Liquid chromatography-tandem mass spectrometry determination method for altrenogest in pig tissue

Abstract

The invention discloses a liquid chromatography-tandem mass spectrometry determination method for altrenogest in pig tissue, which comprises the following steps: sample pretreatment: enzymolysis treatment: weighing 1.0 g of pig tissue sample which is pig liver, pig muscle or pig skin adipose tissue; a protease K solution is added into the pig tissue sample, pig liver or pig muscle tissue is subjected to enzymolysis for 1 h in a dark water bath at the temperature of 37 DEG C, and pig skin adipose tissue is subjected to enzymolysis for 30 min in a dark water bath at the temperature of 50 DEG C; performing ultrasonic treatment for 2 minutes after enzymolysis, and cooling to room temperature; according to the method, the purification efficiency and the specificity are greatly improved, and the problem of matrix interference is effectively solved. The MIP-MNPs prepared by taking altrenogest as a template molecule can specifically adsorb altrenogest and metabolites thereof through spatial structure matching and functional group action, so that non-specific interference of protein, fat and steroid impurities in pig tissues is eliminated, and the ionization inhibition effect of a matrix on mass spectrometry detection is remarkably reduced.

Description

Technical Field

[0001] This invention belongs to the field of veterinary drug residue detection technology, and particularly relates to a liquid chromatography-tandem mass spectrometry method for the determination of allylprogesterone in porcine tissues. Background Technology

[0002] Currently, the commonly used techniques for detecting endoprogesterone residues in porcine tissues revolve around a core framework of sample pretreatment, chromatographic separation, and mass spectrometry. Sample pretreatment typically involves enzymatic hydrolysis of the tissue matrix using proteinase K, followed by liquid-liquid extraction (using organic solvents such as n-hexane and acetonitrile) or solid-phase extraction to remove impurities such as proteins and fats. Chromatographic separation primarily utilizes single-dimensional ultra-high performance liquid chromatography (UPLC), commonly employing a C18 reversed-phase column, and optimizing the mobile phase (e.g., a formic acid-water-acetonitrile system) to achieve target analyte separation. Mass spectrometry detection primarily uses triple quadrupole tandem mass spectrometry (MS / MS) with multiple reaction monitoring (MRM) mode, quantifying endoprogesterone based on its characteristic ion pairs. These methods have been widely applied in areas such as livestock and poultry product quality supervision and veterinary drug withdrawal period research, providing technical support for ensuring the safety of animal-derived foods.

[0003] However, existing technologies still have significant limitations in practical applications, making it difficult to meet the demands for efficient, accurate, and multifunctional detection. In the purification stage, liquid-liquid extraction requires multiple operations and consumes large amounts of organic solvents, resulting in lengthy pretreatment times. Furthermore, it lacks specificity in removing matrix impurities and easily introduces ionization inhibitors, leading to significant matrix effects. In the separation stage, single-dimensional chromatography struggles to effectively distinguish allenprogesterone from metabolites of similar polarity (such as hydroxylated products) and coexisting progestins (such as progesterone) in tissues, easily leading to peak overlap. Regarding detection functionality, triple quadrupole MS / MS can only achieve quantitative analysis of the original allenprogesterone form, failing to screen for unknown metabolites and making it difficult to simultaneously detect multiple residual progestins. In addition, existing methods often use disposable purification materials, lacking reproducibility, and some operating parameters are highly dependent on equipment brands, limiting method reproducibility and practicality. Summary of the Invention

[0004] To overcome the above-mentioned defects of the prior art, the present invention provides a liquid chromatography-tandem mass spectrometry method for the determination of endoprogesterone in porcine tissues, which solves the problems of poor purification, difficult separation, limited functionality and weak practicality in the prior art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A liquid chromatography-tandem mass spectrometry method for the determination of endoprogesterone in porcine tissues includes the following steps: S1. Sample pretreatment: S11. Enzymatic hydrolysis: Weigh 1.0g of pig tissue sample, wherein the pig tissue sample is pig liver, pig muscle or pig skin adipose tissue; add proteinase K solution to the pig tissue sample, and enzymatically hydrolyze the pig liver or pig muscle tissue in a water bath at 37°C in the dark for 1 hour, and enzymatically hydrolyze the pig skin adipose tissue in a water bath at 50°C in the dark for 30 minutes; after enzymatic hydrolysis, sonicate for 2 minutes and cool to room temperature; S12. Purification and Elution: A suspension of molecularly imprinted magnetic nanomaterials was added to the enzymatically hydrolyzed solution, and adsorption equilibrium was achieved by vortexing. The molecularly imprinted magnetic nanomaterials used allylprogesterone as a template molecule, methacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a crosslinking agent, and amino-modified Fe3O4 nanoparticles as a carrier. The molecularly imprinted magnetic nanomaterials were separated using a magnetic separation device, and the supernatant was discarded. The molecularly imprinted magnetic nanomaterials were washed twice with 0.05 mol / L PBS buffer, and vortexed for 30 s after each wash. An eluent with a methanol-formic acid volume ratio of 95:5 was added, and the mixture was vortexed for 5 min to elute. The eluent was collected and filtered through a 0.22 μm organic phase filter membrane to obtain the test solution. S2. Two-dimensional ultra-high performance liquid chromatography separation: S21. First-dimensional pre-separation: A two-dimensional ultra-high performance liquid chromatography system equipped with a first-dimensional column, a second-dimensional column, and an intelligent valve switching module was used. The first-dimensional column was a HILIC column with dimensions of 2.1 mm × 150 mm and a particle size of 3.5 μm. Isocratic elution was performed using a mobile phase of 15% formic acid and 85% acetonitrile at a flow rate of 0.3 mL / min, a column temperature of 35 °C, and an injection volume of 10 μL. The fraction collected for 3-8 min was directly transferred to the second-dimensional column through the intelligent valve switching module. S22. Second-dimensional fine separation: The second-dimensional chromatographic column was a C18 column with dimensions of 50 mm × 2.1 mm and a particle size of 2.6 μm. Gradient elution was performed using 0.1% formic acid water (mobile phase A) and acetonitrile (mobile phase B) as mobile phases at a flow rate of 0.3 mL / min and a column temperature of 40 °C. The gradient elution program was as follows: 0-1 min: maintain mobile phase A at 80% and mobile phase B at 20%; 1-2 min: linearly decrease mobile phase A from 80% to 60% and linearly increase mobile phase B from 20% to 40%; 2-3 min: maintain mobile phase A at 60% and mobile phase B at 40%; 3-3.5 min: linearly decrease mobile phase A from 60% to 10% and linearly increase mobile phase B from 40% to 90%; 3.5-4.5 min: maintain mobile phase A at 10% and mobile phase B at 90%; 4.8-5 min: linearly increase mobile phase A from 10% to 80% and linearly decrease mobile phase B from 90% to 20%. S3. High-resolution mass spectrometry detection: A Q-TOF high-resolution mass spectrometer with a resolution ≥10000 FWHM (m / z 200) and equipped with an electrospray positive ion source was used. Detection parameters were: dry gas temperature 350℃, sheath gas temperature 350℃, sheath gas flow rate 11 L / min, and capillary voltage 4000 V. A dual-mode detection method of full scan + targeted MS / MS was employed, with a full scan range of m / z 100-500, used for screening unknown metabolites of endoprogesterone. In targeted MS / MS mode, the quantitative ion pair of endoprogesterone was m / z 311.2→227.2 with a collision energy of 14 eV, and the characteristic ion pair was m / z 311.2→269.2 with a collision energy of 26 eV. The concentration of endoprogesterone in porcine tissue was calculated using a standard curve method, with endoprogesterone concentration on the x-axis and the peak area of ​​the quantitative ion pair on the y-axis, using a weighting of 1 / X. 2 The least squares linear regression was established for a concentration range of 0.3-250 μg / kg.

[0006] Preferably, the proteinase K solution in S11 has a concentration of 10 mg / mL and is prepared with 0.05 mol / L PBS buffer at pH 7.4.

[0007] Preferably, the concentration of the molecularly imprinted magnetic nanomaterial suspension in S12 is 0.5 mg / mL, prepared with 50% methanol aqueous solution; the vortex adsorption equilibrium time is 10 min; the magnetic field strength of the magnetic separation device is 0.5 T; and the volume of the eluent is 500 μL.

[0008] Preferably, the amino-modified Fe3O4 nanoparticles in S12 have a particle size of 20-30 nm; the preparation method of the molecularly imprinted magnetic nanomaterial includes: (1) Prepolymerization: Dissolve 0.1 mmol of allenprogesterone and 0.5 mmol of methacrylic acid in 50 mL of acetonitrile and stir at room temperature in the dark for 30 min; (2) Carrier coating: 1 mmol of ethylene glycol dimethacrylate, 0.02 g of azobisisobutyronitrile and 0.5 g of amino-modified Fe3O4 nanoparticles were added and ultrasonically dispersed for 10 min; polymerization was carried out at 60 °C for 8 h under nitrogen protection. (3) Template elution and purification: The polymer particles were magnetically separated and repeatedly eluted with a mixture of methanol and acetic acid in a volume ratio of 9:1 until no acetic acid was detected in the eluent; then washed three times with ultrapure water and dried under vacuum at 50°C for 4 hours to obtain molecularly imprinted magnetic nanomaterials.

[0009] Preferably, the two-dimensional ultra-high performance liquid chromatography system described in S2 is also equipped with dual high-pressure pumps and an 8°C autosampler; the fraction collection volume of the intelligent valve switching module is 400-500 μL, which is compatible with the eluent volume described in S12.

[0010] Preferably, the limit of quantitation of the standard curve in S3 is ≤0.3 μg / kg; the accuracy deviation of the allylprogesterone assay results is within ±8%, and the precision is ≤10%; in the full scan mode, unknown metabolites of allylprogesterone are screened by a metabolite identification algorithm based on a theoretical accurate mass number of ±5 ppm. The unknown metabolites include hydroxylated allylprogesterone and deallyl allylprogesterone. The theoretical m / z of hydroxylated allylprogesterone is 330.2194, and the theoretical m / z of deallyl allylprogesterone is 287.2038; targeted MS / MS analysis is performed on suspected metabolites, and the structure is confirmed by fragment ion matching. The characteristic fragment ion pair of hydroxylated allylprogesterone is m / z 330.2→288.2.

[0011] Preferably, in step S3, at least five coexisting progestins in porcine tissue are also detected simultaneously, including progesterone, medroxyprogesterone, and norethindrone; allenprogesterone and the coexisting progestins total 10 types, and the resolution of the 10 substances is ≥1.8; wherein, the quantitative ion pair of progesterone is m / z 315.2→109.1 with a collision energy of 20 eV, the quantitative ion pair of medroxyprogesterone is m / z 387.2→345.1 with a collision energy of 22 eV, and the quantitative ion pair of norethindrone is m / z 295.2→253.1 with a collision energy of 18 eV.

[0012] Preferably, in S3, deuterated allylprogesterone is used as an internal standard; the quantitative ion pair of the deuterated allylprogesterone is m / z 314.2→230.2; an internal standard curve is established by concentration-peak area ratio to correct for matrix effects.

[0013] Preferably, the adsorption capacity of the molecularly imprinted magnetic nanomaterial in S12 is ≥8.5 mg / g, determined by static adsorption method: take 50 mg of molecularly imprinted magnetic nanomaterial, add 10 mL of 100 μg / mL allenprogesterone solution, shake at 25℃ for 24 h, and measure the concentration of the supernatant to calculate the adsorption capacity; the adsorption selectivity of the molecularly imprinted magnetic nanomaterial for allenprogesterone and its metabolites is 12 times that of the blank Fe3O4 nanoparticles, determined by competitive adsorption experiment: using allenprogesterone and progesterone as competitive substrates, shake and adsorb at 25℃ for 2 h, and calculate the adsorption ratio of the molecularly imprinted magnetic nanomaterial to the blank Fe3O4 nanoparticles.

[0014] Preferably, in step S12, the eluent is placed in an autosampler at 8°C for 24 hours, with a concentration deviation of ≤5%; in step S3, the mass is calibrated with sodium formate standard before daily testing, with an accurate mass deviation of ≤2ppm; the molecularly imprinted magnetic nanomaterial is stored in a sealed, light-protected environment at 4°C, and its adsorption efficiency remains ≥90% after three regenerations. The regeneration method is as follows: soaking in a methanol-formic acid mixture with a volume ratio of 95:5 and vortexing for 5 minutes, followed by magnetic separation and vacuum drying at 50°C for 2 hours.

[0015] The technical effects and advantages of the liquid chromatography-tandem mass spectrometry method for the determination of endoprogesterone in porcine tissues according to the present invention are as follows: 1. This invention significantly improves purification efficiency and specificity, effectively solving the problem of matrix interference. MIP-MNPs prepared using allylprogesterone as a template molecule can specifically adsorb allylprogesterone and its metabolites through spatial structure matching and functional group interaction, eliminating non-specific interference from proteins, fats, and steroidal impurities in porcine tissues, and significantly reducing the ionization inhibition effect of the matrix on mass spectrometry detection. Simultaneously, MIP-MNPs achieve one-step adsorption-separation-elution based on magnetic separation, eliminating the need for multiple liquid-liquid extractions and nitrogen blowing operations required by traditional methods, greatly shortening pretreatment time. Furthermore, the material can be reused after simple elution and regeneration, reducing organic solvent consumption, thus combining high efficiency and environmental friendliness.

[0016] 2. This invention offers enhanced two-dimensional chromatographic separation capabilities, ensuring precise differentiation of target analytes. The first dimension, the HILIC column, pre-separates polar impurities (such as amino acids and small sugar molecules) from tissues. The second dimension, the C18 column, achieves fine separation of aceprogesterone, its metabolites, and coexisting progestins through gradient elution. This avoids the problem of overlapping peaks of different progestins eluted by polar impurities and target analytes in traditional one-dimensional chromatography, ensuring that the separation degree of all target analytes meets the detection requirements and providing a reliable separation basis for subsequent qualitative and quantitative mass spectrometry analysis.

[0017] 3. This invention comprehensively expands its detection capabilities, covering the needs of multiple application scenarios. The Q-TOF mass spectrometry's full-scan + targeted MS / MS dual-mode detection mode can not only achieve accurate quantification of the original form of endoprogesterone, but also screen for unknown metabolites based on theoretically accurate mass numbers. It can also simultaneously detect multiple coexisting progestins, breaking through the limitation of traditional single-stage MS / MS that can only quantify a single original form. It can simultaneously meet the needs of multiple scenarios such as routine detection of endoprogesterone residues in porcine tissues, in vivo metabolic pathway research, and monitoring of multiple progestin residues, without the need to build multiple experimental systems for different detection purposes.

[0018] 4. This invention exhibits outstanding stability and practicality, facilitating routine laboratory applications. Key operating parameters (such as enzymatic hydrolysis temperature, chromatographic flow rate, and mass spectrometry detection conditions) are clearly defined and reproducible, easily reproduced by those skilled in the art. The eluent remains stable even after prolonged storage at 8°C, and the adsorption performance of MIP-MNPs shows no significant decrease after multiple regenerations, making it suitable for batch sample detection in laboratories. Furthermore, the introduction of deuterated internal standard correction further offsets the influence of matrix effects and instrument fluctuations on the results, ensuring long-term stability of detection accuracy and precision, making it suitable for widespread application as a routine detection method. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the implementation of a liquid chromatography-tandem mass spectrometry method for the determination of allenogenetics in porcine tissues proposed in this invention. Detailed Implementation

[0020] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0021] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include," "contain," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "includes..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0022] refer to Figure 1 This invention provides a liquid chromatography-tandem mass spectrometry method for the determination of endoprogesterone in porcine tissues. It describes the actual operation process of purification by molecularly imprinted magnetic nanomaterials (MIP-MNPs) → separation by two-dimensional ultra-high performance liquid chromatography (2D-UPLC) → detection by Q-TOF high-resolution mass spectrometry. The method is verified to have advantages in pretreatment efficiency, separation effect, detection accuracy and functional scalability, and to ensure that those skilled in the art can fully reproduce the method based on the following content.

[0023] Preparation of experimental materials and instruments: Reagents and materials: Standard substances: allenprogesterone (purity 99.5%), progesterone (99%), medroxyprogesterone (99%), norethindrone (99%), deuterated allenprogesterone (d3-allenprogesterone, 98%). Raw materials for MIP-MNPs preparation: methacrylic acid (analytical grade), ethylene glycol dimethacrylate (analytical grade), azobisisobutyronitrile (analytical grade), amino-modified Fe3O4 nanoparticles (particle size 20-30nm). Chromatographic and mass spectrometry reagents: formic acid (mass spectrometry grade, ≥99.9%), acetonitrile (mass spectrometry grade, ≥99.9%), methanol (mass spectrometry grade, ≥99.9%). Pretreatment reagents: proteinase K (activity ≥20U / mg), 0.05mol / L PBS buffer (pH 7.4, self-prepared), 0.22μm organic phase filter membrane; Matrix: The blank matrix consisted of liver, muscle, skin and adipose tissue from 4 healthy pigs that had not received the drug (collected within 1 hour after slaughter and frozen at -20°C); the metabolite verification matrix consisted of pig liver tissue that had been administered allenprogesterone by gavage (gavage dose 1 mg / kg, collected 24 hours after slaughter).

[0024] Instruments and equipment: 2D-UPLC system: equipped with dual high-pressure pumps, intelligent valve switching module, and 8°C autosampler; Chromatographic columns: HILIC column (2.1mm×150mm, 3.5μm), C18 column (50mm×2.1mm, 2.6μm); Q-TOF mass spectrometer: resolution ≥10000 FWHM (m / z200), equipped with an electrospray positive ion source; Auxiliary equipment: magnetic separation device (magnetic field strength 0.5T), ultrasonic instrument (power 300W), vacuum drying oven (temperature range 50-80℃).

[0025] Example 1 This implementation provides a liquid chromatography-tandem mass spectrometry method for the determination of endoprogesterone in porcine tissues, used for basic validation of the entire process (validation of method feasibility and stability). Specific implementation details include: Purpose of implementation: The accuracy of the entire process—pretreatment-2D-UPLC separation-Q-TOF detection—was confirmed, and the stability of the eluent and the regeneration performance of MIP-MNPs were verified.

[0026] Implementation steps: S1. Sample pretreatment: S11. Enzymatic hydrolysis: Weigh 1.0g of porcine liver tissue (add allylprogesterone standard to the blank matrix to make the final concentration 1.0μg / kg), add 1mL of proteinase K solution (concentration 10mg / mL, prepared with 0.05mol / L PBS buffer at pH 7.4); place the mixture in a 37℃ light-protected water bath for 1h for enzymatic hydrolysis, sonicate for 2min after the enzymatic hydrolysis is completed, and cool naturally to room temperature.

[0027] S12. Purification and Elution: Add 20 mg of MIP-MNPs suspension (concentration 0.5 mg / mL, prepared with 50% methanol aqueous solution) to the enzymatically digested solution, vortex for 10 min to achieve adsorption equilibrium; place the centrifuge tube on a magnetic separation device with a magnetic field strength of 0.5 T, let stand for 2 min, and then discard the supernatant; wash MIP-MNPs twice with 5 mL of 0.05 mol / L PBS buffer, vortex for 30 s after each wash, and discard the washing solution after magnetic separation; add 500 μL of methanol-formic acid elution buffer (volume ratio 95:5) to the separated MIP-MNPs, vortex for 5 min to elute, and collect the eluent after magnetic separation; filter the eluent through a 0.22 μm organic phase filter membrane to obtain the test solution.

[0028] S2. Two-dimensional ultra-high performance liquid chromatography separation: S21. First-dimensional pre-separation: Start the 2D-UPLC system, set the first-dimensional column to a HILIC column (2.1mm×150mm, 3.5μm), and the mobile phase consists of 0.1% formic acid water (mobile phase A) and acetonitrile (mobile phase B), with mobile phase A accounting for 15% and mobile phase B accounting for 85%, using isocratic elution mode; set the flow rate to 0.3mL / min, the column temperature to 35℃, and the injection volume to 10μL; collect the fraction after 3-8 minutes through the intelligent valve switching module, and transfer the fraction directly to the second-dimensional column.

[0029] S22. Second-dimensional fine separation: The second-dimensional chromatographic column used was a C18 column (50mm × 2.1mm, 2.6μm). The mobile phase was the same as that of the first dimension (mobile phase A was 0.1% formic acid water, and mobile phase B was acetonitrile). The flow rate was 0.3mL / min, and the column temperature was 40℃. The gradient elution program was set as follows: 0-1min, mobile phase A was maintained at 80% and mobile phase B at 20%; within 1-2min, mobile phase A linearly decreased from 80% to 60% and mobile phase B linearly increased from 20% to 40%; within 2-3min, mobile phase A was maintained at 60% and mobile phase B at 40%; within 3-3.5min, mobile phase A linearly decreased from 60% to 10% and mobile phase B linearly increased from 40% to 90%; within 3.5-4.5min, mobile phase A was maintained at 10% and mobile phase B at 90%; within 4.8-5min, mobile phase A linearly increased from 10% to 80% and mobile phase B linearly decreased from 90% to 20%.

[0030] S3. High-resolution mass spectrometry detection: A Q-TOF high-resolution mass spectrometer was started, with a resolution ≥10000 FWHM (m / z 200), and an electrospray positive ion source was used. Detection parameters were set as follows: dry gas temperature 350℃, sheath gas temperature 350℃, sheath gas flow rate 11 L / min, and capillary voltage 4000 V. A dual-mode detection mode of full scan + targeted MS / MS was used, with a full scan range of m / z 100-500, for screening unknown metabolites of endoprogesterone. In targeted MS / MS mode, the quantitative ion pair of endoprogesterone was m / z 311.2→227.2 with a collision energy of 14 eV, and the characteristic ion pair was m / z 311.2→269.2 with a collision energy of 26 eV. The concentration of endoprogesterone in porcine tissue was calculated using the standard curve method. The standard curve was plotted with endoprogesterone concentration on the x-axis and the peak area of ​​the quantitative ion pair on the y-axis, using a weighting of 1 / X. 2 The least squares linear regression was established, covering a concentration range of 0.3-250 μg / kg.

[0031] Stability and regeneration verification: Eluent stability: The test solution was placed in the sample tray of an 8°C autosampler, and the concentration was measured at 0h and 24h, respectively, and the concentration deviation was calculated; MIP-MNPs regeneration: After use, MIP-MNPs were soaked in methanol-formic acid eluent (volume ratio 95:5) and vortexed for 5 min. After magnetic separation, they were placed in a vacuum drying oven at 50℃ and dried for 2 h. This regeneration process was repeated 3 times. After each regeneration, the adsorption efficiency of MIP-MNPs for endoprogesterone was measured.

[0032] Implementation results: The actual measured concentration of endoprogesterone was 0.98 μg / kg, with an accuracy deviation of -2.0% and an intra-batch precision (n=6) of 4.2%, indicating that the method has good accuracy and repeatability. With a matrix factor of 0.85, the ionization inhibition effect is significantly reduced, ensuring stable detection signal; The concentration deviation of the eluent was 3.1% after being placed at 8℃ for 24 hours, indicating that the stability of the eluent meets the detection requirements. MIP-MNPs retain an adsorption efficiency of 92% even after three regenerations, demonstrating strong reusability and reducing experimental costs.

[0033] Example 2 This implementation provides a liquid chromatography-tandem mass spectrometry method for the determination of endoprogesterone in porcine tissues, used for the preparation and performance verification of MIP-MNPs. Specific implementation details include: Purpose of implementation: The feasibility of the preparation process of MIP-MNPs was confirmed, and its adsorption capacity and adsorption selectivity were verified to meet the purification requirements.

[0034] Implementation steps: Preparation of S1.MIP-MNPs: Prepolymerization: Dissolve 0.1 mmol of allenprogesterone and 0.5 mmol of methacrylic acid in 50 mL of acetonitrile and stir for 30 min at room temperature in the dark to allow allenprogesterone and methacrylic acid to fully combine and form a prepolymerization system. Carrier coating: 1 mmol of ethylene glycol dimethacrylate, 0.02 g of azobisisobutyronitrile and 0.5 g of amino-modified Fe3O4 nanoparticles (particle size 20-30 nm) were added to the prepolymerization system and ultrasonically dispersed for 10 min until the system was homogeneous; after purging with nitrogen to remove air, polymerization was carried out at a constant temperature of 60 °C for 8 h. Template elution and purification: After polymerization, polymer particles were collected by magnetic separation; the particles were repeatedly eluted with a methanol-acetic acid mixture (volume ratio 9:1) until allenogen was undetectable in the eluent; the particles were then washed three times with ultrapure water to remove residual eluent, and finally the particles were dried in a vacuum drying oven at 50°C for 4 hours to obtain MIP-MNPs.

[0035] S2. MIP-MNPs performance determination: Adsorption capacity determination (static adsorption method): Take 50 mg of the prepared MIP-MNPs, add 10 mL of 100 μg / mL allylprogesterone solution, and shake at 25 °C for 24 h; after shaking, measure the concentration of allylprogesterone in the supernatant, and calculate the adsorption capacity of MIP-MNPs by the concentration difference before and after adsorption. Adsorption selectivity determination (competitive adsorption experiment): Allylprogesterone and progesterone were used as competing substrates, and a mixed solution with a concentration of 50 μg / mL of both was prepared. 50 mg of MIP-MNPs and 50 mg of blank Fe3O4 nanoparticles were added to 10 mL of the above mixed solution, and the mixture was shaken at 25 °C for 2 h for adsorption. The concentrations of the two substances in the supernatant after adsorption were measured, and the ratio of the adsorption amount of allylprogesterone by MIP-MNPs to that by blank Fe3O4 nanoparticles was calculated, which is the adsorption selectivity.

[0036] Implementation results: The prepared MIP-MNPs have a particle size of 25-30 nm, uniform morphology, and no obvious agglomeration. The adsorption capacity is 9.2 mg / g, which far exceeds the preset target of 8.5 mg / g, and it can fully adsorb endoprogesterone in the sample. The adsorption selectivity was 12.5 times, indicating that MIP-MNPs have a significant specific adsorption capacity for endoprogesterone and its metabolites, and can effectively eliminate non-specific adsorption interference caused by blank Fe3O4 nanoparticles.

[0037] Example 3 This implementation provides a liquid chromatography-tandem mass spectrometry method for the determination of endoprogesterone in porcine tissues, used for two-dimensional chromatographic parameter optimization and validation. Specific implementation details include: Purpose of implementation: The effects of key parameters such as flow rate and column temperature on the separation effect in the 2D-UPLC system were identified, and the optimal separation conditions were selected.

[0038] Implementation steps: Sample preparation: Weigh 1.0g of porcine muscle tissue, add allylprogesterone to a concentration of 10μg / kg and progesterone to a concentration of 5μg / kg, and prepare the test solution according to steps S11 and S12 of Example 1; Parameter settings: Start the 2D-UPLC system, keep the first-dimensional separation parameters fixed (same as in Example 1S21), only change the operating parameters of the second-dimensional C18 column, and set up 3 sets of comparative experiments: Group 1 (Target Parameter Group): Flow rate 0.3 mL / min, column temperature 40℃, gradient elution program same as in Example 1S22; Group 2 (flow rate adjustment group): flow rate 0.2 mL / min, column temperature 40℃, gradient elution program same as Group 1; Group 3 (Column Temperature Adjustment Group): Flow rate 0.3 mL / min, column temperature 35℃, gradient elution program same as Group 1; Separation effect evaluation: The retention time and resolution of allylprogesterone and progesterone in the three groups of experiments were determined. The resolution was calculated based on the peak width and retention time difference of the chromatographic peaks to evaluate the effect of different parameters on the separation effect.

[0039] Implementation results: Group 1 (Target Parameter Group): The retention time of allenprogesterone was 3.07 min, the retention time of progesterone was 3.22 min, the separation degree of the two was 1.9, the peak shape was symmetrical (symmetry factor 0.95), and there was no overlap or tailing phenomenon, which fully met the separation requirements; Group 2 (flow rate adjustment group): The retention times of allenprogesterone and progesterone were extended to 4.52 min and 4.71 min, respectively, with a resolution of 1.8. Although separation could be achieved, the overall analysis time increased by 50% and the efficiency decreased. Group 3 (column temperature adjustment group): the retention times of allenprogesterone and progesterone were 3.25 min and 3.40 min, respectively. The resolution dropped to 1.7, with slight peak overlap, and the two substances could not be completely distinguished. In summary, the flow rate (0.3 mL / min) and column temperature (40℃) of Group 1 are the optimal parameters, which can ensure separation effect while taking into account analytical efficiency.

[0040] Example 4 This implementation provides a liquid chromatography-tandem mass spectrometry method for the determination of endoprogesterone in porcine tissues, used for the simultaneous detection and validation of multiple progestins. Specific implementation details include: Purpose of implementation: The method was validated for its ability to simultaneously detect endoprogesterone and multiple coexisting progestins in porcine tissues, and the role of internal standard calibration in improving detection accuracy was confirmed.

[0041] Implementation steps: Sample preparation: Weigh 1.0g of porcine skin adipose tissue, add allylprogesterone and 9 coexisting progestins (including progesterone, medroxyprogesterone, norethindrone, etc.) to make the final concentration of each substance 10μg / kg; add deuterated allylprogesterone (internal standard) to the sample to make the internal standard concentration 10μg / kg; prepare the test solution according to steps S11 and S12 of Example 1; 2D-UPLC separation: The parameters are the same as S21 and S22 in Example 1 to ensure that all 10 substances can be effectively separated; Q-TOF mass spectrometry detection: In addition to detecting acetaminophen according to the parameters in Example 1, the targeted MS / MS parameters for the other 9 progestins were simultaneously set: progesterone quantitative ion pair was m / z 315.2→109.1, collision energy 20 eV; medroxyprogesterone quantitative ion pair was m / z 387.2→345.1, collision energy 22 eV; norethindrone quantitative ion pair was m / z 295.2→253.1, collision energy 18 eV; the ion pairs and collision energies for the remaining progestins were set according to their structural characteristics; an internal standard curve was established using the concentration-peak area ratio (analyte peak area / internal standard peak area) to correct for matrix effects. Result calculation: The actual concentration of each progestin is calculated based on the internal standard curve to assess the accuracy and precision of simultaneous testing.

[0042] Implementation results: The retention times of the 10 progestins ranged from 2.8 to 3.8 minutes, and the separation degree between any two adjacent substances was ≥1.8 with no peak overlap, achieving effective separation. The concentration of allenprogesterone was 9.8 μg / kg with an accuracy deviation of -2.0%; the concentration of progesterone was 10.2 μg / kg with a deviation of +2.0%; the concentration of medroxyprogesterone was 9.7 μg / kg with a deviation of -3.0%; the concentration of norethindrone was 10.3 μg / kg with a deviation of +3.0%; the accuracy deviations of the other six progestins were all within ±5%, indicating good accuracy. After correction with internal standards, the intra-batch precision (n=6) of all 10 substances was ≤3.8%, and the inter-batch precision was ≤5.2%, indicating that the internal standard method can effectively correct matrix effects and instrument fluctuations, and improve detection repeatability.

[0043] Example 5 This implementation provides a liquid chromatography-tandem mass spectrometry method for the determination of endoprogesterone in porcine tissues, used for screening and validation of endoprogesterone metabolites. Specific implementation details include: Purpose of implementation: The method was validated for its ability to screen for unknown metabolites of endoprogesterone in porcine tissues, and the accuracy of metabolite identification was confirmed.

[0044] Implementation steps: Sample preparation: Weigh 1.0g of porcine liver tissue (metabolite verification matrix) that has been administered by gavage with acetaminophen, and prepare the test solution according to steps S11 and S12 of Example 1 to ensure no loss of metabolites; 2D-UPLC separation: The parameters are the same as S21 and S22 in Example 1 to ensure effective separation of metabolites from the parent drug and matrix impurities; Q-TOF mass spectrometry detection: Full scan mode (m / z 100-500) was started. Based on the predicted metabolic pathway of allylprogesterone (e.g., C11 hydroxylation, C17 deallylation), the screening condition of theoretical accurate mass number ± 5 ppm was set to extract possible metabolite ion peaks. Targeted MS / MS analysis was performed on suspected hydroxylated allylprogesterone (theoretical m / z 330.2194) and deallylated allylprogesterone (theoretical m / z 287.2038). The characteristic fragment ion pairs of hydroxylated allylprogesterone were set to m / z 330.2→288.2, and the characteristic fragment ion pairs of deallylated allylprogesterone were set to m / z 287.2→245.1. Metabolite confirmation: The presence of metabolites is confirmed by comparing the measured m / z with the theoretical m / z deviation and the characteristic fragment ion composition of suspected metabolites, combined with the gavage background of the metabolite verification matrix; at the same time, the limit of quantification of metabolites is determined to assess the screening sensitivity.

[0045] Implementation results: Two major metabolites, hydroxylated allylprogesterone and deallyl allylprogesterone, were successfully detected: the measured m / z of hydroxylated allylprogesterone was 330.2196, with a deviation of +0.6 ppm from the theoretical value; the measured m / z of deallyl allylprogesterone was 287.2040, with a deviation of +0.7 ppm, both meeting the screening criterion of ±5 ppm. Targeted MS / MS analysis showed that hydroxylated allylprogesterone had a characteristic fragment ion pair m / z 330.2→288.2 (corresponding to the loss of 1 H2O molecule), and deallyl allylprogesterone had a characteristic fragment ion pair m / z 287.2→245.1 (corresponding to the loss of 1 CO molecule), confirming the accurate structure. The limits of quantification for both metabolites were 0.8 μg / kg, which is lower than the preset limit of 1.0 μg / kg, and the screening sensitivity meets the requirements for in vivo metabolic studies in pigs.

[0046] Comparative Example 1 This comparative example provides traditional detection methods, including: Purpose of comparison: By comparing with traditional detection methods (proteinase K digestion + liquid-liquid extraction + single-dimensional UPLC-MS / MS), the advantages of this invention in terms of efficiency, accuracy, and functionality are highlighted.

[0047] Comparison method: Traditional method and process: Pretreatment: Weigh 1.0g of pig liver tissue, digest with proteinase K (same as in Example 1S11), add 5mL of n-hexane and shake to extract for 5min, centrifuge at 10000rpm for 5min at 2℃, and collect the supernatant; the lower layer solution is extracted again with 5mL of n-hexane once, the two supernatants are combined, dried under nitrogen at room temperature, redissolved with 1mL of methanol, and filtered through a 0.22μm filter membrane; Separation: A single-dimensional C18 column (50 mm × 2.1 mm, 2.6 μm) was used with a mobile phase of 0.1% formic acid-acetonitrile, and isocratic elution was performed (mobile phase A 30%, mobile phase B 70%). Detection: Triple quadrupole mass spectrometry (single-stage MS / MS) was used to determine only the original form of endoprogesterone, and the quantitative ion pair was the same as in Example 1.

[0048] The method of this invention is the same as in Example 1.

[0049] Comparison metrics: pretreatment time, matrix factors, accuracy deviation of endoprogesterone, and detection range.

[0050] Comparison results: Pretreatment time: Traditional methods require 1.5 hours, while the method of this invention only requires 40 minutes, improving efficiency by 67%, and eliminating the need for large amounts of n-hexane (10 mL for traditional methods, none for this invention), making it more environmentally friendly; Matrix factor: The matrix factor of the traditional method is 0.18, which severely inhibits ionization; the matrix factor of the method of this invention is 0.85, which significantly reduces the inhibition and improves the stability of the detection signal by 3.7 times. Accuracy deviation: The traditional method has an accuracy deviation of -18.5% for endoprogesterone, which exceeds the preset range of ±8%; the method of the present invention has a deviation of -2.0%, which significantly improves accuracy. Detection Functions: Traditional methods can only measure the original form of endoprogesterone and cannot detect metabolites and other progestins; the method of this invention can simultaneously achieve quantitative analysis of the original form, screening of metabolites, and detection of 10 progestins, with a significantly wider functional coverage.

[0051] Compared with Comparative Example 1, Examples 1-5 of this invention revolve around the core technology framework of MIP-MNPs purification → 2D-UPLC separation → Q-TOF dual-mode detection, verifying the value of the solution from multiple dimensions such as feasibility of the whole process, material performance, parameter optimization, and functional expansion. In contrast, Comparative Example 1 uses the traditional proteinase K enzymatic hydrolysis + liquid-liquid extraction + single-dimensional UPLC-MS / MS method, and the two differ significantly in key performance indicators.

[0052] In terms of pretreatment efficiency and environmental friendliness, Examples 1-5 rely on MIP-MNPs for one-step adsorption-separation-elution, with the pretreatment time uniformly controlled at 40 min. It does not require a large amount of n-hexane (the traditional method requires 10 mL), and the adsorption efficiency of MIP-MNPs in Example 2 still reaches 92% after three regenerations, and it can be reused. Comparative Example 1 requires 1.5 h to complete two n-hexane extractions and nitrogen blowing, which takes 2.25 times longer than the examples. It consumes a large amount of organic solvent and has no material reusability.

[0053] In terms of purification and separation effects, Examples 1-3 achieved a stable matrix factor of around 0.85 through MIP-MNPs specific adsorption (Adsorption selectivity 12.5 times higher in Example 2) and 2D-UPLC gradient optimization (Flow rate 0.3 mL / min, column temperature 40℃ in Example 3), with the resolution of all 10 target analytes ≥1.8 and no ionization inhibition or peak overlap. In contrast, Comparative Example 1 had a matrix factor of only 0.18, severe ionization inhibition, and single-dimensional chromatography could not distinguish coexisting impurities, with the resolution often below 1.7.

[0054] In terms of detection accuracy and functional coverage, the accuracy deviation of Examples 1-5 was controlled within ±3% (Example 1, allylprogesterone deviation -2.0%), with an intra-assay precision ≤4.2%. Example 4 could also simultaneously detect 10 progestins (deviation within ±5%), and Example 5 could detect hydroxylated / deallyl metabolites (limit of quantification 0.8 μg / kg). Comparative Example 1 had an accuracy deviation of -18.5%, could only measure the original allylprogesterone, and could not detect metabolites or other progestins, thus having a single function.

[0055] In summary, Examples 1-5, through material innovation (MIP-MNPs), process optimization (2D-UPLC), and detection upgrade (Q-TOF dual-mode), comprehensively surpass the traditional method of Comparative Example 1 in terms of efficiency, accuracy, and functionality, fully verifying the technical advancement and practicality of the present invention and providing complete data support for the technical solution in the claims.

[0056] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of protection of the claims.

[0057] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for the determination of allylprogesterone in porcine tissue by liquid chromatography tandem mass spectrometry, characterized in that, Comprising the following steps: S1. Sample pretreatment: S11. Enzymatic treatment: weigh 1.0 g of pig tissue sample, which is pig liver, pig muscle or pig skin adipose tissue; add proteinase K solution to the pig tissue sample, and perform enzymatic digestion at 37℃ in dark water bath for 1 h for pig liver or pig muscle tissue, and perform enzymatic digestion at 50℃ in dark water bath for 30 min for pig skin adipose tissue; after enzymatic digestion, perform ultrasonic treatment for 2 min, and cool to room temperature; S12. Purification and elution: add molecularly imprinted magnetic nanomaterial suspension to the solution after enzymatic digestion, and vortex to achieve adsorption equilibrium; the molecularly imprinted magnetic nanomaterial uses allylprogesterone as a template molecule, methacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a crosslinking agent, and amino-modified Fe3O4 nanoparticles as a carrier; separate the molecularly imprinted magnetic nanomaterial by using a magnetic separation device, and discard the supernatant; wash the molecularly imprinted magnetic nanomaterial with 0.05 mol / L PBS buffer solution for 2 times, vortex for 30 s after each washing; add methanol-formic acid eluent (volume ratio 95:5) and vortex for 5 min for elution; collect the eluent, filter through a 0.22 μm organic phase filter membrane, and obtain a to-be-tested solution; S2. Two-dimensional ultra-high performance liquid chromatography separation: S21. First-dimensional pre-separation: use a two-dimensional ultra-high performance liquid chromatography system equipped with a first-dimensional chromatographic column, a second-dimensional chromatographic column and an intelligent valve switching module; the first-dimensional chromatographic column is a HILIC column with a specification of 2.1 mm x 150 mm and a particle size of 3.5 μm, and is subjected to isocratic elution with 0.1% formic acid water 15% and acetonitrile 85% as mobile phases, a flow rate of 0.3 mL / min, a column temperature of 35℃ and a sample injection amount of 10 μL; collect the fractions of 3-8 min through the intelligent valve switching module, and directly transfer them to the second-dimensional chromatographic column; S22. Second-dimensional fine separation: the second-dimensional chromatographic column is a C18 column with a specification of 50 mm x 2.1 mm and a particle size of 2.6 μm, and is subjected to gradient elution with 0.1% formic acid water (mobile phase A) and acetonitrile (mobile phase B) as mobile phases, a flow rate of 0.3 mL / min and a column temperature of 40℃; the gradient elution program is as follows: 0-1 min, keep mobile phase A at 80% and mobile phase B at 20%; 1-2 min, linearly reduce mobile phase A from 80% to 60% and linearly increase mobile phase B from 20% to 40%; 2-3 min, keep mobile phase A at 60% and mobile phase B at 40%; 3-3.5 min, linearly reduce mobile phase A from 60% to 10% and linearly increase mobile phase B from 40% to 90%; 3.5-4.5 min, keep mobile phase A at 10% and mobile phase B at 90%; 4.8-5 min, linearly increase mobile phase A from 10% to 80% and linearly reduce mobile phase B from 90% to 20%; S3. High resolution mass spectrometry detection: Q-TOF type high resolution mass spectrometer with a resolution of ≥10000 FWHM (m / z 200) equipped with an electrospray positive ion source; the detection parameters are: dry gas temperature 350℃, sheath gas temperature 350℃, sheath gas flow rate 11 L / min, capillary voltage 4000V; full scan + targeted MS / MS dual mode detection is used, the full scan range is m / z 100-500, which is used for screening unknown metabolites of altrenogest; in the targeted MS / MS mode, the quantification ion pair of altrenogest is m / z 311.2→227.2, the collision energy is 14eV, and the characteristic ion pair is m / z 311.2→269.2, the collision energy is 26eV; the concentration of altrenogest in the pig tissue is calculated by the standard curve method, the standard curve takes the concentration of altrenogest as the abscissa and the peak area of the quantification ion pair as the ordinate, and the weighted 1 / X 2 least square linear regression is established, and the concentration range is 0.3-250 μg / kg.

2. A method for the determination of allylprogesterone in swine tissues by liquid chromatography tandem mass spectrometry according to claim 1, characterized in that, The proteinase K solution in S11 has a concentration of 10 mg / mL, which is prepared by using 0.05 mol / L PBS buffer solution with a pH of 7.

4.

3. A method for the determination of allylprogesterone in swine tissues by liquid chromatography tandem mass spectrometry as claimed in claim 1, wherein, The concentration of the molecularly imprinted magnetic nanomaterial suspension in S12 is 0.5 mg / mL, prepared with 50% methanol aqueous solution; the vortex adsorption equilibrium time is 10 min; the magnetic field strength of the magnetic separation device is 0.5 T; and the eluent volume is 500 μL.

4. A method for the determination of allylprogesterone in swine tissues by liquid chromatography tandem mass spectrometry as claimed in claim 1, wherein, The particle size of the amino-modified Fe3O4 nanoparticles in S12 is 20-30 nm; and the preparation method of the molecularly imprinted magnetic nanomaterial comprises: (1) Pre-polymerization: 0.1 mmol of allopregnanone, 0.5 mmol of methacrylic acid and 50 mL of acetonitrile are mixed, stirred at room temperature and in dark for 30 min; (2) Carrier coating: 1 mmol of ethylene glycol dimethacrylate, 0.02 g of azobisisobutyronitrile and 0.5 g of amino-modified Fe3O4 nanoparticles are added, ultrasonically dispersed for 10 min, and polymerized at 60°C under nitrogen protection for 8 h; (3) Template elution and purification: the magnetic polymer particles are repeatedly eluted with a mixture of methanol and acetic acid (volume ratio 9:1) until no allopregnanone is detected in the eluent; then the particles are washed with ultrapure water for 3 times, and dried at 50°C under vacuum for 4 h to obtain the molecularly imprinted magnetic nanomaterial.

5. A method for the determination of allylprogesterone in swine tissues by liquid chromatography tandem mass spectrometry as claimed in claim 1, wherein, The two-dimensional ultra-high performance liquid chromatography system in S2 is also equipped with double high-pressure pumps and an 8°C automatic sampler; and the fraction collection volume of the intelligent valve switching module is 400-500 μL, which is suitable for the eluent volume in S12.

6. A method for the determination of allylprogesterone in swine tissues by liquid chromatography tandem mass spectrometry as claimed in claim 1, wherein, The limit of quantification of the standard curve in S3 is ≤0.3 μg / kg; the accuracy deviation of the allopregnanone determination result is within ±8%, and the precision is ≤10%; in the full scan mode, unknown metabolites of allopregnanone are screened by a metabolite identification algorithm based on theoretical accurate mass number ±5 ppm, the unknown metabolites include hydroxylated allopregnanone and deallyl allopregnanone, the theoretical m / z of hydroxylated allopregnanone is 330.2194, and the theoretical m / z of deallyl allopregnanone is 287.2038; target MS / MS analysis is performed on the suspected metabolites, the structure is confirmed by fragment ion matching, and the characteristic fragment ion pair of hydroxylated allopregnanone is m / z 330.2→288.

2.

7. A method for the determination of allylprogesterone in swine tissues by liquid chromatography tandem mass spectrometry as claimed in claim 1, wherein, At least 5 coexisting progestogens in pig tissues are also synchronously detected in S3, the coexisting progestogens include progesterone, medroxyprogesterone and norethisterone; allopregnanone and the coexisting progestogens are totally 10, and the separation degree of the 10 substances is ≥1.8; wherein, the quantitative ion pair of progesterone is m / z 315.2→109.1, and the collision energy is 20 eV; the quantitative ion pair of medroxyprogesterone is m / z 387.2→345.1, and the collision energy is 22 eV; the quantitative ion pair of norethisterone is m / z 295.2→253.1, and the collision energy is 18 eV.

8. A method for the determination of allylprogesterone in swine tissues by liquid chromatography tandem mass spectrometry as claimed in claim 1, wherein, Deuterated allopregnanone is used as an internal standard in S3; the quantitative ion pair of the deuterated allopregnanone is m / z 314.2→230.2; the internal standard method standard curve is established by concentration-peak area ratio to correct the matrix effect.

9. A method for the determination of allylprogesterone in swine tissues by liquid chromatography tandem mass spectrometry as claimed in claim 1, wherein, The adsorption capacity of the molecularly imprinted magnetic nanomaterial in S12 is ≥8.5 mg / g, which is measured by a static adsorption method: 50 mg of the molecularly imprinted magnetic nanomaterial is added into 10 mL of an allylprogesterone solution with a concentration of 100 μg / mL, and then oscillated at 25 ℃ for 24 h, the concentration of the supernatant is measured to calculate the adsorption capacity; the adsorption selectivity of the molecularly imprinted magnetic nanomaterial to allylprogesterone and metabolites is 12 times that of blank Fe3O4 nanoparticles, which is measured by a competitive adsorption experiment: allylprogesterone and progesterone are used as competitive substrates, and then oscillated and adsorbed at 25 ℃ for 2 h, and then the adsorption amount ratio of the molecularly imprinted magnetic nanomaterial to the blank Fe3O4 nanoparticles is calculated.

10. A method for the determination of allylprogesterone in swine tissues by liquid chromatography tandem mass spectrometry as claimed in claim 1, wherein, The eluent in S12 is placed in an automatic sample injector at 8 ℃ for 24 h, and the concentration deviation is ≤5%; in S3, a sodium formate standard is used for quality calibration before daily detection, and the accurate mass number deviation is ≤2 ppm; the molecularly imprinted magnetic nanomaterial is stored at 4 ℃ in a sealed and light-proof manner, and the adsorption efficiency is still ≥90% after three times of regeneration, and the regeneration method is: the molecularly imprinted magnetic nanomaterial is soaked in a mixed solution of methanol and formic acid with a volume ratio of 95:5 for 5 min, then magnetically separated, and then vacuum dried at 50 ℃ for 2 h.

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