Special medical food vitamin b12 enrichment and detection material and method based on magnetic molecular imprinting

Abstract

The application relates to the technical field of food detection, in particular to a special medical food vitamin B12 enrichment and detection material and method based on magnetic molecular imprinting. The material is a magnetic molecular imprinting polymer (MMIPs), which takes Fe3O4 as a magnetic core, is coated with a SiO2 layer and a KH570 coupling agent in sequence, and is coupled with a vitamin B12 imprinting polymer layer through surface molecular imprinting technology. The imprinting polymer layer is polymerized from a template molecule vitamin B12 (CNcbl), a functional monomer methacrylic acid (MAA) and a crosslinking agent trimethylolpropane trimethacrylate (TRIM). The material of the application realizes specific recognition through molecular imprinting technology, and simplifies a separation step by using superparamagnetism, so that the cost is significantly reduced and the efficiency is improved.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, specifically to a material and method for the enrichment and detection of vitamin B12 in special medical foods based on magnetic molecular imprinting. Background Technology

[0002] Currently, the national standard GB5009.285-2022 uses immunoaffinity column purification-liquid chromatography to detect vitamin B12 in food. While this method is accurate and reliable, it has significant drawbacks:

[0003] 1. High cost: Immunoaffinity columns rely on monoclonal antibodies, which are difficult to prepare and expensive. They are also for single use, resulting in huge cost pressures when conducting large-scale testing.

[0004] 2. Cumbersome operation: Pretreatment requires multiple steps (such as activation, rinsing, and elution), and the analysis time for a single sample is long and the efficiency is low.

[0005] 3. Poor stability: Biological antibodies are easily inactivated by environmental conditions and have insufficient stability.

[0006] 4. High dependence on equipment: It requires the use of large-scale equipment such as high performance liquid chromatography (HPLC), which limits its widespread application.

[0007] The complex matrix of foods for special medical purposes exacerbates these problems. Therefore, there is an urgent need to develop a low-cost, easy-to-operate, stable, and reusable alternative. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a material and method for the enrichment and detection of vitamin B12 based on magnetic molecularly imprinted polymers. This material achieves specific recognition through molecular imprinting technology and simplifies the separation steps by utilizing superparamagnetism, significantly reducing costs and improving efficiency.

[0009] This invention is achieved through the following technical solution:

[0010] A material for the enrichment and detection of vitamin B12 in special medical foods based on magnetic molecular imprinting is provided. The material is a magnetic molecular imprinted polymer (MMIPs), which uses Fe3O4 as a magnetic core, and is sequentially coated with a SiO2 layer and a KH570 coupling agent. The vitamin B12 imprinted polymer layer is coupled through surface molecular imprinting technology. The imprinted polymer layer is polymerized from the template molecule vitamin B12 (CNcbl), the functional monomer methacrylic acid (MAA), and the crosslinking agent trimethylolpropane trimethacrylate (TRIM).

[0011] In this scheme, the magnetic molecular imprinted material is Fe3O4@SiO2 / KH570@MMIPs, and its structure includes:

[0012] Magnetic core: Fe3O4 nanoparticles, synthesized by co-precipitation method (FeCl3·6H2O and FeCl2·4H2O react with ammonia at 70℃).

[0013] Intermediate layer: SiO2 coating layer (formed by tetraethyl orthosilicate (TEOs) catalyzed by ammonia), coupling agent KH570 is used to enhance the bonding.

[0014] Imprinted layer: Using vitamin B12 as a template molecule, methacrylic acid (MAA) as a functional monomer, and trimethylolpropane trimethacrylate (TRIM) as a cross-linking agent, specific recognition sites are formed through polymerization.

[0015] This invention integrates the functions of both the magnetic core and the imprinted polymer layer through a core-shell structure (Fe3O4@SiO2 / KH570@MMIPs). The SiO2 interlayer plays a crucial role: it protects the magnetic core from chemical corrosion and provides abundant coupling sites through KH570, ensuring the robust growth of the imprinted polymer layer. This design enables the recognition unit (imprinted layer) and the separation unit (magnetic core) to work efficiently together, achieving a smooth process of immediate separation after recognition, producing a convenient effect that cannot be achieved by a single material.

[0016] The principle of this invention is as follows: Using molecular imprinting technology, a functional monomer (MAA) is pre-assembled with a template molecule (vitamin B12), and then frozen within a polymer network using a cross-linking agent (TRIM). After template elution, the resulting cavity is highly complementary to vitamin B12 in size, shape, and chemical functional groups (such as carboxyl groups), fundamentally solving the inherent problems of high cost and poor stability associated with biometric recognition principles. Experiments have demonstrated that using the three-dimensional cross-linking agent TRIM (compared to the traditional chain-like EGDMA) can form a denser and more stable cross-linked network. The three functional groups of TRIM act as multiple anchor points, more firmly fixing the spatial orientation of the functional monomer, making the imprinted cavity less prone to deformation and collapse during elution and use, thereby significantly improving the stability and binding capacity of the recognition site.

[0017] Utilizing the superparamagnetism of the Fe3O4 core, the material is instantly magnetized and directionally moved under an applied magnetic field; after the magnetic field is removed, the magnetism disappears immediately, and the material can be redispersed, achieving "one-click" rapid separation, which greatly simplifies the operation in principle.

[0018] Preferably, the amount of crosslinking agent TRIM added is 4~12 mmol, and the amount of functional monomer MAA added is 2~6 mmol.

[0019] Furthermore, the magnetic core Fe3O4 was prepared by co-precipitation, in which the mass ratio of FeCl3·6H2O and FeCl2·4H2O was (1.8~2.2):1, and it was generated by reacting with ammonia water at 60~80℃.

[0020] Preferably, the SiO2 layer is coated by tetraethyl orthosilicate (TEOs) in an ethanol solution catalyzed by ammonia, with the amount of TEOs added being 3-5 mL.

[0021] A method for enriching and detecting vitamin B12 using the aforementioned magnetic molecular imprinting material, comprising the following steps:

[0022] (a) Sample pretreatment: Weigh the special medical food sample, add an appropriate amount of water to disperse it, and then homogenize it to obtain a uniform sample homogenate; then add protease to perform enzymatic hydrolysis to release bound vitamin B12.

[0023] (b) Enrichment: The enzymatic hydrolysate is mixed with the magnetic molecular imprinted material to carry out an adsorption reaction, and the material is separated by an external magnetic field;

[0024] (c) Elution: The adsorbed vitamin B12 was eluted with methanol-acetic acid solution;

[0025] (d) Detection: The eluent was dried with nitrogen and then reconstituted, and quantitatively detected by high performance liquid chromatography or mass spectrometry.

[0026] Preferably, the protease in step (a) is pepsin or trypsin, and the enzymatic hydrolysis temperature is 37°C.

[0027] Furthermore, in step (b), the adsorption reaction time is 30-60 minutes, and the separation time by the external magnetic field is within 10 seconds.

[0028] Furthermore, in step (c), the volume ratio of the methanol-acetic acid solution is 9:1.

[0029] Furthermore, the magnetic molecular imprinted material can be reused at least 5 times, and the adsorption capacity retention rate is not less than 80% after each use.

[0030] The application of the aforementioned magnetic molecular imprinted material in the detection of vitamin B12 in foods for special medical purposes, including foods for special medical purposes (FSMPs), showed that the spiked recovery rate was 77.84%~116.70%, and the relative standard deviation (RSD) was <10%.

[0031] The beneficial effects of this invention are:

[0032] I. Significantly reduce testing costs

[0033] This invention utilizes synthetic magnetic molecularly imprinted materials (MMIPs) to replace expensive immunoaffinity columns, solving the problem of high costs associated with the latter due to their reliance on monoclonal antibodies. Immunoaffinity columns are typically single-use, while MMIPs can be reused at least five times, maintaining good adsorption capacity retention after repeated use. This significantly reduces the cost per test for samples requiring high-volume analysis.

[0034] II. Simple and efficient operation, improving detection efficiency.

[0035] Utilizing the material's superparamagnetism (saturation magnetization of 12.13 emu / g), one-click solid-liquid separation is achieved in just 10 seconds. This eliminates the cumbersome steps of activation, rinsing, centrifugation, and filtration required in immunoaffinity column methods, significantly reducing the processing time for a single sample and improving detection efficiency.

[0036] III. The material has high stability and a wide range of applications.

[0037] Thermogravimetric analysis (TGA) results showed that MMIPs experienced only slight weight loss in the temperature range of 30–200°C, indicating excellent thermal stability under normal operating temperatures. The organic imprinted layer only underwent concentrated decomposition at around 420°C, demonstrating a compact material structure and uniform coating.

[0038] Specific adsorption experiments have demonstrated that MMIPs have high selectivity for vitamin B12 (CNcbl) (imprint factor IF=3.861), effectively eliminating interference from structural analogs such as thiamine, riboflavin, and folic acid, making them suitable for complex matrix samples such as special medical foods (e.g., infant formula).

[0039] IV. High accuracy and reliability in testing

[0040] In the actual sample testing of special medical foods, the spiked recoveries ranged from 77.84% to 116.70%, with relative standard deviations (RSDs) all <10%, meeting the testing requirements. Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) was used to validate the quality control samples; the measured results were close to the reference values ​​(quality control sample recovery rate 96.49%), further demonstrating the accuracy and applicability of the method.

[0041] V. Improved Environmental Adaptability and Universality

[0042] This method does not require reliance on large, specialized HPLC equipment in a laboratory and can be implemented using various chromatographic or mass spectrometric platforms, thus improving its versatility. By systematically optimizing key parameters such as the functional monomer (MAA, 3 mmol), crosslinking agent (TRIM, 8 mmol), and magnetic support (300 mg), the adsorption capacity and recognition efficiency are maximized, ensuring the stability of the method in different application scenarios.

[0043] In summary, this invention, through material innovation (MMIPs replacing immunoaffinity columns) and process optimization (magnetic separation + parameter control), significantly outperforms existing national standard methods in terms of cost, efficiency, stability, and accuracy, and is particularly suitable for the efficient and low-cost detection of vitamin B12 in special medical foods. Attached Figure Description

[0044] Figure 1 The images show scanning electron microscope (SEM) images of Fe3O4 (a), Fe3O4@SiO2 / KH570 (b), MMIPs (c), and MMIPs (d) prepared in Example 1 of this invention.

[0045] Figure 2 This is a bar chart comparing the adsorption capacity of CNcbl by MMIPs prepared with different amounts of magnetic materials in Embodiment 2 of the present invention.

[0046] Figure 3 This is a bar chart comparing the adsorption capacity of CNcbl by MMIPs prepared from five functional monomers in Example 3 of the present invention.

[0047] Figure 4 This is a graph showing the relationship between the amount of MAA added and the amount of MMIPs adsorbed in Example 4 of the present invention.

[0048] Figure 5 This is a graph showing the relationship between the amount of TRIM added and the amount of MMIPs adsorbed in Example 5 of the present invention.

[0049] Figure 6 This is a comparison chart of the selective adsorption effects of MMIPs and MNIPs on CNcbl and its analogues (thiamine, riboflavin, folic acid, OHcbl, Adocbl) in Example 6 of the present invention.

[0050] Figure 7 This is a thermal stability analysis diagram of MMIPs in Embodiment 6 of the present invention. Figure 7 (a) is a thermogravimetric analysis (TGA) curve. Figure 7 (b) is the derivative thermogravimetric analysis (DTG) curve.

[0051] Figure 8 This is a magnetization curve diagram of Fe3O4, Fe3O4@SiO2 / KH570 and MMIPs in Example 6 of the present invention.

[0052] Figure 9 This is a data table showing the results of the spike recovery experiment of the special medical food sample in Example 7 of the present invention. Detailed Implementation

[0053] To clearly illustrate the technical features of this solution, the following detailed implementation method will be used to explain the solution.

[0054] Example 1: Preparation of magnetic molecularly imprinted materials (Fe3O4@SiO2 / KH570@MMIPs)

[0055] This embodiment provides the specific synthesis steps for the core materials.

[0056] Synthesis of Fe3O4 magnetic nanoparticles: 2g FeCl3·6H2O and 1g FeCl2·4H2O were dissolved in ultrapure water and stirred under nitrogen protection, then heated to 70℃ in a water bath. 25% ammonia solution was added dropwise, and the mixture was refluxed for 1 hour, forming a black precipitate. The nanoparticles were collected using a magnet, washed with water until pH neutral, and dried under vacuum at 60℃.

[0057] SiO2 coating: 1 g of the Fe3O4 nanoparticles prepared above were dispersed in 100 mL of ethanol solution and sonicated for 30 minutes. Then, 3 mL of 25% ammonia water (NH3·H2O) and 4 mL of tetraethyl orthosilicate (TEOs) were added, and the mixture was stirred at room temperature for 12 hours. After the reaction was complete, the product was separated using an external magnetic field, washed three times each with anhydrous ethanol and ultrapure water, and dried under vacuum at 50 °C to obtain Fe3O4@SiO2.

[0058] KH570 coupling agent modification: 1.5 mL of KH570 was added to a mixture of 15 mL of water and 45 mL of anhydrous ethanol, stirred, and allowed to stand for 30 minutes each. The pH was adjusted to 4.4-5.0 with acetic acid. 300 mg of Fe3O4@SiO2 was added, and the mixture was reacted at 65 °C for 6 hours under nitrogen atmosphere. After the reaction, the mixture was washed three times alternately with ethanol and water, and dried to obtain Fe3O4@SiO2 / KH570.

[0059] Surface molecularly imprinted polymerization: 0.05 mmol of vitamin B12 (template molecule) and 3 mmol of methacrylic acid (MAA, functional monomer) were added to a two-necked flask, followed by 50 mL of dimethyl sulfoxide (DMSO) as solvent. Prepolymerization was carried out by stirring at room temperature for 12 hours. Then, 300 mg of Fe3O4@SiO2 / KH570 particles, 8 mmol of trimethylolpropane trimethacrylate (TRIM, crosslinking agent), and 0.18 mmol of initiator AIBN were added, and the mixture was sonicated for 15 minutes to degas. Polymerization was carried out at 65 °C for 12 hours under nitrogen protection. After polymerization, the mixture was cooled to room temperature, and the precipitate was separated using an external magnetic field. Soxhlet extraction was performed with a methanol:acetic acid (9:1, v / v) solution until vitamin B12 was undetectable at 361 nm by HPLC to completely elute the template molecule. Finally, residual acetic acid was washed with methanol, and the product was dried under vacuum at 60 °C for 12 hours to obtain the target product—magnetic vitamin B12 molecularly imprinted polymers (MMIPs).

[0060] The material morphology at key stages of the preparation process was characterized by scanning electron microscopy (SEM), and the results are as follows: Figure 1 As shown. Figure 1 (a) shows that the Fe3O4 nanoparticles prepared by the co-precipitation method are regular spherical with uniform particle size distribution and smooth surface. After SiO2 coating and KH570 coupling, the resulting Fe3O4@SiO2 / KH570 material... Figure 1 (b) While still spherical, the particle size is significantly increased, and the surface is more dense, indicating that the SiO2 layer has been successfully applied. The final MMIPs obtained... Figure 1 (c, d) The surface roughness is significantly increased, exhibiting a porous network structure, confirming that the vitamin B12 molecularly imprinted polymer layer has been successfully grafted onto the surface of the magnetic support. This morphological structure is beneficial for providing a large number of specific recognition sites, which is the structural basis for the material to achieve high adsorption capacity and selectivity.

[0061] Preparation of control material MNIPs: The preparation of magnetic non-molecularly imprinted polymers (MNIPs) is exactly the same as that of MMIPs, except that no template molecule (vitamin B12) is added.

[0062] Example 2: Optimization of Magnetic Carrier Addition Amount

[0063] Keeping other synthesis conditions unchanged, following the steps of Example 1, only changing the amount of magnetic support Fe3O4@SiO2 / KH570, a series of MMIPs were prepared, and their adsorption capacity for CNcbl was tested. The results are as follows... Figure 2As shown, the adsorption capacity of MMIPs on CNcbl is maximized when the magnetic support dosage is 300 mg. At this dosage, the carrier surface forms an imprinted polymer layer of optimal thickness, maximizing the total number of effective imprinted sites. This indicates that the molecularly imprinted polymer layer thickness on the Fe3O4@SiO2 / KH570 surface is optimal at this point. This is because when the magnetic material dosage is low, the number of supports available for loading the imprinted polymer is insufficient, resulting in a smaller total number of MMIPs formed. As the Fe3O4@SiO2 / KH570 dosage increases to 300 mg, the carrier surface provides sufficient sites for the imprinted polymer, allowing more MMIPs to be successfully loaded onto the magnetic particle surface, increasing the total number of effective imprinted sites and thus showing an increase in adsorption capacity. However, when the magnetic support dosage exceeds 300 mg, the adsorption capacity decreases. This may be because excessive magnetic support tends to aggregate in the polymerization system, preventing some support from fully contacting the functional monomers and template molecules, thereby reducing the loading efficiency of the imprinted polymer. Therefore, the addition of 300 mg Fe3O4@SiO2 / KH570 can achieve the maximum adsorption of CNcbl by MMIPs while ensuring good dispersibility and sufficient loading capacity of the carrier. Thus, this addition amount is determined to be the optimal amount of magnetic material.

[0064] Example 3: Functional Monomer Screening

[0065] MMIPs were prepared according to the steps of Example 1, using methacrylic acid (MAA), 3-aminopropyltriethoxysilane (APTES), acrylamide (AM), methyl methacrylate (MMA), and 4-vinylpyridine (4-VP) as functional monomers, respectively, and their adsorption capacity for vitamin B12 (CNcbl) was compared. Figure 3 As shown, the experimental results indicate that MMIPs prepared with MAA as the functional monomer have the best adsorption performance. This is because the carboxyl group in MAA can form strong and stable multiple interactions with multiple active sites in the CNcbl molecule.

[0066] The choice of functional monomer directly affects the quality and quantity of recognition sites in molecularly imprinted polymers. The screening experiment in Example 3 demonstrated that MAA can form the most stable and effective specific binding with vitamin B12 molecules. This experimental result factually supports the superior performance and inventiveness of this invention compared to methods that arbitrarily select functional monomers, and provides solid data support.

[0067] Example 4: Optimization of the amount of functional monomer MAA added

[0068] This embodiment aims to determine the optimal dosage of MAA.

[0069] Keeping other conditions constant, following the steps of Example 1, only the amount of functional monomer MAA added was changed (from low to high) to prepare a series of MMIPs, and their adsorption capacity was tested. Figure 4 As shown, the results indicate that the adsorption capacity of MMIPs for CNcbl first increases and then decreases with increasing MAA addition. The adsorption capacity of MMIPs reaches its maximum when the MAA addition is 3 mmol. At this point, the functional monomer can fully bind with the template molecule to form a sufficient number of specific imprinted sites, while avoiding self-aggregation and non-specific binding caused by excessive MAA.

[0070] Example 5: Optimization of crosslinking agent dosage

[0071] This embodiment aims to determine the optimal dosage of TRIM.

[0072] Keeping other conditions constant, following the steps of Example 1, the amount of crosslinking agent TRIM added was varied (from 4 mmol to 12 mmol) to prepare MMIPs and test the adsorption capacity. Figure 5 As shown, the results indicate that the adsorption capacity of MMIPs for CNcbl reaches its peak when the TRIM addition amount is 8 mmol. At this addition amount, the crosslinking density is moderate, which can form a stable and regular three-dimensional network structure, maximizing the number of effective imprinted sites.

[0073] Example 6: Material property characterization

[0074] This embodiment performs key performance tests on the prepared MMIPs material.

[0075] Specific adsorption experiments: Adsorption experiments were conducted using MMIPs and MNIPs on vitamin B12 (CNcbl) and its analogues (such as thiamine, riboflavin, folic acid, OHcbl, and Adocbl). Figure 6 As shown, the results indicate that MMIPs exhibit high selective adsorption for CNcbl, with an imprinting factor (IF) of 3.861, which is significantly higher than that of other analogs, demonstrating their good specific recognition ability.

[0076] Thermal stability testing (TGA): such as Figure 7 As shown in (a), thermogravimetric analysis revealed that MMIPs exhibited slight weight loss and good thermal stability within the temperature range of 30–200 °C. Significant weight loss (approximately 80%) occurred between 250 and 450 °C, far exceeding the corresponding weight loss of Fe3O4. This phenomenon is primarily attributed to the thermal decomposition of the organic molecularly imprinted polymer layer coating the MMIPs surface at high temperatures. The organic layer is rich in thermally unstable chemical bonds such as C–H, C=O, and C–N, undergoing complex thermal decomposition reactions above 250 °C, including chain breaking, dehydrogenation, carbonization, and volatilization, leading to a significant decrease in the overall material quality. Figure 7As shown in (b), the maximum weight loss rate peak corresponding to the thermal decomposition of the organically imprinted polymer layer appears at approximately 420℃ (corresponding to the steep drop in the TGA curve or the DTG peak), indicating that this temperature is the key temperature point for the concentrated decomposition of the organic polymer layer, further confirming the presence of a large number of organic functional components in the material. In contrast, Fe3O4, as an inorganic magnetic core, exhibits significantly better thermal stability than the organic components, showing only slight weight loss within the same temperature range, with almost no involvement in the decomposition process of organic matter. Furthermore, the TGA curves of MMIPs show that their polymer structure is relatively compact, their thermal decomposition behavior is concentrated, and their peak shape is relatively sharp, indicating that the organically imprinted layer is uniformly coated on the surface of the magnetic nanoparticles and is well-bonded with the magnetic core.

[0077] Magnetic property testing: such as Figure 8 As shown, under an external magnetic field of ±20,000 Oe, the magnetization curve exhibits a near-saturation upward trend. The curve shows no significant remanence or coercivity when crossing the origin, indicating that MMIPs possess superparamagnetism, with Fe3O4 exhibiting a saturation magnetization of 70.62 emu / g. After SiO2 shell modification, the saturation magnetization of Fe3O4@SiO2 / KH570 slightly decreases to 66 emu / g. For MMIPs samples with thicker shells, the saturation magnetization further decreases to 12.13 emu / g, indicating that increasing the thickness of the nonmagnetic shell leads to a gradual decrease in magnetic strength. Despite the decrease in magnetization, the material can still be rapidly attracted and recovered by an external magnetic field within 10 seconds, demonstrating excellent magnetic response performance.

[0078] Example 7: Application of Vitamin B12 Detection in Special Medical Foods

[0079] This embodiment verifies the accuracy and reliability of the method in actual sample testing.

[0080] Sample pretreatment: Weigh three commercially available Foods for Special Medical Purposes (FSMPs) samples and homogenize them with water. Add pepsin / trypsin and enzymatically hydrolyze the samples in a 37°C water bath to release bound vitamin B12.

[0081] Enrichment and Detection: Take the enzymatic hydrolysate, add the MMIPs material prepared in Example 1, and after adsorption for 30-60 minutes, separate using an external magnetic field. Elute with methanol-acetic acid (9:1, v / v) solution, concentrate by nitrogen blowing, and redissolve. Quantitative analysis is performed using high performance liquid chromatography (HPLC) or ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS).

[0082] like Figure 9As shown in the table, the spiked recovery experiment results were obtained by performing spiked recovery experiments on three FSMP samples (spiking levels of 10, 20, and 40 ng / g), with each level measured in triplicate. The results showed that the spiked recoveries ranged from 77.84% to 116.70%, and the relative standard deviations (RSD, n=3) were all less than 10%. The results for the quality control samples (recovery rate of 96.49%) were close to the reference values. The experimental results demonstrate that the vitamin B12 recovery rate is high and the precision is good, proving the accuracy and reliability of this method. Simultaneously, ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS) was used to quantitatively detect vitamin B12 in the quality control samples of infant formula for special medical purposes using the internal standard method. The results in the table show that the measured results are close to the reference values, further proving the applicability of this method, which is suitable for the actual detection of vitamin B12 in special medical foods.

[0083] Of course, the above description is not limited to the examples above. Technical features not described in this invention can be implemented by or using existing technology, and will not be repeated here. The above embodiments and drawings are only used to illustrate the technical solutions of this invention and are not intended to limit this invention. This invention has been described in detail with reference to preferred embodiments. Those skilled in the art should understand that any changes, modifications, additions or substitutions made by those skilled in the art within the scope of this invention do not depart from the spirit of this invention and should also fall within the scope of protection of the claims of this invention.

Claims

1. A material for the enrichment and detection of vitamin B12 in special medical foods based on magnetic molecular imprinting, characterized in that: The material is a magnetic molecularly imprinted polymer (MMIPs), which uses Fe3O4 as a magnetic core, sequentially coated with a SiO2 layer and a KH570 coupling agent, and coupled with a vitamin B12 imprinted polymer layer through surface molecular imprinting technology. The imprinted polymer layer is polymerized from the template molecule vitamin B12 (CNcbl), the functional monomer methacrylic acid (MAA), and the crosslinking agent trimethylolpropane trimethacrylate (TRIM).

2. The material for enriching and detecting vitamin B12 in special medical foods based on magnetic molecular imprinting according to claim 1, characterized in that: The amount of crosslinking agent TRIM added is 4~12 mmol, and the amount of functional monomer MAA added is 2~6 mmol.

3. The material for enriching and detecting vitamin B12 in special medical foods based on magnetic molecular imprinting according to claim 1, characterized in that: The magnetic core Fe3O4 was prepared by co-precipitation, in which the mass ratio of FeCl3·6H2O and FeCl2·4H2O was (1.8~2.2):1, and it was generated by reacting with ammonia water at 60~80℃.

4. The material for enriching and detecting vitamin B12 in special medical foods based on magnetic molecular imprinting according to claim 3, characterized in that: The SiO2 layer coating was achieved by using tetraethyl orthosilicate (TEOs) in an ethanol solution with ammonia as a catalyst, with the amount of TEOs added being 3-5 mL.

5. A method for enriching and detecting vitamin B12 using the magnetic molecular imprinting material according to any one of claims 1 to 4, characterized in that: Includes the following steps: (a) Sample pretreatment: Weigh the special medical food sample, add an appropriate amount of water to disperse it, and then homogenize it to obtain a uniform sample homogenate; then add protease to perform enzymatic hydrolysis to release bound vitamin B12. (b) Enrichment: The enzymatic hydrolysate is mixed with the magnetic molecular imprinted material to carry out an adsorption reaction, and the material is separated by an external magnetic field; (c) Elution: The adsorbed vitamin B12 was eluted with methanol-acetic acid solution; (d) Detection: The eluent was dried with nitrogen and then reconstituted, and quantitatively detected by high performance liquid chromatography or mass spectrometry.

6. The method according to claim 5, characterized in that: In step (a), the protease is either pepsin or trypsin, and the enzymatic hydrolysis temperature is 37°C.

7. The method according to claim 5, characterized in that: In step (b), the adsorption reaction time is 30-60 minutes, and the separation time of the external magnetic field is within 10 seconds.

8. The method according to claim 5, characterized in that: In step (c), the volume ratio of methanol to acetic acid solution is 9:

1.

9. The method according to claim 5, characterized in that: The magnetic molecular imprinted material can be reused at least 5 times, and the adsorption capacity retention rate is not less than 80% after each use.

10. The application of the magnetic molecular imprinting material according to any one of claims 1 to 4 in the detection of vitamin B12 in special medical foods, characterized in that: Foods for special medical purposes (FSMPs) had a spiked recovery rate of 77.84% to 116.70% and a relative standard deviation (RSD) of <10%.

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