Tobacco plant alkaloid ZIF-8 (at) MIP molecularly imprinted polymer as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of analysis and detection materials, and particularly relates to a tobacco plant alkaloid ZIF-8 (at) MIP molecularly imprinted polymer as well as a preparation method and application thereof. According to the preparation method, ZIF-8 is taken as a carrier material, 4-pyrrolidinyl pyridine is taken as an alternative template, alpha-MAA is taken as a functional monomer, EGDMA is taken as a cross-linking agent, AIBN is taken as an initiator, an imprinted polymer layer is synthesized on the surface of ZIF-8, 4-pyrrolidinyl pyridine, alpha-MAA, EGDMA is taken as a cross-linking agent, AIBN is taken as an initiator, and imprinting sites are distributed on the surface of the material, so that the ZIF-8-coated MIP molecularly imprinted polymer for enriching nornicotine is prepared. The prepared ZIF-8 (at) MIP molecularly imprinted polymer is used for preparing a novel separation medium (namely column filler or adsorbent) of an SPE column, nicotine, isonicotine or nornicotine in tobacco products such as a tobacco extracting solution, a reconstituted tobacco extracting solution and a reconstituted tobacco concentrated solution can be specifically separated and enriched, and the detection sensitivity and accuracy are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical and detection materials, and particularly relates to a tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer, a preparation method thereof and an application thereof. Background Art

[0002] Nor-nicotine (Nor-Nic) belongs to tobacco alkaloids and ranks second only to nicotine in tobacco content. It has strong alkalinity and can produce irritating-smelling nitrosamine compounds such as NNK during the tobacco processing. Some studies have shown that since secondary amine-based nor-nicotine is more unstable than tertiary amine-based nicotine and is more prone to nitrosation reactions, the conversion of nicotine to nor-nicotine resulting in an increase in nor-nicotine content is the main factor for the increase in the total amount of NNN (N-nitrosonornicotine) and TSNAs (nitrosamines) in tobacco leaves. Reducing the content of nor-nicotine substances in tobacco products, realizing the precise regulation of nor-nicotine substances in tobacco products, and improving the quality of tobacco products are key problems that the tobacco industry needs to solve.

[0003] At present, the determination methods of nicotine and nor-nicotine in tobacco include gravimetric analysis, spectrophotometry, titration, polarimetry, infrared spectroscopy, atomic absorption spectrophotometry, chromatography, potentiometry, polarography, chromatography, etc. Generally, the alkaloids in tobacco are detected and analyzed by GC-MS coupling technology, but its pretreatment is very cumbersome and time-consuming and laborious. In the process of using HPLC method, the sample can be injected for detection after simple solid-phase extraction pretreatment, but the sample matrix needs to be considered to be relatively complex. Tobacco products such as tobacco leaves, reconstituted tobacco leaves by papermaking method, and tobacco concentrates have complex components. In the determination process of nor-nicotine and nicotine, there are many sample pretreatment steps, many interfering components of the target to be measured, and low concentration levels of secondary tobacco alkaloids, which put higher requirements on sample pretreatment, separation and detection technologies.

[0004] Molecular imprinting is a new technology for separating and purifying organic substances that has emerged in recent years. It has high selective recognition for target molecules and their analogs and can selectively adsorb target molecules. Scientific researchers have synthesized a large number of molecularly imprinted polymers, and many studies have combined MIPs (molecularly imprinted polymers) with SPE, using MIPs as the separation medium in the SPE column to achieve the separation and enrichment of various complex matrices. However, there is currently no relevant report on the surface molecular imprinting separation and enrichment of nor-nicotine in tobacco and its tobacco products.

[0005] Therefore, for tobacco and its tobacco products, it is very important to explore and synthesize a new separation medium as the column packing or adsorption material in SPE for separating and enriching nor-nicotine in complex matrices.

[0006] For surface molecular imprinted polymers (SMIPs), an important issue is what material to use as the core wrapped by the polymer layer.

[0007] Therefore, in the present invention, through screening a variety of new materials, ZIF-8 (Zeolitic Imidazole Frameworks, ZIF) is finally selected as the carrier material, 4-pyrrolidinylpyridine, a structural analog of nicotine, as the alternative template, α-MAA as the functional monomer, EGDMA as the crosslinking agent, and AIBN as the initiator. An imprinted polymer layer is synthesized on its surface, so that the imprinting sites are distributed on the material surface, thereby preparing the ZIF-8@MIP molecularly imprinted polymer for enriching nicotine, isonicotine or nornicotine.

[0008] On this basis, the ZIF-8@MIP molecularly imprinted polymer is used as a new separation medium (i.e., column packing or adsorbent) for the SPE column, and SMIPs is combined with SPE to specifically separate and enrich nornicotine in tobacco products such as tobacco extracts, reconstituted tobacco extracts, and reconstituted tobacco concentrates. Summary of the Invention

[0009] The object of the present invention is to provide a tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer and its preparation method. By adjusting the types and amounts of raw materials such as the carrier material ZIF-8, template molecule, functional monomer, crosslinking agent, and initiator, an imprinted polymer layer is synthesized on its surface, so that the imprinting sites are distributed on the material surface, thereby improving the adsorption performance of the prepared ZIF-8@MIP molecularly imprinted polymer.

[0010] Another object of the present invention is to provide the application of the tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer as a separation medium (adsorbent material) for the SPE column.

[0011] The third object of the present invention is to provide the tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer for highly selective separation or enrichment of nicotine, isonicotine or nornicotine in tobacco and tobacco products, facilitating accurate quantitative and qualitative detection of nicotine, isonicotine or nornicotine in tobacco and tobacco products.

[0012] The present invention can also establish an SPE-UPLC-MS / MS method suitable for detecting the contents of nicotine, isonicotine or nornicotine in tobacco and its tobacco products (tobacco extracts, reconstituted tobacco extracts, reconstituted tobacco concentrates, etc.) by using the tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer.

[0013] Based on the above objects, the present invention adopts the following technical solutions:

[0014] A preparation method of a tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer, comprising the following steps:

[0015] 1) Preparation of ZIF-8:

[0016] Place the metal zinc salt in water and ultrasonically treat it until it is completely dissolved, denoted as solution A; then place 2-methylimidazole in water and ultrasonically treat it until it is completely dissolved, denoted as solution B;

[0017] Add solution A to solution B, stir at room temperature (25±5°C) for 3-7 min, then let it stand and react in a reaction kettle for 20-30 h to obtain a white crystalline solid. Separate the solid from the liquid (by centrifugation), wash, and dry to obtain the metal-organic framework material ZIF-8;

[0018] 2) Synthesis of ZIF-8@MIP:

[0019] Use the metal-organic framework material (ZIF-8) prepared in step 1) as the carrier material; weigh ZIF-8, add it to solvent I, and ultrasonically treat it for 13-18 min to prepare a ZIF-8 dispersion;

[0020] Add the template molecule and the functional monomer to solvent II, ultrasonically treat it for 5-20 min to allow the template molecule and the functional monomer to come into full contact and undergo prepolymerization. Then add the prepared ZIF-8 dispersion, and then successively add the crosslinking agent and the initiator. Stir at room temperature (25±5°C) for 7-15 min, then raise the temperature to a certain value and react for a certain time. After the reaction is completed, cool to room temperature (25±5°C), separate the solid from the liquid (by suction filtration), obtain a solid product, wash, and dry to obtain a powdery solid, which is the surface molecularly imprinted polymer ZIF-8@MIP.

[0021] Specifically, in step 1), the metal zinc salt is any one of zinc acetate, zinc chloride, and zinc nitrate.

[0022] Specifically, in step 1), the concentration of solution A is 0.08-0.15 g / mL.

[0023] Specifically, in step 1), the concentration of solution B is 0.1-0.18 g / mL.

[0024] Specifically, in step 1), the mass ratio of solution A to solution B is 1:(1.5-3), or the volume ratio of solution A to solution B is 1:(2-4).

[0025] Specifically, in step 1), the number of washing times is 3-5 times, the drying temperature is 50-70°C, and the drying time is 10-15 h.

[0026] Specifically, in step 2), the solvent I is any one of methanol, absolute ethanol, acetone, and acetonitrile, preferably methanol.

[0027] Specifically, in step 2), the solvent II is any one of methanol, absolute ethanol, acetone, and acetonitrile, preferably methanol.

[0028] Specifically, in step 2), the concentration of the ZIF-8 dispersion is 0.01 - 0.05 g / mL.

[0029] Specifically, in step 2), the template molecule is pyrrolidinylpyridine, and the functional monomer is α-methylacrylic acid (α-MAA).

[0030] Specifically, in step 2), the cross-linking agent is ethylene glycol dimethacrylate (EGDMA), and the initiator is azobisisobutyronitrile (AIBN).

[0031] Specifically, in step 2), the molar ratio of the template molecule, functional monomer, and cross-linking agent is 1:(3 - 8):(10 - 30).

[0032] Specifically, in step 2), the mass ratio of the template molecule to the initiator is 1:(1 - 2), preferably 1:1.2.

[0033] Specifically, in step 2), the addition amount of the ZIF-8 dispersion is 50 - 100 mL.

[0034] Preferably, in step 2), the volume ratio of the ZIF-8 dispersion to the cross-linking agent is (12 - 28):1.

[0035] Alternatively, in step 2), the mass ratio of ZIF-8 to the template molecule is 1:(0.1 - 0.2), preferably 1:0.15.

[0036] Specifically, in step 2), the reaction temperature is 60 - 100 °C, and the reaction time is 5 - 24 h.

[0037] Specifically, in step 2), the number of washing times is 3 - 5 times, the drying temperature is 50 - 70 °C, and the drying time is 10 - 15 h.

[0038] Further preferably, for the surface molecularly imprinted polymer ZIF-8@MIP prepared in step 2), the template molecule therein is removed by elution with an eluent; the specific elution method is as follows:

[0039] Wrap the prepared surface molecularly imprinted polymer ZIF-8@MIP with filter paper, place it in a Soxhlet extractor, add the eluent, and continuously reflux at 80 - 95 °C for 12 - 24 h until no template molecule can be detected.

[0040] Specifically, after elution with the eluent, the surface molecularly imprinted polymer is refluxed and rinsed with pure methanol for 4 - 8 h to maintain neutrality; finally, it is dried in an oven at 50 - 70 °C for 10 - 15 h.

[0041] Specifically, the eluent is an acidic eluent or a basic eluent. The acidic elution solution is an eluent of methanol / acetic acid (9:1, V / V), and the basic eluent is an eluent of methanol / triethylamine (9:1, V / V).

[0042] Specifically, the dosage ratio of the eluent to the imprinted material is (0.01 - 0.02) g / mL.

[0043] Furthermore, the present invention also provides the tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer prepared by the above method. The tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer has high thermal stability, and the BET specific surface area is 67.39 m 2 ·g -1 , and the tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer has excellent adsorption performance and good selective adsorption for tobacco alkaloids such as nicotine, isonicotine or nornicotine, and can be used as a separation medium (adsorbent material) for preparing a tobacco alkaloid SPE column.

[0044] Furthermore, based on a general inventive concept, the present invention also provides the application of the tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer in the specific separation and enrichment of nicotine, isonicotine or nornicotine in tobacco and tobacco products (tobacco extract, reconstituted tobacco extract, reconstituted tobacco concentrate, etc.).

[0045] Furthermore, based on a general inventive concept, the present invention also provides the application of the tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer as a separation medium (adsorbent material) in a solid phase extraction column (SPE column).

[0046] Specifically, the tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer is used as a separation medium for a solid phase extraction column (SPE) to prepare a tobacco alkaloid SPE column, and can separate or enrich nicotine, isonicotine or nornicotine in tobacco and tobacco products (tobacco extract, reconstituted tobacco extract, reconstituted tobacco concentrate, etc.).

[0047] Furthermore, based on a general inventive concept, the present invention also provides a tobacco alkaloid SPE column prepared by using the tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer.

[0048] Furthermore, based on a general inventive concept, the present invention also provides an application of the tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer in establishing an SPE-UPLC-MS / MS method suitable for detecting the contents of nicotine, isonicotine or nornicotine in tobacco and tobacco products (tobacco extracts, reconstituted tobacco extracts, reconstituted tobacco concentrates, etc.).

[0049] Furthermore, based on a general inventive concept, the present invention also provides a method for preparing a tobacco alkaloid SPE column using the tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer, comprising the following steps:

[0050] SPE column packing and activation: Weigh the ZIF-8@MIP as the adsorbent filler of the SPE column, place it in a prepared empty SPE column, seal both ends of the adsorbent with a sieve plate, and press the filler to a certain height;

[0051] For the prepared SPE column, before use, it is sequentially activated with methanol and water to remove residual impurities and pollutants, so that the surface active sites of the adsorbent in the SPE column are fully exposed, which can enhance the adsorption capacity for the target substance.

[0052] The present invention also establishes an SPE-UPLC-MS / MS method for nornicotine for the prepared tobacco alkaloid SPE column, so as to evaluate the sensitivity and accuracy of the separation medium ZIF-8@MIP prepared by the present invention as an SPE column filler for detecting the contents of nicotine, isonicotine or nornicotine in tobacco and tobacco products (tobacco extracts, reconstituted tobacco extracts, reconstituted tobacco concentrates, etc.).

[0053] Specifically, the optimization of the use conditions of the prepared tobacco alkaloid SPE column includes the following aspects:

[0054] (1) Determination of the sample loading solution concentration: Select the sample loading solvent, prepare sample loading solutions with different concentrations, and detect whether the target substance in the sample loading solution leaks through HPLC-PDA to judge the binding ability of the column filler to target substance solutions with different concentrations, so as to determine the sample loading solution concentration.

[0055] (2) Optimization of the elution solvent: Select different eluent systems. After the SPE column is activated, load the target substance solution, wash, elute, and collect the eluted solution flowing out. Detect the content of the target substance through HPLC-PDA and calculate the recovery rate to determine the eluent system.

[0056] (3) Optimization of the elution volume: Use the optimized elution solvent to elute the SPE column after sample loading and washing, filter and detect and analyze the eluent flowing out each time to determine the optimal volume.

[0057] (4) Determination of the loading capacity of the self-made SPE column: Prepare the target solution as the sample loading solution. After activating the SPE column, load the target solution, process the effluent of each time, and perform detection and analysis by HPLC-PDA to determine the optimal loading capacity.

[0058] Compared with the prior art, the present invention has the following advantages:

[0059] 1. In the present invention, ZIF-8 is used as the carrier material, 4-pyrrolidinopyridine is used as the alternative template, α-MAA is used as the functional monomer, EGDMA is used as the cross-linking agent, and AIBN is used as the initiator. An imprinted polymer layer is synthesized on its surface, so that the imprinting sites are distributed on the material surface, and thus the ZIF-8@MIP molecularly imprinted polymer for enriching nornicotine is prepared.

[0060] 2. The present invention also uses the prepared ZIF-8@MIP molecularly imprinted polymer as a new separation medium (i.e., column packing or adsorbent) for preparing an SPE column, which can specifically separate and enrich nicotine, isonicotine or nornicotine in tobacco products such as tobacco extracts, reconstituted tobacco extracts, and reconstituted tobacco concentrates, and improve the sensitivity and accuracy of detection. Description of the Drawings

[0061] Figure 1 It is the optimization result of the template molecule elution solution in Example 2, where A is: acidic elution solution, B: basic elution solution;

[0062] Figure 2 It is an infrared spectrogram; where a is: ZIF-8, b is: ZIF-8@MIP, c is: MIP;

[0063] Figure 3 It is a scanning electron micrograph; where A and B are: ZIF-8; C and D are: ZIF-8@MIP;

[0064] Figure 4 It is an X-ray diffraction pattern; where a is: ZIF-8; b is: ZIF-8@MIP;

[0065] Figure 5 It is the N2 adsorption-desorption curve and pore size distribution curve of three materials; A and B are respectively the N2 adsorption-desorption curve and HK method micropore size distribution curve of ZIF-8; C and D are respectively the N2 adsorption-desorption curve and BJH mesopore size distribution curve of MIP; E and F are respectively the N2 adsorption-desorption curve and BJH mesopore size distribution curve of ZIF-8@MIP;

[0066] Figure 6 It is a standard curve and an isothermal adsorption curve diagram; where A is: nornicotine concentration-absorbance standard curve; B is: isothermal adsorption curve;

[0067] Figure 7 Adsorption rate curves of ZIF-8@MIP and MIP;

[0068] Figure 8 Selective adsorption of ZIF-8@MIP for two types of substances;

[0069] Figure 9 Diagram of molecular structure analogues and non-analogues;

[0070] Figure 10 Chromatograms of effluents of sample solutions with different concentrations;

[0071] Figure 11 Diagram for optimizing elution solvent;

[0072] Figure 12 Diagram for optimizing elution volume;

[0073] Figure 13 Diagram for determining column capacity;

[0074] Figure 14 Nicotine standard curve;

[0075] Figure 15 Results of reusability of ZIF-8@MIP SPE column. Detailed implementation mode

[0076] In order to make the technical objectives, technical solutions and beneficial effects of the present invention clearer, the following further illustrates the technical solutions of the present invention in combination with specific embodiments. However, the embodiments are intended to explain the present invention and should not be construed as a limitation of the present invention. For those without specific technologies or conditions noted in the embodiments, the technologies or conditions described in the literature in the field or according to the product specifications are followed.

[0077] Reagents, materials, instruments, chromatographic conditions

[0078] 1. Reagents: Zinc acetate dihydrate (Zn·(Ac)2·2H2O, 98%) provides the central ion Zn for preparing ZIF-8 2+, 2-Methylimidazole (98%) provides ligands for the preparation of ZIF-8, α-Methylacrylic acid (α-MAA, 97%) is used as a functional monomer, Ethylene glycol dimethacrylate (EGDMA, 98%) is used as a crosslinking agent, and Azodiisobutyronitrile (AIBN, 98%) is an initiator, all of which are purchased from Tianjin Fuchen Chemical Reagent Factory. 4-Pyrrolidinylpyridine (98%), Nornicotine (Nor-NIC, 98%), Nicotine (NIC), Isonicotine (Iso-NIC, 98%), Chlorogenic acid (CHA, 98%), Rutin (RUT, 98%), 7-Hydroxycoumarin (7-HC, 98%), Methanol (AR), Absolute ethanol (AR), Acetic acid (AR), Triethylamine (AR), Formic acid (HPLC), Ammonium acetate (HPLC), Methanol (HPLC) are purchased from Tianjin Siyou Fine Chemical Co., Ltd. Except for the reagents whose sources are already indicated, the rest of the reagents are purchased from Shanghai Macklin Biochemical Co., Ltd.

[0079] 2. Materials: Florisil packing SPE column and Silica packing SPE column are purchased from Hangzhou Micronpai Technology Co., Ltd.

[0080] 3. Instruments: Fourier transform infrared spectrometer Invenio (Bruker, Germany); X-ray diffractometer Minifle600 (Rigako, Japan); Scanning electron microscope ZEISS Gemini SEM300 (ZEISS, Germany); Malvern particle size analyzer Malvern2000 (Malvern Panalytical, UK); Thermogravimetric analyzer TG 209F3 (NETZSCH, Germany); Specific surface area analyzer BK100C (Beijing Jingwei Gaobo Instrument Co., Ltd., China); UV-visible spectrophotometer TU-1810 (Beijing Purkinje General Instrument Co., Ltd., China); High performance liquid chromatograph - diode array detector HPLC-PDA 2695-2988 (Waters, USA); UPLC-Xevo tandem quadrupole liquid chromatography-mass spectrometry Xevo-TQD H-Class (Waters, USA).

[0081] 4. Liquid chromatography conditions:

[0082] Chromatographic column: Diamonsil Plus 5μm C18, 150mm×4.6mm;

[0083] Mobile phase: methanol / (0.1% formic acid - water) (50:50, V / V);

[0084] Flow rate: 1mL·min -1 ;

[0085] Sample injection volume: 10μL;

[0086] Column temperature: 30°C;

[0087] Ultraviolet detection wavelength: 259nm.

[0088] Ultra - performance liquid chromatography - mass spectrometry detection conditions:

[0089] Chromatographic column: 2.6μm EVO C18 150×3.0mm

[0090] Mobile phase A: methanol, B: 0.1% ammonium acetate - water, gradient elution program: 0 - 1min, 2% A, 1 - 7min, A increases from 2% to 90%, 7 - 10min, A remains at 90%, 10 - 12min, A decreases from 90% to 2%, 12 - 15min, A remains at 2%, ending at the 15th minute, flow rate is 0.3mL·min -1 . Column temperature is maintained at 30°C, sample injection volume is 2μL.

[0091] 5. Mass spectrometry conditions: Electrospray ionization source ESI+; Multiple reaction monitoring (MRM) mode, monitoring parameters of each component (Table 1); Ion source voltage: 3.0kV; Ion source temperature: 150°C; Nebulizing gas pressure: 1000L·h -1 ; Auxiliary gas pressure: 50L·h -1 .

[0092] Table 1 Composition of nicotine - like substances and instrument detection parameters

[0093]

[0094] Example 1

[0095] Example 1 provides a preparation method of nicotine - reducing ZIF - 8@MIP molecularly imprinted polymer, and the specific steps are as follows:

[0096] 1) Synthesis of metal - organic framework material ZIF - 8:

[0097] Add 2.19 g (10 mmol) of zinc acetate dihydrate (Zn·(Ac)2·2H2O) to a 50 mL conical flask, then add 20 mL of deionized water, and sonicate for 10 min (ultrasonic frequency is 40 kHz) until it is completely dissolved, denoted as solution A;

[0098] Accurately weigh 6.48 g (80 mmol) of 2-methylimidazole into a 100 mL conical flask, then add 40 mL of deionized water, and sonicate for 10 min (ultrasonic frequency is 40 kHz) to obtain solution B after complete dissolution;

[0099] Quickly pour solution A into solution B (where the mass ratio of solution A to solution B is 22.19:46.48, or the volume ratio is 20:60), stir at room temperature (25 ± 5 °C) for 5 min, then let it stand for 24 h (to achieve sufficient growth of ZIF-8 crystals), obtain a white solid precipitate, centrifuge, and wash it 3 times with water and absolute ethanol respectively, then place it in an oven at 60 °C for 12 h to obtain a white solid powder, which is ZIF-8. Keep it dry for later use;

[0100] 2) Synthesis of surface molecularly imprinted polymer (ZIF-8@MIP):

[0101] Add 1.00 g of the metal-organic framework material (ZIF-8) prepared in step 1) and 50 mL of methanol to a 150 mL conical flask in sequence, then sonicate for 15 min (ultrasonic frequency is 40 kHz) to obtain a ZIF-8 dispersion;

[0102] Add 0.15 g (1 mmol) of 4-pyrrolidinopyridine and 100 mL of methanol to a 500 mL flask in sequence, sonicate for 5 min (ultrasonic frequency is 40 kHz) until completely dissolved, then add 340 μL (4 mmol) of α-MAA reagent, continue to sonicate for 10 min (ultrasonic frequency is 40 kHz) to allow the template molecule and the functional monomer to come into full contact and undergo pre-polymerization to obtain a pre-polymerization reaction system. Add the ZIF-8 dispersion (the addition amount is 70 mL) to the pre-polymerization reaction system, then add 3.77 mL (20 mmol) of EGDMA and 0.18 g of thermal initiator AIBN in sequence, continue to sonicate for 5 min (ultrasonic frequency is 40 kHz) to ensure complete dissolution; then stir at room temperature (25 ± 5 °C) for 10 min, then raise the temperature of the system to 83 °C and react for 6 h, then cool to room temperature (25 ± 5 °C), perform vacuum filtration to obtain a solid product, wash it 5 times with methanol and water in sequence, and dry it in an oven at 60 °C for 12 h to obtain the surface molecularly imprinted polymer (ZIF-8@MIP). Keep it dry for later use.

[0103] Comparative Example 1

[0104] The present invention also made Comparative Example 1 to synthesize the surface non-imprinted material ZIF-8@NIP. The difference between Comparative Example 1 and Example 1 is that in step 2), the template molecule 4-pyrrolidinylpyridine is not added, and the remaining operations are the same as those in Example 1.

[0105] Comparative Example 2

[0106] The present invention also made Comparative Example 2 to synthesize the non-surface imprinted material MIP. The difference between Comparative Example 2 and Example 1 is that in step 2), the carrier metal-organic framework material (ZIF-8) is not added, and the remaining operations are the same as those in Example 1.

[0107] Example 2

[0108] Elution of the template molecule in the non-surface imprinted material MIP of Comparative Example 2 and the surface molecularly imprinted polymer ZIF-8@MIP of Example 1:

[0109] Adopt the dynamic elution mode of a Soxhlet extractor. Wrap the prepared non-surface imprinted material MIP or surface molecularly imprinted polymer ZIF-8@MIP with filter paper, put it into the Soxhlet extractor, pour in the prepared acidic elution solution methanol / acetic acid (9:1, V / V), and continuously reflux at 90 °C for 24 h. During this period, take the eluate every 2 h until the template molecule cannot be detected, and measure its ultraviolet absorbance. The amount of the acidic elution solution used is 250 to 300 mL, and the amount of the imprinted material used is 3.5 g to 4.5 g.

[0110] Meanwhile, use the basic elution solution methanol / triethylamine (9:1, V / V) as the basic elution control, and keep other operations the same to determine the optimal elution solution and elution time.

[0111] After elution with the elution solution, reflux and rinse the imprinted material with pure methanol for 6 h to keep it neutral. Finally, dry it in an oven at 60 °C for 12 h for standby.

[0112] Experimental results of Example 2:

[0113] By comparing the elution of the template molecule by the acidic elution solution methanol / acetic acid (9:1, V / V) and the basic elution solution methanol / triethylamine (9:1, V / V), from Figure 1As can be seen, the template molecules could not be detected in the acidic elution solution after 10 h of elution, indicating that the template molecules in the polymer had been completely eluted, while the basic elution solution required a longer time to completely elute the template molecules. Therefore, the acidic elution solution had a better elution effect. By using an eluent with a stronger polarity to break the hydrogen bonds between the functional monomers and the template molecules in the polymer, the template molecules would be eluted, and then some spatial positions with imprinting functions would be generated, which were called imprinting sites. Generally, these imprinting sites matched the size of the eluted template molecules, had specific recognition effects, and could also recognize molecules with similar structures to the template molecules well.

[0114] Material Characterization

[0115] The surface molecularly imprinted polymer ZIF-8@MIP obtained in Example 1, and Comparative Example 1 and Comparative Example 2 were characterized by SEM, FT-IR, XRD, PSD, specific surface area, etc., to fully explore the morphology and internal structural characteristics of the surface molecularly imprinted polymer ZIF-8@MIP. Some of the characterization results are as follows:

[0116] 1. FT-IR Characterization

[0117] Fourier Transform Infrared Spectroscopy (FTIR) has a very wide range of applications in material characterization, and can provide valuable information in many aspects for inferring the chemical structure, composition and surface characteristics of materials, and is an important tool for studying materials. The Fourier transform infrared spectrometer was used to scan ZIF-8, ZIF-8@MIP and MIP in the wavenumber range of 4000~400 cm -1 , and then the functional group structure in the material was analyzed according to the infrared spectrum information.

[0118] Figure 2 shows the FT-IR information of ZIF-8, ZIF-8@MIP, MIP in the range of 4000-400 cm -1 . First, the spectral information of ZIF-8 was seen in Figure 2 (a). The strong absorption peak at the wavenumber of 450~420 cm -1 in the fingerprint region represented the overall vibration of metal atoms and ligand skeletons. The obvious and strong absorption peak at 760~690 cm -1 was the vibration of the Zn-N bond, which was considered to be the key characteristic peak of ZIF-8. The absorption peak near 950 cm -1 was caused by the out-of-plane bending vibration of C-H on the imidazole ring (the out-of-plane vibration mode of the ring), and the absorption peak at 1300~1250 cm -1 was the in-plane bending vibration of the imidazole ring, and the absorption peak at 1450~1350 cm-1 The absorption peak represents the C-N stretching vibration in the imidazole ring, which can confirm the existence of the ligand ring. The absorption peak appearing near 1580 cm -1 represents the C=N symmetric stretching vibration in the imidazole ring, further indicating the integrity of the ligand structure. In the range of 3130 - 2920 cm -1 , the absorption peaks of C-H stretching vibration from the methyl group and the imidazole ring can be seen. Through these obvious characteristic peaks, it is shown that the carrier ZIF-8 has been successfully synthesized. Then observe Figure 2 (b) The spectrum of ZIF-8@MIP. It can be learned that the obvious absorption peak appearing near 1250 cm -1 is consistent with that in ZIF-8 and is generated by the in-plane bending vibration of the imidazole ring. And near 420 cm -1 and near 690 cm -1 , absorption peaks corresponding to the same positions in ZIF-8 appear, and the intensity is significantly weakened, indicating the existence of the ZIF-8 carrier in ZIF-8@MIP. Due to the polymer covering its surface, the characteristic absorption peaks of ZIF-8 are weakened. In addition, the strong absorption peak appearing near 1730 cm -1 is from the C=O double bond vibration of the cross-linking agent in the material. This characteristic peak appears at the same position in Figure 2 (c) The spectrum of MIP. The obvious absorption of the C=C bond that does not appear at 1630 cm -1 indicates that the functional monomers have been completely polymerized. Since no carrier is added, no characteristic peaks of ZIF-8 appear.

[0119] 2. SEM Characterization

[0120] Scanning Electron Microscope (SEM) has high resolution, versatility and wide applicability for the surface morphology characterization of material samples. Especially for the characterization of nanomaterials, SEM can observe the size, morphology and distribution of nanoparticles. ZIF-8 and ZIF-8@MIP are characterized by scanning electron microscope, and the images at magnifications of 20000 times and 50000 times are selected for analysis respectively.

[0121] Figure 3 (A) and (B) show the SEM effects of ZIF-8 at magnifications of 50k X and 20k X respectively. It can be seen that the carrier material particles are clear, with distinct edges and corners, and most of them are uniform dodecahedrons, indicating that the ZIF-8 prepared by the room temperature hydration method is very successful. And through Figure 3 (C) and (D), it is found that compared with the carrier ZIF-8, the edges and corners of these particles become blurred, indicating that its surface is covered with polymer, indicating the successful synthesis of ZIF-8@MIP.

[0122] 3. XRD Characterization

[0123] X-ray Diffraction (XRD) is based on the interaction between X-rays and atoms in crystalline materials. Due to the strong periodic structure of crystalline materials, when X-rays irradiate on the crystal, the atoms will cause it to scatter, and enhance each other in a certain direction to form diffraction peaks. Generally speaking, the intensities of diffraction peaks at different positions are different, and these parameters can reflect the structural characteristics of crystalline materials. The ZIF-8 and ZIF-8@MIP were scanned using an X-ray diffractometer in the range of 2θ from 5° to 80°.

[0124] Figure 4 The XRD patterns of the carrier material ZIF-8 and the imprinted material ZIF-8@MIP are shown. By Figure 4 (a) Analyzing the X-ray diffraction peaks of ZIF-8, it was found that obvious characteristic peaks appeared near 2θ = 7.3°, 10.4°, 12.7°, 14.7°, 16.4° and 18.0°. These characteristic peaks are consistent with the ZIF-8 crystal diffraction data provided in the CCDC database (602542), and it can be seen in Figure 4 (a) that the peak at 2θ = 7.3° is the main peak, and compared with other characteristic peaks, its intensity is the highest. Figure 4 These characteristic peaks in it confirm the successful synthesis of ZIF-8 crystals. In Figure 4 (b), it can be seen that the characteristic peaks of ZIF-8@MIP are consistent with those of ZIF-8, and the intensities of all characteristic peaks have decreased. The main peak is still the strongest compared with other peaks, indicating that the polymer has successfully adhered to the surface of the carrier and caused a certain degree of weakening of the ZIF-8 crystal diffraction peaks.

[0125] 4. Specific Surface Area Measurement

[0126] The specific surface area analyzer combined with the BET method is an effective tool for measuring the specific surface area of solid materials and is widely used in the fields of materials science and chemical engineering. Using a specific surface area analyzer, the N2 adsorption-desorption tests of ZIF-8, ZIF-8@MIP and MIP were carried out by the static volumetric method with N2 as the adsorption medium, and the specific surface area can be calculated according to the BET equation.

[0127] By Figure 5From the N₂ adsorption-desorption isotherm curves and pore size distribution curves of the three materials shown, it can be seen that the carrier material ZIF-8 has a high N₂ adsorption capacity. The N₂ adsorption capacity of MIP is worse than that of ZIF-8, while the N₂ adsorption capacity of ZIF-8@MIP has been significantly improved compared with MIP. The specific surface areas of the three materials calculated by the BET equation are summarized in Table 2 below, and some pore size data of the three materials are also shown in Table 2. By comparing the specific surface areas of the three materials, it is known that ZIF-8@MIP prepared with ZIF-8 with a high specific surface area as the carrier material has a specific surface area of 67.39 m 2 ·g -1 which is nearly three times that of the non-surface imprinted material MIP. This fully shows that using ZIF-8 as the support carrier can greatly improve the specific surface area of the imprinted material. Through the analysis of the pore size distribution, it can be understood that the pore size of ZIF-8 is less than 2 nm, belonging to microporous materials, while the two imprinted materials MIP and ZIF-8@MIP contain more high molecular polymers, so the pore size is greater than 2 nm, belonging to mesoporous materials.

[0128] Table 2 Specific surface area and pore size data of three materials

[0129]

[0130] Determination of the adsorption performance of the material in Example 3

[0131] The present invention also studied the adsorption capacity and mass transfer rate of ZIF-8@MIP through adsorption rate and isothermal adsorption experiments. Specifically as follows:

[0132] 1.1 Isothermal adsorption experiment of ZIF-8@MIP

[0133] Two groups of 4 mL of the same series of concentration gradients (5, 10, 15, 20, 25, 30, 35 μg·mL -1 ) of the target solution were respectively prepared in 5 mL capped centrifuge tubes. For one group of solutions, the absorbance was measured by an ultraviolet spectrophotometer, and an absorbance-concentration standard curve was plotted.

[0134] To each solution in the other group, 10 mg of ZIF-8@MIP was added as an adsorbent. After vortexing for 3 - 5 s, it was left to adsorb statically for 1 h. The supernatant was taken for treatment and its absorbance was measured. The adsorption capacity was calculated according to formula (2-1), and an isothermal adsorption curve of ZIF-8@MIP for the nicotine solution was plotted.

[0135]

[0136] In formula (2-1), Q is the mass of the target adsorbed by the adsorbent per unit mass (μg·mg -1), where m is the mass of the adsorbent added to the target solution (mg), c0 is the concentration before adsorption of the solution (μg·mL -1 ), and c1 is the concentration of the solution after adsorption (μg·mL -1 )(calculated according to the above standard curve equation), and V represents the volume of the solution before adsorption (mL).

[0137] At the same time, a control experiment of MIP was conducted.

[0138] 1.2 ZIF-8@MIP Adsorption Rate Experiment

[0139] Accurately weigh 10 mg of ZIF-8@MIP into a 5 mL centrifuge tube, and add 4 mL of 25 μg·mL -1 target solution. After adsorption for different times, measure the absorbance of the supernatant after treatment, calculate the adsorption capacity, plot the adsorption rate curve, and linearly fit the experimental data and calculation results using the pseudo-second-order kinetic adsorption model (2-4).

[0140]

[0141] In Equation 2-2, t is the adsorption time, and Q t is the adsorption capacity of the adsorbent at time t (μg·mg -1 ), Q e is the equilibrium adsorption capacity of the adsorbent (μg·mg -1 ), and k2 is the rate adsorption constant (mg·μg -1 ·min -1 ).

[0142] At the same time, a control experiment of MIP was conducted.

[0143] 1.3 ZIF-8@MIP Selective Adsorption Experiment

[0144] ZIF-8@MIP uses 4-pyrrolidinylpyridine with a molecular structure similar to that of Nor-NIC as a substitute template. To determine the adsorption selectivity for Nor-NIC, the adsorption of two structurally similar NIC and Iso-NIC was measured, and the adsorption capacities of ZIF-8@MIP were compared. ZIF-8@MIP and ZIF-8@NIP were used to measure the adsorption of multiple molecular structure analogs or non-analogs NIC, Nor-NIC, Iso-NIC, CHA, RUT, and 7-HC (structures are as Figure 9 shown), and after treating the supernatant, the measurement was carried out. The imprinting factor (Impact factor, IF) was calculated according to Equation 2-3 to evaluate its selectivity.

[0145]

[0146] In Formula 2-3, Q M is the adsorption capacity of ZIF-8@MIP, and Q N is the adsorption capacity of ZIF-8@NIP.

[0147] Experimental results:

[0148] 2.1 Isothermal adsorption

[0149] From Figure 6 the isothermal adsorption curves, it can be seen that MIP and ZIF-8@MIP adsorb the nicotine solution with a concentration of 5 - 35 μg·mL -1 . Their adsorption capacities increase with the increase of substrate concentration, and the adsorption capacity of ZIF-8@MIP is significantly higher than that of MIP. When the substrate concentration is 35 μg·mL -1 , the adsorption capacity of MIP reaches 2.19 μg·mg -1 , which is still less than 2.62 μg·mg of ZIF-8@MIP -1 . According to the previous description, since ZIF-8 is a typical zeolitic imidazolate MOF material with a high specific surface area and adsorption capacity, the ZIF-8@MIP synthesized in this invention further confirms the excellent performance of ZIF-8. Comparing the adsorption capacity differences between the prepared MIP and ZIF-8@MIP, it shows that due to the support provided by the carrier ZIF-8 in ZIF-8@MIP, the imprinting sites are distributed on the surface, making its adsorption of target molecules more effective. Therefore, ZIF-8@MIP has better adsorption performance.

[0150] 2.2 Adsorption rate

[0151] In the experiment of measuring the adsorption rates of the two materials, their adsorption rate curves are as Figure 7 shown. It can be easily seen that the time taken for MIP to reach adsorption equilibrium during the adsorption process is close to 7 minutes, while ZIF-8@MIP reaches the adsorption-desorption equilibrium state within 5 minutes, indicating that the latter has a faster adsorption rate. In addition, it shows that the addition of the carrier ZIF-8 not only enables more imprinting sites to be exposed on the surface of the imprinted material supported by the carrier material, but also provides more interactions for the adsorption process, such as π-π stacking and electrostatic attraction. The adsorption of the imprinted material itself is mainly based on hydrogen bonds. These combined effects accelerate the adsorption of the adsorbent to the target. Compared with MIP, the adsorption rate of ZIF-8@MIP has been greatly improved. This is very beneficial for its practical application as an adsorption material.

[0152] From Figure 7 it can also be seen that the adsorption rate of the surface-imprinted material is faster than that of the non-surface-imprinted material. Therefore, when the main force for the adsorption of the target in the solution by the imprinted material is relatively strong, it is closer to chemical adsorption.

[0153] 2.3 Adsorption selectivity analysis

[0154] ZIF-8@MIP was used to adsorb substances similar and dissimilar to the template molecule structure respectively. By comparing the adsorption amounts of ZIF-8@MIP for the two types of substances, the adsorption selectivity of the material can be evaluated. According to Figure 8 As shown, the adsorption amounts of ZIF-8@MIP for nicotine such as NIC, Nor-NIC and Iso-NIC with similar structures are significantly higher than those for substances such as CHA, 7-HC and RUT with relatively large structural differences. This difference may be due to the fact that the imprinted cavity of the imprinted material was predetermined by the template molecule during the preparation process, and 4-pyrrolidinylpyridine was used as the substitute template molecule for Nor-NIC, and their spatial sizes, functional groups, etc. are very similar. Combining Figure 9 with the molecular structures of the two types of substances shown in, it can be explained that due to the imprinted cavities matching the original molecular spatial structure generated after the prepared imprinted material was eluted, these imprinted cavities provide more adsorption possibilities for analogues, while for the RUT molecule with a relatively large space, the adsorption process is difficult to carry out, so the adsorption amount of RUT is very low. In addition, it is noted that the adsorption amount of the material for 7-HC with a spatial size close to the template molecule is also very low. It is not difficult to find that the functional groups on the 7-HC molecule are too different from those of the substitute template molecule, indicating that its adsorption amount is more affected by the functional groups.

[0155] In addition, ZIF-8@NIP was used to adsorb 6 substances respectively, and the imprinting factors of ZIF-8@MIP for the 6 substances were calculated according to formula 2-3 and summarized in Table 3. As can be seen from Table 3, the imprinting factors of the 3 substances with similar structures are Nor-NIC (2.21), NIC (1.47) and Iso-NIC (1.59) respectively, which are all greater than the 3 substances CHA (0.62), 7-HC (0.51) and RUT (0.38) with large structural differences from the template molecule, indicating that ZIF-8@MIP has good adsorption selectivity.

[0156] Table 3 Selective adsorption of ZIF-8@MIP and ZIF-8@NIP

[0157]

[0158] Example 4

[0159] The surface molecularly imprinted polymer (ZIF-8@MIP) prepared in Example 1 was used as the separation medium of a solid-phase extraction column (SPE column) to separate and enrich nornicotine in the reconstituted tobacco extract sample, and UPLC-MS / MS was used for its quantitative detection. The specific steps are as follows:

[0160] Preparation and Optimization of Usage Conditions of SPE Column

[0161] 1.1 Packing and Activation of SPE Column

[0162] Weigh 100 mg of ZIF-8@MIP as the adsorbent filler of the SPE column, and directly pour it into the prepared empty SPE column. Seal both ends of the adsorbent with a sieve plate, gently tap the outer wall, and press the filler to a certain height. For the prepared SPE column, before use, it needs to be activated successively with 6 mL of methanol and 10 mL of water to remove residual impurities and pollutants, so that the surface active sites of the adsorbent in the SPE column are fully exposed, which can enhance the adsorption capacity for the target substance.

[0163] 1.2 Determination of Sampling Solution Concentration

[0164] Select methanol: water (1:9, V / V) as the sampling solvent, and prepare three different concentrations (5 μg·mL -1 , 10 μg·mL -1 , 20 μg·mL -1 ) of the sampling solution. Load 1 mL of each sampling solution onto the activated SPE column, process the effluent collected each time, and use HPLC-PDA to detect whether the target substance leaks to judge the binding ability of the column filler to different concentrations of nicotine solution.

[0165] 1.3 Optimization of Elution Solvent

[0166] Solid-phase extraction is one of the commonly used techniques in sample pretreatment, aiming to effectively remove impurities that affect the detection of the target substance in the actual sample to reduce the background value of instrument measurement. Using the eluent to treat the SPE column after sampling can remove impurities that affect the experiment while retaining the target substance in the adsorbent. The role of the eluent is to destroy the interaction between the target substance enriched in the SPE column and the adsorbent to ensure that the target substance is eluted and then collected for detection and analysis after treatment.

[0167] The eluent plays a key role in the use of SPE. To ensure the efficient elution of the target substance and improve the recovery rate, selecting a suitable eluent is an important part of optimizing the usage conditions of the SPE column. Different eluent systems have different elution effects on the target substance. Select two eluent systems of 0.1% formic acid-methanol / water and 0.1% formic acid-acetonitrile / water for comparison. Prepare 12 self-made SPE columns. After the SPE columns are activated, load 1 mL of the target substance solution with a concentration of 5 μg·mL -1 . Use pure water as the eluent, and then use elution solutions with different organic ratios (50%, 60%, 70%, 80%, 90%, 100%) for elution. Collect the eluted eluent, filter it and load it into a liquid phase vial, and use HPLC-PDA to detect the content of the target substance and calculate the recovery rate to screen out a suitable eluent.

[0168] 1.4 Elution Volume Optimization

[0169] To achieve better separation and enrichment of samples by the prepared SPE column, it is necessary to optimize the dosage of the elution solution. The elution volume is one of the important indicators reflecting the enrichment effect of the SPE column. An appropriate dosage of the elution solution can weaken the background influence of the sample matrix while reducing the use of organic solvents and improving the enrichment efficiency. Using the optimized elution solvent, the SPE column after loading and rinsing is eluted. Each time, 1 mL of the elution solution is used. After continuously eluting 5 mL, the effluent of each elution is filtered and then loaded into a liquid-phase vial for detection and analysis.

[0170] 1.5 Determination of the Loading Capacity of the Self-made SPE Column

[0171] The maximum loading capacity of the SPE column for the target substance in the sample is the main condition restricting the sample loading amount. If it exceeds the column capacity, it will have a serious impact on the detection and analysis of the target substance. Therefore, it is very crucial to explore the column capacity of the self-made SPE column. Prepare 30 mL of a target substance solution with a concentration of 10 μg·mL -1 as the sample loading solution. After activating the SPE column, 1 mL is loaded each time. After the previous sample loading solution has drained completely, the next sample loading is carried out. The effluent of each treatment is loaded into a liquid-phase vial and detected and analyzed by HPLC-PDA.

[0172] 1.6 Method Evaluation

[0173] Combined with the optimized self-made SPE column mentioned above, a liquid-phase detection method for nornicotine is established to evaluate the detection method established by using the separation medium ZIF-8@MIP prepared in the present invention as the SPE column packing. It is necessary to measure the linear range, sensitivity, and accuracy of this method. Finally, this method can be applied to the treatment of actual samples.

[0174] 1.6.1 Method Linearity and Sensitivity

[0175] By plotting a standard curve and calculating the correlation coefficient, the linear relationship between the response value of the instrument and the concentration of the target substance within a specific concentration range can be evaluated. According to the equation of the plotted standard curve, the lowest concentration that the method can detect for the sample target substance, that is, the limit of detection (LOD) of the method, can be calculated. In addition, the lowest concentration at which the method can accurately quantify the sample target substance, that is, the limit of quantification (LOQ) of the method, can be calculated.

[0176] 1.6.2 Method Accuracy

[0177] The measured results can be compared with the reference values through spiking experiments, and the recovery rate can be calculated to evaluate the accuracy of the method. In the experiment, three groups of spiked samples with different concentrations (5 μg·mL -1 , 10 μg·mL -1 , 20 μg·mL -1 ) were prepared. 18 self-made SPE columns were prepared, with 6 columns in each group, corresponding to samples with different concentrations respectively. Each group of samples was processed according to the optimized conditions. Finally, each eluate was detected by HPLC-PDA, and the recovery rates at three spiking levels were calculated. By calculating the relative standard deviation of the recovery rate of each group, the accuracy of the method can be intuitively evaluated.

[0178] 1.6.3 Exploration of the number of reuse times

[0179] If the self-made SPE column filled with ZIF-8@MIP has reusability, its overall use cost will be greatly reduced, which is very important for its future marketization. In the experiment, 3 self-made SPE columns were taken for repeated use. According to the aforementioned optimized conditions, taking (activation - sample loading - elution - elution) as a cycle, each SPE column experienced 5 cycles. The eluates of each cycle were collected, processed and loaded into liquid phase vials, and the recovery rate was detected and calculated.

[0180] Experimental results:

[0181] 2.1 Optimization of the packing method of the SPE column

[0182] The solid-phase extraction adsorbent can be loaded into the empty column in two ways, namely dry packing and wet packing. Dry packing is to directly pour the dry adsorbent such as ZIF-8@MIP prepared in this study into the empty column, and then gently tap and compact it. The operation of the whole process is very simple, but it will also cause voids or channeling, resulting in uneven column efficiency and other consequences. Wet packing means that the adsorbent material is first dispersed into a uniform paste in a solvent (such as methanol), and then transferred to the prepared empty column. Immediately afterwards, the paste-like packing is made more compact by gravity sedimentation or pressurization. For the SPE column prepared by this method, because the column bed is uniform, it can reduce the dead volume and improve the extraction efficiency. However, the disadvantages are also obvious. The packing process is cumbersome, it needs to be used immediately, and a large amount of organic solvents are required for assistance.

[0183] Therefore, the present invention summarizes the two column packing methods and combines their advantages, and proposes a more convenient packing method. First, the adsorbent is poured into the empty column in a powder state by dry column packing, and both ends are sealed but not compacted. When in use, it is wetted with a small amount of methanol in advance, and the outer wall is tapped to make the adsorbent compact.

[0184] 2.2 Optimization of the sample loading concentration

[0185] Optimizing the concentration of the sample solution can provide data support for subsequent experiments. An overly high concentration in the sample solution may cause the target substance to flow out, reducing the recovery rate and even affecting the results of subsequent experiments. As can be seen from Figure 10 , when using target solutions of 5 μg·mL -1 , 10 μg·mL -1 , and 20 μg·mL -1 for sample loading and detecting the eluate, no leakage occurred. These results indicate that selecting a sample loading concentration of 5 μg·mL -1 for the ZIF-8@MIP column can ensure that the target substance does not leak.

[0186] 2.3 Optimization of the elution solvent

[0187] SPE is one of the commonly used techniques in sample pretreatment, aiming to effectively remove impurities in the actual sample that affect the detection of the target substance to reduce the background value of instrument measurement. Treating the SPE column after sample loading with the eluent can remove the impurities that affect the experiment while retaining the target substance in the adsorbent.

[0188] The role of the elution solvent is to disrupt the interaction between the target substance enriched in the SPE column and the adsorbent to ensure that the target substance is eluted and then collected for detection and analysis after treatment. To ensure the efficient elution of the target substance and improve the recovery rate, selecting the appropriate eluent is an important part of optimizing the use conditions of the SPE column.

[0189] Different elution systems have different elution effects on the target substance. In the preliminary experiment, different ratios of methanol-water and acetonitrile-water were used to elute the self-made column after sample loading, but the results failed to elute it. This indicates that the adsorbent has a strong adsorption effect on the target substance Nor-NIC. Therefore, different ratios of 0.1% formic acid-methanol / water and 0.1% formic acid-acetonitrile / water were further used as the elution solvent, and the results are as Figure 11 shown. When using the elution solvent composed of 0.1% formic acid-methanol / water (8:2, V / V), the target substance can be eluted to the greatest extent. During the elution process, due to the addition of formic acid, the pH of the elution solvent tends to be acidic, promoting the protonation of Nor-NIC and enhancing its polarity, making it easier to be eluted in the above mixed solvent.

[0190] 2.4 Optimization of the elution volume

[0191] To make the self-made SPE column have a better separation and enrichment effect on the sample, it is necessary to optimize the dosage of the elution solution. The elution volume is one of the important indicators reflecting the enrichment effect of the SPE column. An appropriate dosage of the elution solution can reduce the background influence of the sample matrix while reducing the use of organic solvents and improving the enrichment efficiency.

[0192] The optimized elution solvent (80% organic phase) was used to elute the target compound, and then every 1 mL of the eluate was detected. As Figure 12 shown, the target compound was detected in both the 2nd mL and the 3rd mL, but not in the 4th mL. In addition, it was found in the experiment that the target compound was not detected in the 1st mL either, indicating that the 1st mL of the elution solvent might first wet the adsorption material. Further, a small amount of formic acid protonated the target compound, creating a good elution environment for the subsequent elution solvent. In conclusion, it was clear that only 3 mL of the elution solvent was able to completely elute the target compound.

[0193] 2.5 Determination of the capacity of the self-made SPE column

[0194] The maximum loading capacity of the SPE column for the target compound in the sample is the main condition restricting the sample loading amount. If the column capacity is exceeded, it will have a serious impact on the detection and analysis of the target compound. Therefore, it is very crucial to explore the column capacity of the self-made SPE column. To avoid leakage of the self-made solid-phase extraction column (ZIF-8@MIP / SPE column) during the sample loading process, a Nor-NIC solution with a concentration of 10 μg·mL -1 was selected as the sample loading solution, and 1 mL was loaded each time for 30 consecutive times. The eluate after each sample loading was collected and detected by HPLC-PDA. As Figure 13 shows the detection of the eluate from the 27th and 28th sample loadings. It can be found that there was no leakage in the 27th mL, but a large amount of the target compound leaked in the 28th mL, indicating that after continuously loading 27 mL, the ZIF-8@MIP SPE column had reached the maximum loading capacity for the target compound. Therefore, it was determined that the column capacity of the self-made column was 270 μg.

[0195] 2.6 Method evaluation

[0196] 2.6.1 Method linearity and sensitivity

[0197] As Figure 14 shown and calculated, the linear range for the determination of nornicotine by UPLC-MS / MS was 20 - 2000 ng·mL -1 , the linear equation was y = 458.69x + 1397.76, and the correlation coefficient R 2 was 0.9999, indicating a good correlation between the solution concentration and the peak area within this range. The calculated limit of detection (LOD) value was 7.57 ng·mL -1 , and the limit of quantitation (LOQ) value was 25.25 ng·mL -1 . The established method had low LOD and LOQ values and could be used for the detection of actual samples.

[0198] 2.6.2 Method Accuracy

[0199] Performing a standard addition recovery experiment on the target in the sample matrix can reflect the applicability of the analytical method. Using diluted tobacco solution as the blank sample matrix, standard solutions of Nor-NIC with concentrations of 100 ng·mL -1 , 500 ng·mL -1 , and 1000 ng·mL -1 were added respectively to prepare the samples to be measured. The results of the standard addition recovery are shown in Table 4. The relative standard deviation (RSD) of the recovery rate is relatively high at low concentration levels, while when the concentration increases, the RSD value decreases significantly, indicating that the detection error will be significantly reduced at higher concentrations.

[0200] Table 4 Standard Addition Recovery

[0201]

[0202] 2.6.3 Reusability of Self-made SPE Column

[0203] A repeatability experiment was carried out on the self-made ZIF-8@MIP SPE column, and continuous sample loading and elution were performed 4 times according to the optimized usage conditions. The results are as Figure 15 shown. The recovery rate obtained from the 4th elution is still above 92%, indicating that the adsorbent is less damaged by the adsorption-desorption of the target, and the self-made SPE column can be reused at least multiple times.

[0204] Conclusion

[0205] 3.1 To develop a sample pretreatment technology for the detection of tobacco solution, the prepared separation material ZIF-8@MIP was loaded into the SPE column as the adsorption packing material, and then the usage conditions of the self-made SPE column were optimized. This process was combined with HPLC-PDA detection, and finally the best parameters were obtained. After loading the sample, the SPE column was rinsed with pure water. When the elution solvent was a mixed solution composed of 0.1% formic acid-methanol / water (8:2, V / V), 3 mL of the elution solvent could elute all the target substances. By continuously loading the sample, the maximum loading capacity of the 100 mg ZIF-8@SPE column was measured to be 270 μg, and the self-made column had sufficient column capacity to process actual samples.

[0206] 3.2 The detection method established by using the self-made SPE column combined with UPLC-MS / MS was verified, and it was found that there was a good linear relationship for Nor-NIC concentrations in the range of 20 ng·mL -1 to 2000 ng·mL -1 . The LOD of the method was calculated to be 7.57 ng·mL -1 , and the LOQ was 25.25 ng·mL-1 , the accuracy of the method was evaluated by the 3-level 6-parallel experiment method. At the levels of 100 ng·mL -1 , 500 ng·mL -1 , 1000 ng·mL -1 , the recovery rate of the method was between 90.21% and 100.94%, and the RSD value was lower than 3.19%, indicating that the detection method established by the present invention is accurate and reliable. Subsequently, the ZIF-8@MIP SPE column was compared with two commercially available solid-phase extraction columns. The results showed that the self-made column has the ability to selectively enrich nicotine substances, while the Florisil SPE column and the Silica SPE column have poor solid-phase extraction effects on nicotine substances in tobacco liquid. Therefore, the imprinted separation material ZIF-8@MIP prepared by the present invention has appropriate adsorption-desorption ability and high selectivity, and can be used as the adsorption filler of the SPE column and applied in the sample pretreatment of tobacco liquid.

Claims

1. A preparation method of a tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer, characterized in that, It includes the following steps: 1) Preparation of ZIF-8: Dissolve metal zinc salt in water, denoted as solution A; then dissolve 2-methylimidazole in water, denoted as solution B; Add solution A to solution B, stir at room temperature for 3 - 7 min, then let it stand and react for 20 - 30 h to obtain a white crystalline solid. Separate the solid from the liquid, wash, and dry to obtain the metal-organic framework material ZIF-8; 2) Synthesis of ZIF-8@MIP: Use the metal-organic framework material ZIF-8 prepared in step 1) as the carrier material; weigh ZIF-8, add it to solvent I, and ultrasonically treat for 13 - 18 min to prepare a ZIF-8 dispersion; Add the template molecule and functional monomer to solvent II, ultrasonically treat for 5 - 20 min to allow the template molecule and functional monomer to fully contact and undergo prepolymerization. Then add the prepared ZIF-8 dispersion, and successively add the crosslinking agent and initiator. Stir at room temperature for 7 - 15 min, then raise the temperature to a certain value and react for a certain time. After the reaction, cool to room temperature, separate the solid from the liquid to obtain a solid product, wash, and dry to obtain a powdery solid, which is the surface molecularly imprinted polymer ZIF-8@MIP.

2. The preparation method according to claim 1, characterized in that, In step 1), the metal zinc salt is any one of zinc acetate, zinc chloride, and zinc nitrate; in step 1), the concentration of solution A is 0.08 - 0.15 g / mL; in step 1), the concentration of solution B is 0.1 - 0.18 g / mL; In step 1), the mass ratio of solution A to solution B is 1:(1.5 - 3), or the volume ratio of solution A to solution B is 1:(2 - 4).

3. The preparation method according to claim 1, wherein In step 2), the concentration of the ZIF-8 dispersion is 0.01 - 0.05 g / mL; In step 2), the template molecule is pyrrolidinylpyridine, the functional monomer is α-methylacrylic acid; in step 2), the crosslinking agent is ethylene glycol dimethacrylate, and the initiator is azobisisobutyronitrile; In step 2), the molar ratio of the template molecule, functional monomer, and crosslinking agent is 1:(3 - 8):(10 - 30); In step 2), the mass ratio of the template molecule to the initiator is 1:(1 - 2); In step 2), the volume ratio of the ZIF-8 dispersion to the crosslinking agent is (12 - 28):

1.

4. The preparation method according to claim 1, characterized in that, In step 2), the reaction temperature is 60 - 100 °C, and the reaction time is 5 - 24 h.

5. The tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer prepared by the method according to any one of claims 1 - 4.

6. Use of the tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer according to claim 5 in the specific separation and enrichment of nicotine, isonicotine, or nornicotine in tobacco and tobacco products.

7. Use of the tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer according to claim 5 as a separation medium for a solid-phase extraction column.

8. A tobacco alkaloid SPE column prepared by using the tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer according to claim 5.

9. Use of the tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer according to claim 5 in establishing an SPE-UPLC-MS / MS method applicable to detecting the contents of nicotine, nornicotine or anatabine in tobacco and tobacco products.

10. A method for preparing a tobacco alkaloid SPE column using the tobacco alkaloid ZIF-8@MIP molecularly imprinted polymer according to claim 5, comprising the following steps: Weigh the ZIF-8@MIP as the adsorbent filler of the SPE column, place it in a prepared empty SPE column, seal both ends of the adsorbent with sieve plates, and press the filler to a certain height; For the prepared SPE column, it is sequentially activated with methanol and water before use to remove residual impurities and pollutants, so that the surface active sites of the adsorbent in the SPE column are fully exposed, which can enhance the adsorption capacity for the target substance.

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