Hypoletin surface imprinted polymer as well as preparation method and application thereof

Abstract

The invention discloses a scopoletin surface imprinted polymer as well as a preparation method and application thereof, and belongs to the technical field of analysis and detection materials. A metal organic framework MIL-96 is used as a carrier and is combined with a molecular imprinting technology, an MIL-96-coated MIPs surface molecularly imprinted polymer is synthesized, a solid-phase extraction column is prepared, the lowest detection limit of scopoletin is 12.14 ng mL <-1 >, and the lowest quantitation limit of scopoletin is 40.46 ng mL <-1 >. The method for detecting the polyphenols in the tobacco extracting solution is established by combining the high performance liquid chromatography to detect the polyphenols, and the adding standard recovery rate is in a range of 95-102.6%. The method is simple to operate, good in reproducibility and low in cost.

Description

Technical Field

[0001] The present invention belongs to the field of removing harmful substances in tobacco, and particularly relates to a scopoletin surface imprinted polymer, a preparation method thereof and an application thereof. Background Art

[0002] Scopoletin (SCP) belongs to coumarin polyphenols in tobacco. As a secondary metabolite, it plays an important role in aspects such as the growth and development of tobacco, modulation characteristics, leaf color, smoke aroma and taste, and smoke physiological strength of tobacco leaves, and is an important factor in measuring tobacco quality. Its degradation products can increase the aroma of tobacco and significantly improve the quality of tobacco products. Scopoletin widely exists in various functional plants, such as Angelica dahurica, Artemisia annua, Artemisia capillaris, Erycibe obtusifolia, Morinda officinalis, etc. Scopoletin has a wide range of pharmacological activities, including expelling wind and reducing phlegm, dilating blood vessels, anticoagulating, anticancer, antioxidant, anti-hyperglycemic, anti-pain, anti-endotoxin, anti-inflammatory, antibacterial, antifungal, anti-tuberculosis and anti-diabetes, etc. Research also shows that scopoletin also has effects such as anti-hepatotoxicity, anti-thyroid, anti-hypertension, anti-proliferation, anti-inflammatory, nervous system regulation, anti-dopaminergic and anti-adrenergic, etc., and also shows significant effects in anti-tumor, antioxidant and anti-aging aspects. However, although scopoletin has many benefits in the fields of plants and medicine, its presence in tobacco products (such as cigarettes) may also bring potential hazards. During the combustion of tobacco, the pyrolysis of scopoletin will produce carcinogenic substances such as hydroquinone and resorcinol, and will damage the respiratory system and cardiovascular system, posing a threat to human health. In addition, scopoletin will also have a synergistic effect with other tobacco components (such as nicotine and tar), further enhancing its toxic effect. Therefore, the content of scopoletin in tobacco is not the higher the better. While improving the quality of tobacco, it is also necessary to pay attention to the possible health risks and reduce its negative impact by optimizing the planting and processing technology and developing low-harm tobacco products.

[0003] SCP and its structural analogue 7-hydroxycoumarin (7-HC) are as follows,

[0004]

[0005] Using 7-HC as the alternative template and the MOF material MIL-96 as the carrier material, a molecularly imprinted composite material (MIL-96@MIPs) was prepared. After 7-HC was eluted, adsorption experiments were carried out to study the binding ability of the material to SCP, and the adsorption data were fitted to explore the adsorption model. Selective adsorption of the polymer was carried out to explore the selectivity of the polymer for the template molecule and its analogs. Taking MIL-96@MIPs as the solid-phase extraction (SPE) adsorbent, the loading concentration of the self-made SPE column and the composition of the eluent were explored, and the adsorption capacity, regeneration stability and spiked recovery efficiency of the self-made SPE column were investigated. At the same time, it was compared with a commercial SPE column to verify its actual application effect in the enrichment and detection of tobacco polyphenolic compounds, and the accuracy and reproducibility of this method were comprehensively evaluated. Summary of the Invention

[0006] The purpose of the present invention is to provide a scopoletin surface imprinted polymer, its preparation method and application.

[0007] Based on the above purpose, the present invention adopts the following technical solutions:

[0008] A preparation method of a scopoletin surface imprinted polymer is as follows:

[0009] (1) Disperse the carrier MIL-96 in methanol;

[0010] (2) Dissolve 7-hydroxycoumarin and MAA in methanol, then add a cross-linking agent and an initiator, and mix well; the molar ratio of 7-hydroxycoumarin, MAA, and the cross-linking agent is 1:(3-5):(15-25), and the addition amount of the initiator is 2-5% of the sum of the masses of MAA and the cross-linking agent;

[0011] (3) Mix the solution in step (1) and the solution in step (2), react at 75-85 °C for 4-6 h, filter, and dry the solid to obtain a polymer; the ratio of the carrier MIL-96 to 7-hydroxycoumarin is 1 g:(0.5-1.5) mmol;

[0012] (4) Use an eluent to elute and remove 7-hydroxycoumarin from the polymer in step (3), and dry it to obtain the scopoletin surface imprinted polymer, denoted as MIL-96@MIPs.

[0013] Further, in step (1), the concentration of the carrier MIL-96 in methanol is 15-25 g / L; the preparation process of the carrier MIL-96 is as follows: Dissolve Al(NO3)3·9H2O and 1,3,5-benzenetricarboxylic acid in deionized water, stir evenly, transfer to a reaction kettle, and react at 180-220 °C for 10-15 h. After the reaction, cool to room temperature, centrifuge, and wash and dry the solid to obtain it; the molar ratio of Al(NO3)3·9H2O to 1,3,5-benzenetricarboxylic acid is (1.5-1.8):1.

[0014] Further, in step (2), the crosslinking agent is ethylene glycol dimethacrylate, and the initiator is azobisisobutyronitrile.

[0015] Further, in step (4), the eluent is a mixed solvent of methanol and acetic acid with a volume ratio of (3-5):1. Wrap the polymer with filter paper and place it in a Soxhlet extractor, add the eluent, and elute at 85-95 °C until no 7-hydroxycoumarin is detected. Then replace the eluent with methanol, reflux for 20-25 h, and dry to obtain it.

[0016] Further, in step (4), the ratio of the polymer to the eluent is 1 g:(130 mL-150 mL), and the drying temperature is 50-60 °C.

[0017] The scopoletin surface imprinted polymer prepared by the above preparation method.

[0018] Application of the above scopoletin surface imprinted polymer as an SPE adsorbent in adsorbing polyphenols in tobacco.

[0019] Further, soak the MIL-96@MIPs with methanol. The ratio of MIL-96@MIPs to methanol is (9-11) mg:1 mL. Load it into an SPE column with a polyethylene sieve plate at the bottom, cover the top with a polyethylene sieve plate, activate with methanol, and then add water to wash away the methanol to obtain it.

[0020] Further, the polyphenols are caffeic acid, scopoletin, and chlorogenic acid.

[0021] Further, the loading solution is an aqueous solution of (5-10) v% methanol, the eluent is an aqueous solution of (60-70) v% methanol, the loading amount of MIL-96@MIPs in the SPE column is 80-100 mg / mL, and the eluent flow rate is 0.5-2 mL min -1 .

[0022] The present invention has the following advantages:

[0023] This application uses the MOF material MIL-96 as a carrier and 7-hydroxycoumarin as a substitute template to synthesize MIL-96@MIPs at a temperature of 80 °C, avoiding the disadvantages of traditional MIPs, such as deep imprinting sites and low adsorption capacity.

[0024] The polymer is used as the packing material of the MIL-96@MIPs·SPE column, and detection is carried out using a high-performance liquid chromatograph to optimize the conditions of the self-made SPE column. 5% methanol - 95% water (V / V) is selected as the sample loading solution, and 70% methanol - 30% water (V / V) is selected as the eluent. When the elution volume is 2 mL, scopoletin can be completely eluted. The lowest detection limit for scopoletin is 12.14 ng mL -1 , and the lowest quantification limit is 40.46 ng mL -1 .

[0025] The reusability and column capacity of the self-made column are determined, and it is confirmed that the column can be reused 9 times, has good regeneration performance, and the loading capacity reaches 2.89 mg, showing a very considerable loading capacity for the target substance. Description of the Drawings

[0026] Figure 1 XRD patterns of MIL-96 and MIL-96@MIPs;

[0027] Figure 2 FT-IR spectra of MIL-96, MIPs, and MIL-96@MIPs;

[0028] Figure 3 PSD diagrams of MIL-96 and MIL-96@MIPs (a: MIL-96; b: MIL-96@MIPs);

[0029] Figure 4 SEM images of MIL-96 and MIL-96@MIPs (A: MIL-96; B: MIL-96@MIPs);

[0030] Figure 5 Isothermal adsorption diagrams of MIL-96@MIPs and MIPs;

[0031] Figure 6 Adsorption rate diagrams of MIL-96@MIPs and MIPs;

[0032] Figure 7 Optimization results of the sample loading solution for the SPE column;

[0033] Figure 8 Optimization results of the elution solution for the SPE column;

[0034] Figure 9 Optimization results of the elution volume for the SPE column;

[0035] Figure 10 The determination results of the column capacity of the SPE column;

[0036] Figure 11 The results of the reusability of the SPE column;

[0037] Figure 12 The comparison between the MIL-96@MIPs·SPE column and three commercial columns (a: Silica·SPE column; b: Florisil·SPE column; c: C18·SPE column; d: MIL-96@MIPs·SPE column; d: 5 μg mL -1 of the SCP standard solution);

[0038] Figure 13 The determination results of the actual samples (A: Determination of the extract of stems and leaves (a: extract of stems and leaves b: MIL-96@MIPs·SPE column) B: Determination of the tobacco concentrate (a: tobacco concentrate b: MIL-96@MIPs·SPE column) C: Primary extract (a: primary extract b: MIL-96@MIPs·SPE column), CA represents caffeic acid, SCP is scopoletin, and CGA is chlorogenic acid). Detailed implementation manners

[0039] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0040] Example 1

[0041] (I) Preparation of MIL-96@MIPs

[0042] 1. Synthesis of MIL-96

[0043] Add 4.1264 g of Al(NO3)3·9H2O (11 mmol) and 1.387 g of 1,3,5-benzenetricarboxylic acid (6.6 mmol) to 60 ml of deionized water, stir at room temperature for 50 minutes, transfer the mixture to a 100 mL polytetrafluoroethylene reaction kettle after mixing evenly, and react at 200 °C for 12 hours. After the reaction is completed, let the reaction kettle cool naturally to room temperature. The solid product is obtained by centrifugal separation. The product is washed several times with N,N-dimethylformamide (DMF) and absolute ethanol in turn to remove unreacted raw materials and impurities. The washed product is placed in a vacuum drying oven and dried at 65 °C to finally obtain the MIL-96 support.

[0044] 2. Synthesis of MIL-96@MIPs

[0045] (1) Place the carrier MIL-96 (1 g) into a 150 mL conical flask, add 50 mL of methanol, seal the flask, and ultrasonically oscillate for 30 min. During this period, continuously shake the conical flask to prevent the carrier material from sinking to the bottom, resulting in uneven mixing.

[0046] (2) Place 1 mmol (0.1621 g) of 7-hydroxycoumarin and 4 mmol (0.3730 g) of MAA (α-methylacrylic acid) in a 250 mL three-necked flask, add 130 mL of methanol, and ultrasonically oscillate (40 kHz) for 10 min to dissolve and mix evenly. Then add 20 mmol of EDMA (3.9644 g, ethylene dimethacrylate) and 0.1735 g of AIBN (azobisisobutyronitrile, the mass of the initiator is 4% of the sum of the masses of the functional monomer and the crosslinking agent), and continue to ultrasonically oscillate (40 kHz) for 10 min to dissolve and mix evenly.

[0047] (3) Transfer the mixed solution in (1) to the three-necked flask in (2), first mechanically stir at room temperature for 30 min to allow the reactants in (1) and (2) to fully contact. Then raise the temperature to 80 °C and continue the reaction for 5 h. Filter the obtained product and dry it in an oven at 65 °C to obtain the required polymer.

[0048] Synthesis of surface non-molecularly imprinted polymer MIL-96@NIPs: Except for not adding the template molecule, the remaining steps are the same as those of MIL-96@MIPs.

[0049] Synthesis of molecularly imprinted polymer MIPs: Except for not adding the carrier, the remaining steps are the same as those of the polymer.

[0050] 3. Removal of template molecules

[0051] Add 150 mL of an eluent of methanol:acetic acid = 4:1 (V / V) to a round-bottom flask. Wrap 1 g of the polymer with filter paper and place it in a Soxhlet extractor for elution, controlling the temperature at about 90 °C. After eluting for 48 h, take out 4 ml of the solution with a pipette and use a UV-spectrophotometer to detect. If there is a UV absorption peak of 7-hydroxycoumarin, elute until no absorption peak appears. After the template molecule is completely eluted, change the methanol:acetic acid = 4:1 (V / V) solution to pure methanol to remove acetic acid and avoid the influence of acidity on subsequent experiments. After refluxing for 24 h, place the polymer in an oven to dry at a temperature of 55 °C to obtain the surface molecularly imprinted polymer (MIL-96@MIPs).

[0052] The elution method of MIPs is the same as above.

[0053] (2) Characterization indicators

[0054] 1. X-ray diffraction (XRD) of MIL-96 and MIL-96@MIPs

[0055] XRD characterization of MIL-96 and MIL-96@MIPs is as follows Figure 1 shown. It can be seen from the data that MIL-96 has diffraction peaks at 7.11°, 7.59°, 8.94°, 11.04°, 13.33°, 15.31°, 16.49°, 18.11°, 20.74°, 22.17°, 25.70°. The same peaks can also be found in MIL-96@MIPs, but the intensity is weaker than that of MIL-96@MIPs, indicating that the polymer has been successfully coated on the surface of the carrier.

[0056] 2. Infrared spectra (FT-IR) of MIL-96 and MIL-96@MIPs

[0057] In the range of 400 - 4000 cm -1 scanning of MIL-96, MIL-96@MIPs and MIPs gives infrared spectra, as shown in detail Figure 2 . According to the infrared data of MIL-96, the bands observed at 768 cm -1 and 735 cm -1 are due to the C–H bending vibration in the aromatic ring, and they appear in both MIL-96 and MIL-96@MIPs, indicating that MIL-96 participates in the synthesis of MIL-96@MIPs. The presence of the above characteristic peaks indicates the successful synthesis of MIL-96 and MIL-96@MIPs.

[0058] 3. PSD characterization

[0059] The prepared MOF material MIL-96 and the new composite material prepared by precipitation polymerization are subjected to PSD detection, and the PSD characterization results are as follows Figure 3 shown. Figure 3 Figures 3a and 3b are the particle size distribution maps of MIL-96 and MIL-96@MIPs respectively. It can be observed from them that the particle size distribution ranges of MIL-96 and MIL-96@MIPs are between 0.182 - 19.953 μm and 1.441 - 60.256 μm respectively, and the average particle sizes are 4.771 μm and 12.784 μm respectively. The particle size of MIL-96@MIPs is larger than that of the carrier material, indicating that the synthesized composite material MIL-96@MIPs is coated on the surface of MIL-96.

[0060] 4. Scanning electron microscopy (SEM) of ZIF67 and MIL-96@MIPs

[0061] SEM characterization of MIL-96 and MIL-96@MIPs is as follows Figure 4 shown. As can be seen from Figure 4It can be seen that MIL-96 presents an obvious rhombic icosahedron structure, further proving the successful synthesis of the carrier material. And Figure 4 B shows that the MIL-96@MIPs polymer is wrapped on the surface of MIL-96. Combining with the FT-IR characterization in 2 can prove the successful synthesis of the polymer.

[0062] (III) Adsorption performance test of MIL-96@MIPs

[0063] 1. Isothermal adsorption experiment of MIL-96@MIPs

[0064] Take 16 centrifuge tubes, divide them into two groups and number them. Accurately weigh 10 mg of MIPs and MIL-96@MIPs and load them into the centrifuge tubes respectively. Add 4 mL of scopoletin standard solution with concentration gradients of 0.5, 1, 5, 10, 15, 20, 25, 30 μg mL -1 (the solvent is methanol: deionized water = 1:9, V / V) to the two groups of centrifuge tubes, vortex to disperse evenly, and adsorb for 1 h at room temperature. Shake 5 times during the process to enable the polymer to fully adsorb the target substance. Use a 2 mL syringe to aspirate the supernatant and filter it with a filter head. Detect it with a UV-visible spectrophotometer, and calculate the adsorption capacity Q (μg mg -1 ) of MIL-96@MIPs and MIPs for the template molecule through formula (1).

[0065] Q = (C0 - C) * V / m (1)

[0066] C0 (μg mL -1 ) is the initial concentration; C (μg mL -1 ) is the equilibrium concentration; V (mL) is the volume of the prepared solution; m (mg) is the mass of the polymer.

[0067] The results are shown in Figure 5 . The experimental data of isothermal adsorption show that when the concentration of the scopoletin standard solution ranges from 0.5 μg mL -1 to 30 μg mL -1 , the adsorption capacity increases significantly, from 0.17 μg mg -1 to 7.28 μg mg -1 , and its adsorption isotherm still shows a continuous growth trend. The adsorption capacity of MIPs is only 4.66 μg mg -1 when the concentration of the scopoletin solution is 30 μg mL -1 . This significant performance difference is due to the unique structural advantages of MIL-96@MIPs.

[0068] 2. Determination of the adsorption rate of MIL-96@MIPs

[0069] Eight centrifuge tubes were numbered and filled with 10 mg of accurately weighed MIL-96@MIPs. 4 mL of scopoletin solution with a concentration of 20 μg mL -1 (the solvent was methanol: deionized water = 1:9, V / V) was added to each centrifuge tube. The adsorption time gradient was set as 1, 2, 5, 10, 15, 20, 30, and 60 min. The supernatant was aspirated with a 2 mL syringe and filtered through a filter head into a centrifuge tube. It was detected using a UV-visible spectrophotometer, and the adsorption capacity Q (μg mg -1 ) of MIL-96@MIPs for scopoletin at different times was calculated through formula (1).

[0070] The kinetic adsorption test showed that the adsorption exhibited stage characteristics. As Figure 6 shown, within the first 5 min, the adsorption capacities of both MIL-96@MIPs and MIPs increased rapidly, indicating that the mass transfer process had high efficiency in the initial stage. As the contact time extended to the 10 - 20 min interval, the adsorption amount was still increasing but the rate slowed down significantly, suggesting that the active sites were gradually occupied, leading to an increase in mass transfer resistance. When the contact time between the adsorbent and scopoletin was in the 20 - 60 min interval, the adsorption amount of MIL-96@MIPs stabilized at around 4.81 μg mg -1 , and the adsorption amount of MIPs was only 3.34 μg mg -1 , indicating that MIL-96@MIPs had better adsorption performance than MIPs

[0071] 3. Selective adsorption determination of MIL-96@MIPs

[0072] The synthesized MIL-96@MIPs and MIL-96@NIPs were used for the adsorption of scopoletin and its structural analogs and non-analogs to compare the selectivity of the polymers. Using methanol: water = 1:9 (V / V) as the solvent, solutions with a concentration of 20 μg mL -1 of scopoletin, 7-hydroxycoumarin, 4-hydroxycoumarin, nicotine, and nornicotine were prepared respectively. Among them, 7-hydroxycoumarin, 4-hydroxycoumarin, and scopoletin are structural analogs, and nicotine and nornicotine are structural non-analogs. Five 10 mL centrifuge tubes were taken, and 10 mg of MIL-96@MIPs was weighed and placed in each of them. 4 mL of the above-prepared solution was added to each centrifuge tube. After the centrifuge tubes were vortexed for 1 min, the polymer was evenly dispersed. After 2 h, centrifugation was carried out, and the supernatant after filtration was measured. The adsorption capacity of MIL-96@NIPs was measured in the same way.

[0073] The specificity of MIL-96@MIPs for scopoletin was evaluated through the Imprinting Factor (IF):

[0074] IF = Q M / Q N (2)

[0075] Q M and Q N (μg mg -1 ) represent the adsorption amounts of MIL-96@MIPs and MIL-96@NIPs respectively. The results are shown in Table 1 for details.

[0076] Table 1 Selective adsorption

[0077]

[0078] As can be seen from Table 1, compared with MIL-96@NIPs, the synthesized MIL-96@MIPs have good selectivity for SCP. For the structural analogues of scopoletin, 7-hydroxycoumarin and 4-hydroxycoumarin, the adsorption amounts of MIL-96@MIPs are significantly higher than those of MIL-96@NIPs. The IF values of scopoletin, 7-hydroxycoumarin and 4-hydroxycoumarin are 2.22, 2.39 and 2.01 respectively, all higher than those of the non-structural analogues nicotine and nornicotine (1.11 and 1.15 respectively). It shows that the imprinted material MIL-96@MIPs with template molecules added during the synthesis process has better adsorption ability and selectivity for the target substances than MIL-96@NIPs without template molecules. Since after washing away the template molecules with methanol / acetic acid (4:1, V / V), the cavities formed by MIL-96@MIPs are similar in structure to SCP, and can specifically recognize and select scopoletin and its structural analogues.

[0079] (IV) Condition optimization and performance test of SPE (MIL-96@MIP) column

[0080] 1. Preparation of SPE column

[0081] Use the synthesized MIL-96@MIPs as the SPE adsorbent. For wet packing of the column, accurately weigh 100 mg of MIL-96@MIPs and place it in a small beaker, moisten it with 10 mL of methanol, and completely transfer it into an SPE column with a polyethylene sieve plate at the bottom, so that the packing height of MIL-96@MIPs in the column is 1.3 cm, and then cover and seal it with a polyethylene sieve plate. Press with appropriate force to keep a proper distance between the polymer particles, and at the same time keep the self-made SPE column with a low column pressure to maintain a suitable flow rate of the solution through the column. Activate the self-made column with 5 mL of methanol to keep it in a wet state, and then add 10 mL of water to wash away the excess methanol. Keep the flow rate in a stable state during this period. After 5 mL of methanol + 10 mL of water completely flow out of the self-made column, carry out the subsequent experiment.

[0082] 2. Optimization of the sample solution

[0083] The solubility of scopoletin in solutions with different organic phase ratios is different. The loading solution should not only completely dissolve the target substance, but also not carry away the target substance due to excessive organic phase ratio. Therefore, it is crucial to select a suitable loading solution. Nine SPE (MIL-96@MIPs) columns were prepared and activated with 5 mL of methanol before use. Using scopoletin as the solute, loading solutions with a concentration of 5 μg mL -1 were prepared with different ratios of chromatographic grade methanol-water and acetonitrile-water (organic phase ratio: 5%, 10%, 15%, 20%, V / V) and pure water respectively. 1 mL of the loading solution was applied to each self-made column, and the flow rate was controlled at 1 mL min -1 . The effluent was collected with a centrifuge tube and filtered through a 0.22 μm filter head, and detected by HPLC-PDA. The binding rate of the SPE column to scopoletin was calculated to determine the optimal organic phase ratio for loading. The results are as Figure 7 shown.

[0084] To enable the target substance to better contact and adsorb with the polymer and exert the adsorption capacity of the column, different loading solutions were used to load the column, and the effluent was collected for HPLC-PDA detection. The binding rate of the target substance to the SPE column was calculated based on the content of the target substance in the effluent. The results are as Figure 7 shown. It can be seen from the data that regardless of whether the organic phase is a methanol system or an acetonitrile system, the binding rate of the target substance to the polymer is 100%, that is, scopoletin was not detected in the effluent, indicating that template molecule leakage does not occur when using the above solvents. Considering that acetonitrile is more toxic and methanol is economical and practical, 5% methanol-water (V / V) was selected as the loading solution.

[0085] 3. Optimization of elution solvent

[0086] To obtain the highest recovery rate and reduce the interference of impurities on the detection results, it is necessary to optimize the elution solvent. In the experiment, 14 SPE columns (MIL-96@MIPs) were prepared and activated with 5 mL of methanol before loading. Subsequently, 20 mL of a loading solution with a concentration of 5 μg mL-1 was prepared according to the optimized conditions, and 1 mL of the loading solution was applied to each SPE column. At the same time, 4 mL of different ratios of chromatographic grade methanol-water and acetonitrile-water solutions (organic phase ratio of 50%-100%, V / V) were prepared respectively. In addition, two SPE columns were used as blank controls and loaded with a loading solution without template molecules. After loading, 4 mL of methanol-water and acetonitrile-water solutions were added as eluents respectively. The effluents of the 14 SPE columns were collected, the flow rate was controlled at 1 mL min-1, and detected after filtering through a 0.22 μm filter head. By calculating the recovery rate, the optimal elution solvent was finally determined.

[0087] Methanol-water and acetonitrile-water solutions with 50% - 100% were selected as eluents for comparison. The results are as Figure 8As shown in the figure. The recovery rate of the methanol-water system is generally higher than that of the acetonitrile-water system. The recovery rate ranges from 77.9% to 97.1%, and the recovery rate of the acetonitrile-water system is maintained within the range of 62.6% to 75.4%. Among them, the recovery rate is the highest when the 70% methanol-water (V / V) solution is used as the eluent, reaching 97.1%. Therefore, the 70% methanol-water (V / V) solution is selected for the next optimization.

[0088] 4. Optimization of the eluent volume

[0089] When eluting the column, it is necessary to ensure the complete elution of the target molecule and improve the elution efficiency to find the optimal eluent volume. Six SPE (MIL-96@MIPs) columns were prepared. After activation, a 5 μg mL -1 scopoletin solution (the solvent is 5v% methanol-water) was loaded onto it. After the solution completely flowed out, 1, 2, 3, and 4 mL of eluent were added to each SPE column in turn, and the flow rate was controlled at 1 mL min -1 , and during this period, the column should be kept moist to prevent cracks from appearing due to the drying of the polymer, which may affect the experimental results. The effluent was collected with a centrifuge tube for HPLC-PDA detection. According to the peak appearance situation, the optimal eluent volume was judged. Determining the elution volume helps to improve the experimental efficiency and save reagents, and find the minimum volume that can completely elute the target compound through experiments. The results are as Figure 9 shown. It can be seen that when 1 mL of eluent was added, most of the scopoletin was eluted; when 2 mL of eluent was added, a small amount of SCP could be detected, while when 3 mL of eluent was added, scopoletin could not be detected. This indicates that 2 mL can already completely elute the target substance. Therefore, the elution volume was determined to be 2 mL.

[0090] 5. Determination of column capacity

[0091] If the sample loading amount exceeds the maximum loading capacity of MIL-96@MIPs, it will cause the leakage of the target substance and affect the experimental results. Therefore, it is necessary to determine the column capacity. Three SPE (MIL-96@MIPs) columns were prepared. One self-made column was used as a blank control, and one self-made column was used as a parallel control. A scopoletin loading solution with a concentration of 10 μg mL -1 was prepared with the optimized sample loading solvent. The previous activation steps were the same as above. Each time, 1 mL of sample was loaded, and this was done continuously 300 times. During this period, the self-made column should be kept moist to avoid cracks that may affect the experiment. The flow rate was controlled at 1 mL min -1 throughout the process. The effluent was collected with a centrifuge tube and filtered with a 0.22 μm filter head, and then detected by HPLC-PDA.

[0092] To verify the enrichment ability of the column for the target substance and ensure the effective adsorption of the target compound, it is necessary to determine the capacity of the self-made column. Using 10 μg mL-1 The SCP solution was loaded onto the MIL-96@MIPs·SPE column, and the effluent was collected in a centrifuge tube, filtered and transferred to a vial for HPLC-PDA determination. The results are as Figure 10 shown. The target peak of SCP appeared at 290 mL, indicating that SCP leaked from the MIL-96@MIPs·SPE column. Therefore, the column capacity was 2.89 mg.

[0093] 6. Determination of the reusability of the self-made column

[0094] To test the stability of the self-made column, a reusability experiment was carried out. Three SPE (MIL-96@MIPs) columns were prepared, one self-made column as a blank control and one self-made column as a parallel control. From activation to elution was taken as one cycle (the concentration of the scopoletin solution was 5 μg mL -1 ), and the experiment was repeated for 9 cycles on the three self-made columns. HPLC-PDA was used for detection to determine the recovery rate of the polymer in each cycle to judge the reusability of the column.

[0095] Although the traditional SPE column can also achieve the purpose of adsorption, it can only be used once, increasing the experimental cost and making the steps more cumbersome. However, MIL-96@MIPs has excellent thermal stability and chemical stability, and the self-made SPE column with MIL-96@MIPs as the column packing can be reused. To determine the stability of the column, a reusability experiment was carried out on it, and the results are as Figure 11 shown. Using the optimized conditions, the self-made column was recycled 9 times. It can be seen that the recovery rate decreased. The reason may be that the repeated adsorption and desorption caused partial damage to the structure of the polymer, resulting in a decrease in the recovery rate, but it still remained above 92%. This indicates that the self-made SPE (MIL-96@MIPs) column in this experiment has good reusability and can be reused 9 times.

[0096] (V) Method validation

[0097] 1. Linear range

[0098] Using scopoletin as the solute and 5 v% methanol-aqueous solution as the solvent, solutions with concentration gradients of 200, 500, 1000, 1500, 2000, 5000, 10000, 20000 ng mL -1 were prepared, and HPLC-PDA was used for determination to make a standard curve and determine the linear range. Three times the signal-to-noise ratio was taken as the limit of detection (LOD) to detect the minimum detectable amount of scopoletin by this method, and ten times the signal-to-noise ratio was taken as the limit of quantification (LOQ) to indicate the lowest content required for scopoletin to be quantitatively determined by this method.

[0099] The linear range, detection limit, and quantification limit of scopoletin were determined. In the concentration range of 200 - 20000 ng mL -1 , the linear equation was y = 14.41x + 8408.4, (R 2 = 0.9991). The lowest detection limit was 12.14 ng mL -1 , and the lowest quantification limit was 40.46 ng mL -1 .

[0100] 2. Method accuracy

[0101] Under the optimized SPE column conditions, the recoveries of scopoletin at three concentration levels were determined. Activate 18 SPE (MIL-96@MIPs) columns for standby, divide them into three groups on average and label them. Prepare three groups of scopoletin solutions with concentrations of 20, 40, and 60 μg mL -1 (using 5v% methanol-aqueous solution as the solvent) to load onto the columns, and load 1 mL onto each column. Elute the columns with the optimized solution. The columns must not crack throughout the process, control the flow rate at 1 mL min -1 , collect the effluent with a centrifuge tube, and filter it with a 0.22 μm filter head for HPLC-PDA detection. Calculate the recoveries at different concentrations to verify the accuracy and precision of the method. The results are shown in Table 2. It can be seen that at the three loading concentration levels, the recoveries of scopoletin by the self-made column are in the range of 98.66% - 102.7%, and the RSD values are between 1.09% - 2.45%. The experimental results are within the allowable range of detection, proving that the developed method has good accuracy and precision.

[0102] Table 2 Spiked recovery experiment

[0103]

[0104] 3. Commercial columns

[0105] After diluting the primary tobacco extract (from Henan Cigarette Industry Tobacco Sheet Co., Ltd.) until the target substance could not be detected, it was used as a solvent to prepare a 5.0 μg·mL -1 scopoletin solution. Activate SPE (MIL-96@MIPs) and 3 commercial columns (C 18 , Florisil, Silica) respectively. After activation, load 1 mL of the 5.0 μg·mL -1 scopoletin spiked solution, and elute it with the optimized eluent. Filter it with a 0.22 μm filter head for HPLC-PDA detection, calculate the recoveries of the four solid-phase extraction columns, and evaluate the actual performance of the self-made column. The results are shown in Figure 12 .

[0106] It can be seen from Figure 12 that the MIL-96@MIPs·SPE column has the best retention effect on scopoletin. Because specific imprinting sites are left after the replacement template molecule is eluted. When scopoletin passes through the self-made SPE column, the target compound is retained by the imprinting sites through hydrogen bonding. Using the optimized eluent, scopoletin in the MIL-96@MIPs·SPE column can be completely eluted, and the recovery rate is as high as 99.37%, indicating that the method is reliable, the results are accurate and credible, and it is applicable to the analysis of actual samples.

[0107] C 18 ·The packing material of the SPE column consists of octadecyl (C 18 H 37 ) long-chain alkyl groups bonded to the surface of the silica matrix. These alkyl chains have strong hydrophobicity. When the sample solution passes through the C 18 ·SPE column, non-polar or weakly polar compounds will bind to the C 18 alkyl chains through hydrophobic interaction and thus be retained in the column. The recovery rate of the C 18 column is 74.73%, which is relatively ideal compared with the other two commercial columns. The packing material of the Florisil column has a porous structure and adsorbs non-polar or weakly polar compounds through van der Waals forces. Its retention effect on SCP is weak, and the recovery rate is 43.28%. The packing material of the Silica column consists of silica (SiO2·nH2O). The silica surface has a rich pore structure, which can provide a large adsorption area. Its retention ability for non-polar or weakly polar compounds is weak, and the recovery rate is only 35.93%.

[0108] 4. Determination of actual samples

[0109] To detect the use of the self-made SPE (MIL-96@MIPs) column in actual samples, the tobacco extract (the stem and leaf extract, tobacco concentrate and primary extract from Henan Cigarette Industry Tobacco Thin Sheet Co., Ltd.) was filtered with qualitative filter paper. The filtered tobacco extract was diluted 80 times with 5v% methanol-water and then 1 mL of it was loaded onto the SPE (MIL-96@MIPs) column to detect the enrichment ability of the column. The column was eluted according to the optimized conditions, during which the flow rate was controlled at 1 mL min -1 , the eluate was collected, filtered with a 0.22 μm filter head, and used for HPLC-PDA detection. The results are as Figure 13 shown.

[0110] The components in tobacco, namely the stem and leaf extract, tobacco concentrate and primary extract, were enriched by the MIL-96@MIPs·SPE column. The self-made column was loaded with actual samples, and the eluate was added for elution. The eluate was determined by UPLC-MS / MS. The results are as Figure 13As shown in Figure 13(A), in the LC-MS spectrum of the leaf and stem extract, a is the LC-MS spectrum of the leaf and stem extract, and b is the LC-MS spectrum of the leaf and stem extract after enrichment and separation by the MIL-96@MIPs·SPE column. It can be concluded that after treatment with the self-made column, the contents of various polyphenol components in the leaf and stem extract have decreased to varying degrees.

[0111] In Figure 13(B), a is the LC-MS spectrum of the tobacco concentrate, and b is the LC-MS spectrum of the concentrate after treatment with the MIL-96@MIPs·SPE column. After enrichment and separation by the self-made column, the contents of polyphenols such as CA, CGA, and SCP in the primary extract can be retained by the column to a certain extent, indicating that the self-made column can be used for the enrichment of polyphenols in tobacco.

[0112] In Figure 13(C), a is the LC-MS spectrum of the primary tobacco extract, and b is the LC-MS spectrum of the primary extract after adsorption by the MIL-96@MIPs·SPE column. It can be seen that the self-made column has a good enrichment effect on the polyphenols in the primary extract and will not be completely adsorbed, which can meet the requirement of reducing the impact on human health without affecting the taste of tobacco products.

[0113] In summary, in this application, the MOF material MIL-96 is used as the carrier and 7-hydroxycoumarin is used as the alternative template to synthesize MIL-96@MIPs at a temperature of 80 °C, avoiding the disadvantages of traditional MIPs with deep imprinting sites and low adsorption capacity. The carrier MIL-96, the imprinted material MIPs, and MIL-96@MIPs are characterized by techniques such as XRD, FT-IR, PSD, and SEM.

[0114] From the dynamic adsorption experiment, it can be seen that the adsorption capacity of MIL-96@MIPs increases rapidly within 5 min, with a fast mass transfer ability, which forms the basis for subsequent detection of actual samples. When the concentration of SCP is 30 μg mL -1 the adsorption capacity is 7.28 μg mg -1 , while the adsorption capacity of MIPs is only 4.66 μg mg -1 , indicating that the addition of the carrier makes MIL-96@MIPs have a larger adsorption capacity. Through the selective adsorption experiment, the IF values of scopoletin and its structural analogs are all ≥2.01, while the IF values of non-structural analogs are 1.11 and 1.15 respectively, indicating that MIL-96@MIPs has good selectivity.

[0115] The polymer was used as the packing material of the MIL-96@MIPs·SPE column, and detection was carried out using a high-performance liquid chromatograph to optimize the conditions of the self-made SPE column. 5% methanol-95% water (V / V) was selected as the sample loading solution, and 70% methanol-30% water (V / V) was selected as the eluent. When the elution volume was 2 mL, scopoletin could be completely eluted. The reusability and column capacity of the self-made column were determined, and it was confirmed that the column could be reused 9 times, with good regeneration performance. The loading capacity reached 2.89 mg, and the loading capacity for the target substance was very considerable.

[0116] The quantitative accuracy of the method was verified by the matrix spike method. In the concentration gradient range of 20-60 μg kg -1 , the quantitative recovery efficiency of the target substance was in the range of 95.2%-102.6% (n = 6), and the relative standard deviation (RSD = 1.09%-2.45%)

[0117] The self-made MIL-96@MIPs·SPE column was compared with three commercial columns (C 18 ·SPE column, Florisil·SPE column and Silica·SPE column). The self-made SPE column had the best retention effect on SCP, and the recovery rate could reach 99.37%. The C 18 ·SPE column also had a good recovery effect, with a recovery rate of 74.73%. The recovery rates of the Florisil·SPE column and the Florisil·SPE column were 43.28% and 35.93% respectively, and the retention effects were poor.

[0118] The MIL-96@MIPs·SPE column was used for the enrichment and separation of polyphenols in tobacco midrib leaves, concentrates and primary extracts. It was found that MIL-96@MIPs·SPE had good enrichment and adsorption ability for polyphenols in tobacco, and would not be completely adsorbed, which could meet the requirement of reducing the impact on human health without affecting the taste of tobacco products. Moreover, it had the advantages of stable performance, simple operation and low cost, and could be used in the practical application of tobacco products, and might have good commercial value in the future.

Claims

1. A preparation method of scopoletin surface imprinted polymer, characterized in that, The process is as follows: (1) Disperse the carrier MIL-96 in methanol; (2) Dissolve 7-hydroxycoumarin and MAA in methanol, then add a crosslinking agent and an initiator, and mix well; the molar ratio of 7-hydroxycoumarin, MAA, and the crosslinking agent is 1:(3 - 5):(15 - 25), and the addition amount of the initiator is 2 - 5% of the sum of the masses of MAA and the crosslinking agent; (3) Mix the solution in step (1) and the solution in step (2), react at 75 - 85 °C for 4 - 6 h, filter, and dry the solid to obtain a polymer; the ratio of the carrier MIL-96 to 7-hydroxycoumarin is 1 g:(0.5 - 1.5) mmol; (4) Use an eluent to elute and remove 7-hydroxycoumarin from the polymer in step (3), and dry it to obtain the scopoletin surface imprinted polymer, denoted as MIL-96@MIPs.

2. The preparation method of the scopoletin surface imprinted polymer according to claim 1, characterized in that, In step (1), the concentration of the carrier MIL-96 in methanol is 15 - 25 g / L; the preparation process of the carrier MIL-96 is as follows: Dissolve Al(NO3)3·9H2O and 1,3,5-benzenetricarboxylic acid in deionized water, stir evenly, then transfer to a reaction kettle and react at 180 - 220 °C for 10 - 15 h. After the reaction, cool to room temperature, centrifuge, and wash and dry the solid to obtain it; the molar ratio of Al(NO3)3·9H2O to 1,3,5-benzenetricarboxylic acid is (1.5 - 1.8):

1.

3. The preparation method of the scopoletin surface imprinted polymer according to claim 1, wherein, In step (2), the crosslinking agent is ethylene glycol dimethacrylate, and the initiator is azobisisobutyronitrile.

4. The preparation method of the scopoletin surface imprinted polymer according to claim 1, characterized in that, In step (4), the eluent is a mixed solvent of methanol and acetic acid with a volume ratio of (3 - 5):

1. Wrap the polymer with filter paper and place it in a Soxhlet extractor, add the eluent, and elute at 85 - 95 °C until no 7-hydroxycoumarin is detected. Then replace the eluent with methanol, reflux for 20 - 25 h, and dry to obtain it.

5. The preparation method of the scopoletin surface imprinted polymer according to claim 4, characterized in that, In step (4), the ratio of the polymer to the eluent is 1 g:(130 mL - 150 mL), and the drying temperature is 50 - 60 °C.

6. The scopoletin surface imprinted polymer prepared by the preparation method according to any one of claims 1 to 5.

7. Application of the scopoletin surface imprinted polymer according to claim 6 as an SPE adsorbent for adsorbing polyphenolic substances in tobacco.

8. The application according to claim 7, wherein Wet MIL-96@MIPs with methanol, pack them into an SPE column with a polyethylene sieve plate at the bottom, then cover and seal it with a polyethylene sieve plate, activate with methanol, and then add water to wash away the methanol to obtain it.

9. The application according to claim 7, characterized in that, The polyphenolic substances are caffeic acid, scopoletin, and chlorogenic acid.

10. The application according to claim 7, characterized in that, The sample loading solution is an aqueous solution of (5-10) v% methanol, the eluent is an aqueous solution of (60-70) v% methanol, the loading amount of MIL-96@MIPs in the SPE column is 80-100 mg / mL, and the liquid flow rate of the column is 0.5-2 mL min -1 .

Images