers. This achievement is not a simple superposition of the two features of virtual template and hollow structure, but rather stems from the precise synergistic design of the two: The hollow structure ensures that the virtual template can be completely washed away, and the use of the virtual template allows the rapid mass transfer advantage of the hollow structure to be fully used for the adsorption / desorption of the target analyte, rather than to deal with the historical burden caused by its own template leakage.

[0027] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. 7-Ketolithocholic Acid Molecularly Imprinted Polymer Microspheres for Solid-Phase Extraction, characterized in that, The microspheres have a hollow structure, and the microspheres are hollow microspheres with a particle size of 3-5 μm. The hollow structure is formed by dissolving a silica core. The molecularly imprinted polymer shell is formed by thermally initiated free radical copolymerization using lithocholic acid as a template molecule. The hollow structure works synergistically with the template molecule, enabling the microspheres to achieve both template molecule leakage-free operation and rapid adsorption and elution of 7-ketolithocholic acid when used for solid-phase extraction.

2. The 7-ketolithocholic acid molecularly imprinted polymer microspheres according to claim 1, characterized in that, The molecularly imprinted polymer shell is copolymerized from template molecules, functional monomers and crosslinking agents; The functional monomers are 4-vinylphenylboronic acid and acrylamide; The crosslinking agent is ethylene glycol dimethacrylate.

3. The 7-ketolithocholic acid molecularly imprinted polymer microspheres according to claim 2, characterized in that, The molar ratio of the template molecule, 4-vinylphenylboronic acid, acrylamide and crosslinking agent is 1:1:2:

20.

4. The 7-ketolithocholic acid molecularly imprinted polymer microspheres according to claim 1, characterized in that, The particle size distribution index (PDI) of the microspheres is 0.05-0.

08.

5. A method for preparing 7-ketolithocholic acid molecularly imprinted polymer microspheres as described in any one of claims 1-4, characterized in that, Includes the following steps: S1, prepare monodisperse silica microspheres as the core; S2, on the surface of the silica core, the template molecules, functional monomers and crosslinking agents undergo a thermally initiated free radical copolymerization reaction in the presence of an initiator to form a molecularly imprinted polymer shell, thereby obtaining core-shell structured composite microspheres; S3, the silica core in the composite microspheres obtained in step S2 is dissolved by dilute hydrofluoric acid solution or strong alkali solution, and the template molecules are simultaneously eluted to obtain hollow 7-ketolithocholic acid molecularly imprinted polymer microspheres.

6. The preparation method according to claim 5, characterized in that, In step S2, the initiator for the thermally initiated free radical copolymerization reaction is azobisisobutyronitrile, and the reaction temperature is 60-70℃.

7. A solid-phase extraction column, comprising a column tube, sieve plates located at both ends of the column tube, and packing material filled inside the column tube, characterized in that, The packing material is the 7-ketolithocholic acid molecularly imprinted polymer microspheres according to any one of claims 1-4, and the solid phase extraction column can specifically enrich and purify 7-ketolithocholic acid from serum or bile samples, and there is no template molecule leakage during operation.

8. The application of the 7-ketolithocholic acid molecularly imprinted polymer microspheres according to any one of claims 1-4 as a solid-phase extraction adsorbent for the enrichment and purification of 7-ketolithocholic acid from serum or bile samples, characterized in that, The application can avoid detection interference caused by template molecule leakage.

9. The application according to claim 8, characterized in that, Includes the following steps: Activation and equilibration: a solid-phase extraction apparatus containing the molecularly imprinted polymer microspheres was equilibrated with methanol and water. Sample loading: The serum or bile sample solution containing 7-ketolithocholic acid is loaded onto the equilibrated solid-phase extraction device. Rinsing was performed using an acetonitrile-water solution with a volume ratio of 9:

1. Elution was performed using a methanol solution containing 0.5-1% acetic acid as the eluent to elute and collect the specifically adsorbed 7-ketolithocholic acid.

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