Covalent organic framework material solid phase microextraction fiber and application thereof in alkaloid metabolism and neurotransmitter analysis

Abstract

The invention discloses a covalent organic framework material solid-phase microextraction fiber and application thereof in alkaloid metabolism and neurotransmitter analysis. The covalent organic framework material (COFs) comprises repeated structural units of 4, 4 ', 4' '-(1, 3, 5-triazine-2, 4, 6-triyl) tris [benzaldehyde], ethidium bromide and 2, 5-diaminobenzene sulfonic acid, which take an imine bond as a connection mode. The COFs material is used as an adsorbent of a solid-phase microextraction fiber surface coating, and the surface of a stainless steel wire fiber is coated with COFs through polyacrylonitrile glue. The COFs solid-phase microextraction fiber can be used for extracting coptis alkaloid and neurotransmitter in the brain and blood of a living rat, after sampling and extraction, the COFs solid-phase microextraction fiber is directly combined with LC-MS / MS for mass spectrometry analysis, and the COFs solid-phase microextraction fiber has extremely high sensitivity and can be used for rapid, accurate and sensitive analysis of trace coptis alkaloid metabolite and neurotransmitter in a living body.

Description

Technical Field

[0001] The invention relates to a covalent organic skeleton material solid phase microextraction fiber and application thereof in drug metabolism analysis, belonging to the field of solid phase microextraction. Background Art

[0002] Pharmacokinetics is a discipline that quantitatively studies the absorption, distribution, metabolism and excretion of drugs in organisms. It has become an important part of preclinical and clinical drug research. Exploring the metabolic kinetic behavior of drugs in brain tissue can determine whether they can pass through the blood-brain barrier and whether they can reach the effective concentration. It is of great significance in the pharmacodynamics and toxicology research of drugs for the prevention and treatment of brain diseases. The collection of biological samples is the primary link in pharmacokinetic research. The traditional sampling method is mainly to collect whole blood from animals at a specific time point or to kill animals and collect tissues for homogenization and post-processing analysis. This method has the disadvantages of a large number of animals required, complex pre-processing, and the analysis results are mostly non-free drug concentrations. It cannot accurately reflect the real-time changes of components in the body. Therefore, the research and development of new sampling techniques is of great significance for pharmacokinetic research.

[0003] Solid-Phase Microextraction (SPME) is an efficient and sensitive sample pretreatment technology that integrates sampling, extraction, concentration and injection. It can be used in conjunction with high-efficiency analytical detection methods such as chromatography, electrophoresis, and mass spectrometry, and is widely used in analytical detection in the fields of environment, food, and medicine. SPME technology is a solvent-free or low-solvent extraction technology that uses a trace enrichment medium as the core. Compared with traditional extraction methods, it has the advantages of low cost, ease of use, and environmental friendliness. It is an emerging efficient separation and enrichment technology. The SPME sampling device has the characteristics of miniaturization and minimally invasiveness, and the coating volume is small and the enrichment efficiency is high. It is very suitable for the separation and enrichment of trace targets in small-volume biochemical samples. It is easy to realize in-situ and minimally invasive analysis of living biological samples, giving it a unique advantage in live tracking sampling. Moreover, the non-destructive SPME in situ sampling method can minimize interference with organisms. Unlike traditional lethal methods, it can ensure the survival status of experimental subjects during the monitoring period. Long-term in vivo tracking studies of single biological individuals during the dosing cycle can not only reduce the loss of animal samples in the research, but also avoid the influence of individual differences, so as to obtain the most realistic metabolic data of experimental animals.

[0004] The core point of SPME technology development is coating. The defects of commercial SPME coating such as low specific surface area, high price and poor selectivity for target molecules limit its wide application. Therefore, the development of SPME coating materials with simple preparation, high adsorption performance and good selectivity, regular coating morphology and low cost is the development direction that scientific researchers continue to work hard on. Covalent organic frameworks (COFs) are a new type of crystalline porous organic materials with excellent properties such as low density, fine structure, adjustable pore size and structure, diverse synthesis methods and easy functional modification. Although there are many studies on solid phase microextraction coatings, many reported solid phase microextraction coatings do not have biocompatibility and protein exclusion effect, making it difficult to achieve direct extraction of target substances in animal blood and tissues. Therefore, the preparation of coating materials with biocompatibility, protein exclusion effect and high extraction efficiency is of great significance for pharmacokinetic research.

[0005] According to the ancient book "Rihuazi Bencao", Coptis chinensis can treat five kinds of fatigue and seven kinds of injuries, replenish qi, stop heart and stomach pain, palpitations, irritability, and moisten the heart and lungs; "Yao Xing Fu" records that Coptis chinensis is bitter, flat, cold, non-toxic, and can purge heart fire and eliminate the feeling of fullness under the heart. Based on the fact that Coptis chinensis purges heart fire, because the heart governs the mind and fire governs the heart, purging fire must first purge the heart, and when the heart fire is calm, the fire of all meridians will naturally decrease. Modern studies have shown that Coptis chinensis detoxification soup can effectively improve learning and memory functions and the clinical symptoms of patients with senile dementia. Pharmacological studies have shown that it can improve depression, Alzheimer's and other brain disease symptoms by regulating the central monoamine neurotransmitters (NE, 5-HT, DA) of the brain. Therefore, the establishment of a new method for the rapid separation and analysis of the effective components and neurotransmitter markers of Chinese medicine Coptis chinensis in biological samples (blood, tissues, etc.) can provide a basis for the development and clinical application of new drugs of Chinese medicine Coptis chinensis.

[0006] Based on this, the present invention uses commercial 4,4',4"-(1,3,5-triazine-2,4,6-triyl)tri[benzaldehyde] (TTB), ethidium bromide (EB) and 2,5-diaminobenzenesulfonic acid (DABSA) as monomers, and prepares a new EB-TTB-DABSA-COF material with both sulfonic acid group and quaternary ammonium group zwitterions, large specific surface area and good dispersibility through Schiff base polymerization reaction. Summary of the invention

[0007] In view of the shortcomings of the prior art in detecting drug metabolites, such as long preparation time, high detection cost, inability to extract in real time in situ, and inconvenience in long-term in vivo tracking of a single animal individual, the main purpose of the present invention is to provide a functionalized covalent organic framework material solid phase microextraction fiber and its application in the detection of various active ingredients of traditional Chinese medicine (coptis alkaloids) and neurotransmitters, which can effectively solve the above problems, has the advantages of short detection method time, high sensitivity and low detection cost, and can realize the study of the pharmacokinetics of coptis alkaloids and changes in neurotransmitters.

[0008] In order to achieve the above-mentioned purpose, the technical solution of the present invention is, on the one hand, to provide a covalent organic framework material, comprising repeating structural units of 4,4',4"-(1,3,5-triazine-2,4,6-triyl)tri[benzaldehyde], ethidium bromide and 2,5-diaminobenzenesulfonic acid connected by imine bonds.

[0009] On the other hand, the technical solution of the present invention is to provide a method for preparing a covalent organic framework material, comprising the following steps:

[0010] a. Mix 4,4',4"-(1,3,5-triazine-2,4,6-triyl)tris[benzaldehyde], ethidium bromide and 2,5-diaminobenzenesulfonic acid;

[0011] b. Add solvent, dissolve and mix evenly by ultrasonic;

[0012] c. Add acetic acid aqueous solution, mix thoroughly, and react;

[0013] d. Separate and purify to obtain a covalent organic framework material.

[0014] Furthermore, in step a, the molar ratio of 4,4',4"-(1,3,5-triazine-2,4,6-triyl)tris[benzaldehyde], ethidium bromide and 2,5-diaminobenzenesulfonic acid is 4:3 to 4:3 to 4.

[0015] Furthermore, the solvent in step b is a mixed solvent of 1,3,5-trimethylbenzene and 1,4-dioxane, and the volume ratio of the two is 2:3.

[0016] Furthermore, in step c, the concentration of the acetic acid aqueous solution is 12M, the molar ratio of 4,4',4"-(1,3,5-triazine-2,4,6-triyl)tri[benzaldehyde] to acetic acid is 1:8-24; the reaction temperature is 100-150°C, and the reaction time is 48-72h.

[0017] Furthermore, the specific method of separation and purification in step d is: centrifugation to obtain a precipitate, then washing it alternately with tetrahydrofuran, ultrapure water and anhydrous ethanol, and vacuum drying.

[0018] On the other hand, the technical solution of the present invention is to provide a covalent organic framework adhesive material, firstly preparing polyacrylonitrile glue by using polyacrylonitrile and N,N-dimethylformamide, and then adding the covalent organic framework material.

[0019] On the other hand, the technical solution of the present invention is to provide a method for preparing a covalent organic framework bonding material, comprising the following steps:

[0020] a. Mix polyacrylonitrile and N,N-dimethylformamide, and react them in a closed manner to obtain polyacrylonitrile glue;

[0021] b. Add the covalent organic framework material according to claim 1 to polyacrylonitrile glue and shake and mix to obtain a covalent organic framework bonding material.

[0022] Furthermore, in step a, the ratio of polyacrylonitrile to N,N-dimethylformamide is 1:100-150 (w / v); the reaction temperature is 80-100° C., and the reaction time is 1-2 h.

[0023] Furthermore, in step b, the mass ratio of the covalent organic framework material to the polyacrylonitrile glue is 1:1-2.

[0024] On the other hand, the technical solution of the present invention is to provide a covalent organic framework material solid phase microextraction fiber coated with the covalent organic framework adhesive material.

[0025] On the other hand, the technical solution of the present invention is to provide a method for preparing a covalent organic framework material solid phase microextraction fiber, wherein a stainless steel wire is pulled at a uniform speed and impregnated with the covalent organic framework adhesive material, air-dried and then dried, and the above steps are repeated to obtain a solid phase microextraction fiber with a uniform coating.

[0026] Furthermore, the diameter of the stainless steel wire is 0.2-0.3 mm, the length is 5-7 cm, and the coating thickness is 0.02-0.08 mm.

[0027] On the other hand, the technical solution of the present invention is to provide an application of the covalent organic framework material, the covalent organic framework adhesive material, and the covalent organic framework material solid phase microextraction fiber in the metabolism of Coptis chinensis alkaloids and the detection of neurotransmitters.

[0028] The COFs material of the present invention can adsorb coptis alkaloids and neurotransmitters through hydrophobic interaction, electrostatic interaction, π-π interaction and van der Waals force. The COFs material can be used to prepare solid phase microextraction fiber, and is used in conjunction with LC-MS / MS to study the changes of coptis alkaloids and neurotransmitters in the brain and blood of living rats. The method has extremely high sensitivity and can be used for rapid, accurate and sensitive analysis of trace coptis alkaloid metabolites and neurotransmitters in living bodies. The extraction process does not require killing rats, and the extraction and detection process can be completed within 1 hour. It has high sensitivity and low detection cost, while effectively reducing the loss of experimental animals in the study, and can also avoid the influence of individual differences to obtain the most realistic metabolic data of experimental animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a scanning electron microscope image of EB-TTB-DABSA-COF fiber.

[0030] Figure 2This is the EDS element map of EB-TTB-DABSA-COF material.

[0031] Figure 3 This is the XPS overall spectrum of EB-TTB-DABSA-COF material.

[0032] Figure 4 High-resolution spectra of EB-TTB-DABSA-COF materials in C1s, N1s, O1s and S2p channels.

[0033] Figure 5 This is the protein exclusion efficiency diagram of EB-TTB-DABSA-COF material and COFs-SPME fiber.

[0034] Figure 6 Comparison of the adsorption efficiency of COFs-SPME fiber and three commercial material fibers.

[0035] Figure 7 To optimize the adsorption time of COFs solid phase microextraction fiber in serum matrix.

[0036] Figure 8 Optimization of elution solvent after COFs solid phase microextraction fiber extraction.

[0037] Fig. 9 To optimize the elution time after COFs solid phase microextraction fiber extraction.

[0038] Fig.10 Optimization of the adsorption time of COFs solid phase microextraction fibers in brain surrogate matrices.

[0039] Fig.11 This is a blood curve of Chinese medicine in rats after intragastric administration of Coptis chinensis (n=5). In the figure, Model represents the model group and Control represents the control group.

[0040] Fig.12 The neurotransmitter content in the blood of rats within 10 hours after intragastric administration of Coptis chinensis (n=5). In the figure, Model represents the model group and Control represents the control group.

[0041] Fig.13 This is a Chinese medicine curve diagram of the brain tissue of rats after intragastric administration of Coptis chinensis (n=5). In the figure, Model represents the model group and Control represents the control group.

[0042] Fig.14 The neurotransmitter content in the rat brain tissue within 10 hours after oral administration of Coptis chinensis (n=5). In the figure, Model represents the model group, and Control represents the control group (* represents P<0.05, ** represents P<0.01). DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited to the following scope. The reagents used in the examples that are not specified are all commercially available conventional products and can be purchased.

[0044] Example 1: Preparation of covalent organic framework solid phase microextraction fibers

[0045] (1) Preparation of covalent organic framework (EB-TTB-DABSA-COF) materials

[0046] a. Mix 4,4',4"-(1,3,5-triazine-2,4,6-triyl)tris[benzaldehyde] (TTB), ethidium bromide (EB) and 2,5-diaminobenzenesulfonic acid (DABSA), the amounts of which are 0.3 mmol, 0.225 mmol and 0.225 mmol, respectively;

[0047] b. Add 4 mL of a mixed solvent of 1,3,5-trimethylbenzene and 1,4-dioxane (the ratio of the two is 2:3, v / v), dissolve and mix evenly by ultrasonication;

[0048] c. Add 12M acetic acid aqueous solution (the molar ratio of TTB to acetic acid is 1:16), mix thoroughly, and react at 120°C for 60h;

[0049] d. The precipitate was obtained by centrifugation, and then washed alternately with tetrahydrofuran, ultrapure water and anhydrous ethanol, and dried in vacuum at 80° C. to obtain a covalent organic framework material.

[0050] (2) Preparation of solid phase microextraction fiber

[0051] a. Mix polyacrylonitrile and N,N-dimethylformamide (1:100, w / v), and react in a sealed container at 90°C for 1h to obtain polyacrylonitrile glue;

[0052] b. adding the covalent organic framework material to the polyacrylonitrile glue and oscillating and mixing to obtain a covalent organic framework adhesive material; wherein the mass ratio of the covalent organic framework material to the polyacrylonitrile glue is 1:1;

[0053] c. Cut a stainless steel wire with a diameter of 0.2 mm into small sections with a length of 7 cm, wash it with a 50% (v / v) methanol aqueous solution and then dry it; vertically insert it into the covalent organic framework bonding material by the pulling and dipping method, and the coating length is 6 mm. After the surface material is air-dried, bake it at 90°C for 60 seconds to fix the coating. Repeat the dipping, air-drying and baking steps to make the coating as evenly wrapped on the stainless steel wire as possible to obtain a solid phase microextraction fiber (COFs-SPME fiber) with a uniform coating, and the final coating thickness is 0.02 mm.

[0054] Example 2: Preparation of covalent organic framework solid phase microextraction fibers

[0055] (1) Preparation of covalent organic framework materials

[0056] a. Mix 4,4',4"-(1,3,5-triazine-2,4,6-triyl)tris[benzaldehyde], ethidium bromide and 2,5-diaminobenzenesulfonic acid, the amounts of the three substances being 0.3 mmol, 0.3 mmol and 0.225 mmol respectively;

[0057] b. Add 5 mL of a mixed solvent of 1,3,5-trimethylbenzene and 1,4-dioxane (the ratio of the two is 2:3, v / v), dissolve and mix evenly by ultrasonication;

[0058] c. Add 12M acetic acid aqueous solution (the molar ratio of TTB to acetic acid is 1:8), mix thoroughly, and react at 100°C for 72h;

[0059] d. The precipitate was obtained by centrifugation, and then washed alternately with tetrahydrofuran, ultrapure water and anhydrous ethanol, and dried in vacuum at 80° C. to obtain a covalent organic framework material.

[0060] (2) Preparation of solid phase microextraction fiber

[0061] a. Mix polyacrylonitrile and N,N-dimethylformamide (1:150, w / v), and react at 80°C in a sealed container for 2h to obtain polyacrylonitrile glue;

[0062] b. adding the covalent organic framework material to the polyacrylonitrile glue and oscillating and mixing to obtain a covalent organic framework adhesive material; wherein the mass ratio of the covalent organic framework material to the polyacrylonitrile glue is 1:1.5;

[0063] c. Cut the stainless steel wire with a diameter of 0.2 mm into small sections with a length of 6 cm, wash it with 50% (v / v) methanol aqueous solution and then dry it; vertically insert it into the covalent organic framework bonding material by the pulling and dipping method, and the coating length is 5 mm. After the surface material is air-dried, bake it at 60°C for 90 seconds to fix the coating. Repeat the dipping, air-drying and baking steps to make the coating as evenly wrapped on the stainless steel wire as possible to obtain a solid phase microextraction fiber with a uniform coating. The final coating thickness is 0.06 mm.

[0064] Example 3: Preparation of covalent organic framework solid phase microextraction fibers

[0065] (1) Preparation of covalent organic framework materials

[0066] a. Mix 4,4',4"-(1,3,5-triazine-2,4,6-triyl)tris[benzaldehyde], ethidium bromide and 2,5-diaminobenzenesulfonic acid, the amounts of the three substances are 0.3 mmol, 0.225 mmol and 0.3 mmol respectively;

[0067] b. Add 6 mL of a mixed solvent of 1,3,5-trimethylbenzene and 1,4-dioxane (the ratio of the two is 2:3, v / v), dissolve and mix evenly by ultrasonication;

[0068] c. Add 12M acetic acid aqueous solution (the molar ratio of TTB to acetic acid is 1:24), mix thoroughly, and react at 150°C for 48h;

[0069] d. The precipitate was obtained by centrifugation, and then washed alternately with tetrahydrofuran, ultrapure water and anhydrous ethanol, and dried in vacuum at 80° C. to obtain a covalent organic framework material.

[0070] (2) Preparation of solid phase microextraction fiber

[0071] a. Mix polyacrylonitrile and N,N-dimethylformamide (1:120, w / v), and react in a sealed container at 100°C for 1h to obtain polyacrylonitrile glue;

[0072] b. adding the covalent organic framework material to the polyacrylonitrile glue and oscillating and mixing to obtain a covalent organic framework adhesive material; wherein the mass ratio of the covalent organic framework material to the polyacrylonitrile glue is 1:2;

[0073] c. Cut a stainless steel wire with a diameter of 0.3 mm into small sections with a length of 5 cm, wash it with 50% (v / v) methanol aqueous solution and then dry it; vertically insert it into the covalent organic framework bonding material by the pulling and dipping method, and the coating length is 4 mm. After the surface material is air-dried, bake it at 100°C for 40 seconds to fix the coating. Repeat the dipping, air-drying and baking steps to make the coating as evenly wrapped on the stainless steel wire as possible to obtain a solid phase microextraction fiber with a uniform coating. The final coating thickness is 0.08 mm.

[0074] Example 4: Characterization of COFs-SPME Fibers

[0075] Firstly, the morphology and microstructure of COFs-SPME fibers were studied using scanning electron microscopy. Figure 1 As shown in Figs. a and 1b, irregular rod-like substances are stacked together on the surface of the synthesized EB-TTB-DABSA-COF material to form a stamen-like structure. This unique structure is conducive to increasing the specific surface area and adsorption capacity of the EB-TTB-DABSA-COF material, thereby achieving effective adsorption of the target. At the same time, it can be observed that the coated fiber surface is evenly coated, the shape is consistent, the dispersion is good, and the coating thickness is about 20 μm ( Figure 1c and 1d). The surface of the coating is wrapped with a mesh PAN polymer layer, which allows small molecules to enter while effectively excluding macromolecular proteins and other substances, and has excellent biocompatibility in the extraction of biological complex matrix samples. Figure 2 The elemental analysis results show that the COFs material is mainly composed of C, N, O and S elements, with contents of 71.33%, 15.72%, 8.64% and 4.31% respectively, and the distribution of each element is uniform.

[0076] In order to further investigate whether the synthesis of EB-TTB-DABSA-COF material was successful, XPS was used to characterize the synthesized material. Figure 3 It can be seen that there are five peaks in the wide spectrum, namely C1s, N1s, O1s, S2p and Br3d. Then, the high-resolution spectra of the four peaks of C1s, N1s, O1s and S2p were further analyzed. The results are as follows: Figure 4 As shown in a-4d, in the C1s spectrum, two obvious peaks appear, corresponding to CC with a binding energy of 284.3eV and CO with a binding energy of 286.38eV, indicating that the triazine compound TTB is bound to COFs. In the high-resolution spectrum of N1s, the peaks with binding energies of 398.3, 399 and 400.63eV correspond to NH, NN and NC, respectively; in the high-resolution spectra of O1s and S2p, there are C=O and RSR with binding energies of 531.16 and 167.35eV, respectively. The above three spectra indicate that DABSA is bound to the COFs material. At the same time, based on Figure 3 The presence of Br3d peaks in the 2D images, as well as NH and NC in the high-resolution spectra of N1s, indicated that EB was bound to COFs. The above results confirmed that EB-TTB-DABSA-COF was successfully synthesized.

[0077] Example 5: Evaluation of protein exclusion capacity

[0078] There are a large number of protein interferences in the body, and protein exclusion capacity is an important indicator for evaluating the performance of adsorption materials. Based on this, albumin was first used as a probe protein to measure the exclusion capacity of the material. 2 mg of the prepared EB-TTB-DABSA-COF material and COFs-SPME fiber were added to different concentrations of albumin solutions (100, 200 and 300 μg mL -1 ) was added. The mixture was shaken at 500 rpm for 2 h at room temperature, and the absorbance of the supernatant was measured at a wavelength of 280 nm. The protein exclusion rate was calculated as follows: Protein exclusion rate = C s / C 0 × 100%, where C s is the concentration of the remaining protein in the solution after adsorption, C 0is the initial protein concentration in the solution. Figure 5 As shown in the figure, with the increase of albumin concentration, the protein exclusion rates of EB-TTB-DABSA-COF material and COFs-SPME fiber are between 90.93-94.59% and 91.50-95.16%, respectively. At the same time, in order to further clarify the protein exclusion effect, the exclusion effects of different proteins (hemoglobin, β-lactoglobulin and actual serum samples) were also analyzed. The exclusion efficiency of EB-TTB-DABSA-COF material and COFs-SPME fiber for different proteins is between 91.95-97.38% and 95.98-97.72%, respectively. The above results show that the designed EB-TTB-DABSA-COF material and COFs-SPME fiber show excellent exclusion effects on proteins of different concentrations and types. Therefore, EB-TTB-DABSA-COF material can effectively reduce the interference of macromolecular proteins in the matrix, which is conducive to the extraction and analysis of targets in complex samples.

[0079] Experimental verification

[0080] The performance of the prepared COFs-SPME fiber was studied below. The in vivo detection method of coptis alkaloids and neurotransmitters in rat brain and blood based on solid phase microextraction (SPME) technology was as follows:

[0081] (1) Extraction of rat striatum: After anesthetizing rats with isoflurane, fix them to a stereotaxic apparatus, separate the skin and subcutaneous tissue near the fontanelle, and expose the bone surface. Locate the striatum region according to the coordinates (AP: 0.43-0.73 mm, ML: 2.7-3.3 mm). Drill holes on the bone surface with a dental drill, implant a cannula in the striatum region, and tighten the dust cap after fixation. Suture the wound and apply roxithromycin to the wound to prevent infection. Feed the rats for 5 days to restore normal physiological status. The COFs-SPME fiber was activated with 50% (v / v) methanol-water for 20-30 min before use, and the fiber was inserted into the striatum through the brain cannula for extraction for 2-15 min.

[0082] Rat blood extraction: First, insert a No. 7 needle into the tail vein, and then pass the fiber into the blood vessel through the needle. Pull out the needle, and then extract the fiber in the tail vein for 2 to 15 minutes.

[0083] The extracted fibers were first washed with ultrapure water by shaking for 30 to 60 seconds, and then eluted with 0.2% formic acid-methanol:water (1:1, v / v) for 10 to 60 minutes.

[0084] (2) Instrumental analysis: The obtained eluate was detected by liquid chromatography tandem mass spectrometry (LC-MS / MS), model: Thermo Ultimate 3000 ultra-high performance liquid chromatography detection system; the chromatographic column was Agilent UPLC SB-C8 (2.1 mm×100 mm, 1.8 μm); the column temperature was 30°C, the injection volume was 5 μL; the mobile phase was 0.1% formic acid-water (A) and acetonitrile (B); the flow rate was 0.2 mL·min -1 ; Gradient program:

[0085] 0-2min, mobile phase B changes from 2% to 30% at a constant rate;

[0086] 2-8 min, mobile phase B 30%;

[0087] 8-10 min, mobile phase B changes from 30% to 95% at a constant rate;

[0088] 10-12 min, mobile phase B was changed from 95% to 2% at a constant speed;

[0089] 12-15 min, mobile phase B is 2%.

[0090] Mass spectrometry conditions: Model: TSQ Altis high-resolution mass spectrometer; ion source in positive ion mode; scanning mode: SRM; source region condition parameters: spray voltage 3.5 kV, capillary temperature 325 °C, heating temperature 325 °C, sheath gas 30 Arb, auxiliary ventilation 10 Arb, backflush gas 0; nitrogen was used as collision gas and C-trap damping gas.

[0091] (3) The method for quantifying the alkaloids and neurotransmitters of Rhizoma Coptidis is as follows: 0-200 ng / mL is added to the diluted blank serum (or brain substitute matrix). -1 Neurotransmitters and 0~10ng·mL -1 Alkaloids. With the abscissa (x) as the spike concentration and the ordinate (y) as the corresponding peak area, a standard curve was established to quantify the concentrations of Rhizoma Coptidis alkaloids and neurotransmitters in the eluate.

[0092] The detection method of coptis alkaloids and neurotransmitters in serum and brain substitute matrix is ​​basically the same as the in vivo detection method in rats, the difference is that the fiber is directly inserted into the serum or brain substitute matrix for extraction.

[0093] Verification experiment 1: Comparison of the performance of COFs-SPME fiber and commercial SPME in the extraction of alkaloids and neurotransmitters

[0094] This experiment measured the extraction capabilities of the COFs-SPME fiber prepared in Example 1 of the present invention and the solid phase microextraction fiber coated with commercial WAX, PXC, and C18 materials for Coptis chinensis alkaloids and neurotransmitters.

[0095] The COFs-SPME fiber prepared in Example 1 of the present invention and the solid phase microextraction fiber coated with commercial WAX, PXC and C18 materials were used to extract 100 ng·mL -1 Various neurotransmitters (NTs) and 10 ng·mL -1 Phosphate buffer (PBS, 0.01 M, pH 7.4) containing various alkaloids. Before extraction, each fiber was activated in 50% methanol-water for 30 min. The adsorption time of SPME was 30 min, the adsorption volume was 0.5 mL, and the elution condition was 100 μL 0.2% FA-MeOH:ACN:H 2 O (1:1:1, v / v / v), elution time is 30min. The desorption solution enters the LC-MS / MS instrument for analysis, and the elution conditions of each substance are compared, thereby comparing the adsorption capacity of different solid phase microextraction fibers for the analytes. The results are shown in Figure 6 As shown. Figure 6 It can be seen that the COFs-SPME fiber prepared in the present invention has a better extraction effect on coptisine, desmethenberine, berberine, epiberberine, jatrorrhizine, tertrenol, magnolia alkaloids, berberine oxide, palmatine and neurotransmitters (glycine, γ-aminobutyric acid, dopamine, 5-hydroxytryptamine and adrenaline) in the coptis alkaloids than the commercial material fiber, indicating that the COFs-SPME fiber has good extraction performance for alkaloids and neurotransmitter targets.

[0096] Verification experiment 2: Optimization of COFs-SPME fiber conditions for extracting coptis alkaloids and neurotransmitters from blood

[0097] The COFs-SPME fiber prepared in Example 1 of the present invention was used to simultaneously spike 100 ng·mL -1 Various neurotransmitters and 10 ng·mL -1 Before extraction, the COFs-SPME fiber was activated with 50% methanol-water, and then statically adsorbed with 0.5 mL spiked serum for 2 to 15 min; after extraction, it was washed with ultrapure water for 30 s, and then washed with 100 μL methanol (MeOH), acetonitrile (ACN), 0.2% formic acid-methanol (0.2% FA-MeOH), 0.2% formic acid-acetonitrile (0.2% FA-ACN), 0.2% formic acid-methanol water (0.2% FA-MeOH:H 2 O, 1:1, v / v), 0.2% formic acid-methanol water (0.2% FA-MeOH:H 2 O, 6:4, v / v), 0.2% formic acid-acetonitrile water (0.2% FA-ACN:H 2O, 1:1, v / v) and 0.2% formic acid-methanol-acetonitrile water (0.2% FA-MeOH:ACN:H 2 The eluate was analyzed by LC-MS / MS instrument, and the neurotransmitters and alkaloids in the eluate were quantified.

[0098] In order to more intuitively reflect the extraction results, substances with similar adsorption amounts are displayed in the same figure according to the differences in the adsorption amounts of the target substances, so they are divided into Figure a and Figure b.

[0099] The results of extraction time optimization are as follows Figure 7 As shown in Figures 7a and 7b, the extraction effect of COFs-SPME fiber is the best when the extraction (adsorption) time is 8 min.

[0100] The optimization results of elution solvent are as follows Figure 8 As shown in Figures 8a and 8b, the elution effect of COFs-SPME fiber is the best when the elution solvent is 0.2% formic acid-methanol water (1:1, v / v).

[0101] The optimization results of elution time are as follows Fig. 9 As shown in Figures 9a and 9b, the elution effect of COFs-SPME fiber is the best when the elution time is 10 min.

[0102] Verification experiment 3: Optimization of conditions for extraction of Coptis chinensis alkaloids and neurotransmitters in brain surrogate matrix using COFs-SPME fiber

[0103] First, a brain substitute matrix was prepared. Agar gel can be used as biological interstitial fluid. Agar gel (2% agar dispersed in PBS buffer, w / v) and brain homogenate (sheep brain: PBS buffer 1:10, w / v) were mixed in a 1:1 ratio (v / w) as a brain substitute matrix for rat SPME. The COFs-SPME fiber prepared in Example 1 of the present invention was used to simultaneously spike 100 ng·mL -1 Various neurotransmitters and 10 ng·mL -1In brain substitute matrix of various alkaloids. Before extraction, COFs-SPME fiber was activated with 50% methanol-water, and then statically adsorbed with 0.5 mL spiked brain substitute matrix for 2-15 min; after extraction, it was washed with ultrapure water for 30 s, and then eluted with 100 μL methanol, acetonitrile, 0.2% formic acid-methanol, 0.2% formic acid-acetonitrile, 0.2% formic acid-methanol water (1:1, v / v), 0.2% formic acid-methanol water (6:4, v / v), 0.2% formic acid-acetonitrile water (1:1, v / v) and 0.2% formic acid-methanol acetonitrile water (1:1:1, v / v / v) for 10-60 min, and the eluate was analyzed in LC-MS / MS instrument, and the neurotransmitters and alkaloids in the eluate were quantified.

[0104] The results of extraction time optimization are as follows Fig.10 As shown in Figures 10a and 10b, the extraction effect of COFs-SPME fiber is the best when the extraction time is 4 min.

[0105] The optimization results of elution solvent and elution time were the same as those of verification experiment 2.

[0106] Verification experiment 4: Verification of the COFs-SPME fiber method for extracting coptis alkaloids and neurotransmitters from serum

[0107] This experiment determines the linear correlation of the extraction effect of the COFs-SPME fiber prepared in Example 1 of the present invention on alkaloids and neurotransmitters in serum.

[0108] The COFs-SPME fiber prepared in Example 1 of the present invention was used to simultaneously spike blank serum with multiple neurotransmitters (concentrations of 1, 25, 50, 75, 100, 150 and 200 ng·mL -1 ) and various alkaloids (at concentrations of 0.5, 1, 2, 4, 6, 8, and 10 ng mL -1 ) extraction, select the concentration range with good linearity according to the extraction results, and calculate the detection limit and quantification limit in turn. The signal-to-noise ratios of the detection limit (LOD) and the quantification limit (LOQ) are selected as 3 and 10, respectively. The linear range is shown in Table 1. The linear range of neurotransmitters in serum is 1.0-200.0 ng·mL -1 The detection limit and quantification limit were 3.3×10 -3 ~0.05ng·mL -1 and 0.01~0.16ng·mL -1 The linear range of alkaloids is 0.5~10.0ng·mL -1 The detection limit and quantification limit were 0.10×10 -3 ~0.21ng·mL -1 and 0.3×10 -3~0.50ng·mL -1 This indicates that the constructed COFs-SPME fiber can achieve a wide range of alkaloids and neurotransmitters in serum with high sensitivity.

[0109] Table 1 Linear regression equations of target substances in serum

[0110]

[0111]

[0112] Validation experiment 5: Validation of the COFs-SPME fiber method for extracting coptis alkaloids and neurotransmitters in brain surrogate matrix

[0113] This experiment determined the linear correlation of the extraction effect of the COFs-SPME fiber prepared in Example 1 of the present invention on alkaloids and neurotransmitters in a brain substitute matrix.

[0114] The COFs-SPME fiber prepared in Example 1 of the present invention was used to simultaneously spike a variety of neurotransmitters (concentrations of 1, 25, 50, 75, 100, 150 and 200 ng·mL) in a brain surrogate matrix. -1 ) and various alkaloids (at concentrations of 0.5, 1, 2, 4, 6, 8, and 10 ng mL -1 ) extraction, select the concentration range with good linearity according to the extraction results, and calculate the detection limit and quantification limit in turn. The signal-to-noise ratios of the detection limit and quantification limit are selected as 3 and 10, respectively. The linear range is shown in Table 2. The linear range of neurotransmitters in brain surrogate matrix is ​​1.0-200.0 ng·mL -1 The detection limit and quantification limit were 4.40×10 -3 ~0.07ng·mL -1 and 0.01~0.24ng·mL -1 The linear range of alkaloids is 0.5~10.0ng·mL -1 The detection limit and quantification limit were 0.09×10 -3 ~0.12ng·mL -1 and 0.30×10 -3 ~0.38ng·mL -1 This indicates that the COFs-SPME fiber constructed in the present invention can achieve wide range and high sensitivity detection of alkaloids and neurotransmitters in brain tissue samples.

[0115] Table 2 Linear regression equations for targets in brain surrogate matrix

[0116]

[0117] Verification experiment 6: Investigation of the precision and accuracy of COFs-SPME fiber extraction of Coptis chinensis alkaloids and neurotransmitters

[0118] This experiment determined the accuracy and precision of the COFs-SPME fiber prepared in Example 1 of the present invention for the analysis of coptis alkaloids and neurotransmitters in blood and brain surrogate matrices.

[0119] The concentrations of 1.0, 50.0, and 200.0 ng·mL were added to serum and brain substitute matrix, respectively. -1 of various neurotransmitters and concentrations of 0.5, 2.0, and 10.0 ng·mL -1 The accuracy and precision of the method were verified by using a variety of alkaloids. The results are shown in Table 3. In the brain substitute matrix, the accuracy of the method was between 84.7% and 115.7%, and the RSDs were between 0.9% and 18.1%; in serum, the accuracy of the method was between 85.5% and 120.0%, and the RSDs were between 0.9% and 19.8%. This shows that the method has good accuracy and precision and was successfully established.

[0120] Table 3 Precision and accuracy of targets at different concentrations

[0121]

[0122]

[0123] Verification experiment 7: Application of COFs-SPME fiber in the extraction of Coptis chinensis alkaloids and neurotransmitters in blood

[0124] This experiment is the pharmacokinetic analysis of coptis alkaloids and the changes in neurotransmitters in the tail vein of Parkinson's (PD) rats using the COFs-SPME fiber prepared in Example 1 of the present invention.

[0125] The COFs-SPME fiber prepared in Example 1 of the present invention was used to extract the metabolites of alkaloids and neurotransmitters of Coptis chinensis in the tail vein blood of PD rats. After the rats were fasted for 12 hours, the control group (normal rats without treatment) and the PD model group were gavaged with 0.48 g·kg Coptis chinensis extract. -1 , coptis alkaloids and neurotransmitters in the blood at various time points (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8 and 10 h) after administration were extracted through the tail vein. The extraction time was 8 min. After elution with ultrapure water for 30 s, 0.2% formic acid-methanol water (1:1, v / v) was used for 10 min. The eluate was entered into LC-MS / MS to analyze the pharmacokinetics of coptis alkaloids and the changes in the content of neurotransmitters in the blood of PD rats. The specific results are as follows Fig.11 and Fig.12 As shown. Fig.11It can be seen that the nine alkaloids basically reach a peak quickly at 0.5h or 1h, and then gradually decrease, and a second peak phenomenon will occur at 4-6h, which is inferred to have an enterohepatic circulation effect. At the same time, it can be observed that the drug concentration in PD model animals is generally higher than that in the control group, especially after enterohepatic circulation, which is significantly higher than the control group, indicating that the body improves drug utilization in the disease state; Fig.12 The change trend of neurotransmitters in the PD model group and the control group animals was shown. Compared with the control group, the neurotransmitters in the blood of PD rats showed a slight increase, but there was no significant difference. From the above results, it can be seen that the method established by the present invention can effectively track the changes of drugs and disease markers in the body, and provide a reference for disease prevention and treatment and the material basis of the efficacy of new Chinese medicine prescriptions.

[0126] Verification experiment 8: Application of COFs-SPME fiber in the extraction of Coptis chinensis alkaloids and neurotransmitters in rat striatum

[0127] This experiment is the pharmacokinetic analysis of coptis alkaloids and the changes of neurotransmitters in the striatum of PD rats using the COFs-SPME fiber prepared in Example 1 of the present invention.

[0128] The COFs-SPME fiber prepared in Example 1 of the present invention was used to extract the metabolites of alkaloids and neurotransmitters of Rhizoma Coptidis in the striatum of PD rats. After the rats were fasted for 12 hours, the control group (normal rats without treatment) and the PD model group were intragastrically administered with 0.48 g·kg Rhizoma Coptidis. -1 , the alkaloids and neurotransmitters in the striatum of the brain were extracted by stereotaxic positioning and implanted cannula at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8 and 10 h after gavage. The extraction time was 8 min. After 30 s of ultrapure water oscillation elution, 0.2% formic acid-methanol water (1:1, v / v) elution was performed for 10 min. The pharmacokinetics of alkaloids and the changes in neurotransmitter content in the striatum of PD rats and control groups were analyzed by LC-MS / MS technology. The results are shown in Fig.13 and Fig.14 As shown. Fig.13 It can be seen that the nine kinds of Coptis chinensis alkaloids basically reached their peak at 1.5 hours, and then decreased. Due to the existence of systemic circulation, the second absorption peak appeared at 4 hours. Different from the blood, in the model group, the drug concentration in the brain tissue was significantly higher than that in the control group, indicating that the PD rats induced targeted drug aggregation in the disease state and exerted better drug effects. Fig.14The results showed that in the model animals, the levels of neurotransmitters dopamine, 5-hydroxytryptamine and adrenaline increased, indicating that the pharmacodynamic effect effectively restored the pathological indicators of the model animals, verifying the effectiveness of coptis chinensis alkaloids. The above results show that the method established by the present invention can effectively track drugs and disease markers in target organs of brain tissue, providing a method for the prevention and treatment of brain diseases and the material basis of the efficacy of new Chinese medicine prescriptions.

Claims

1. A covalent organic framework material, characterized in that: It contains repeating structural units of 4,4',4"-(1,3,5-triazine-2,4,6-triyl)tris[benzaldehyde], ethidium bromide and 2,5-diaminobenzenesulfonic acid connected by imine bonds.

2. A method for preparing a covalent organic framework material as claimed in claim 1, characterized in that: The following steps are involved: a. Mix 4,4',4"-(1,3,5-triazine-2,4,6-triyl)tris[benzaldehyde], ethidium bromide and 2,5-diaminobenzenesulfonic acid; b. Add solvent, dissolve and mix evenly by ultrasonic; c. Add acetic acid aqueous solution, mix thoroughly, and react; d. Separate and purify to obtain a covalent organic framework material.

3. The preparation method according to claim 2, characterized in that: In step a, the molar ratio of 4,4',4"-(1,3,5-triazine-2,4,6-triyl)tris[benzaldehyde], ethidium bromide and 2,5-diaminobenzenesulfonic acid is 4:3-4:3-4; The solvent in step b is a mixed solvent of 1,3,5-trimethylbenzene and 1,4-dioxane, and the volume ratio of the two is 2:3; In step c, the concentration of the acetic acid aqueous solution is 12M, the molar ratio of 4,4',4"-(1,3,5-triazine-2,4,6-triyl)tri[benzaldehyde] to acetic acid is 1:8-24; the reaction temperature is 100-150°C, and the reaction time is 48-72h; The specific method of separation and purification in step d is: centrifugation to obtain a precipitate, then washing it alternately with tetrahydrofuran, ultrapure water and anhydrous ethanol, and vacuum drying.

4. A covalent organic framework bonding material, characterized in that: First, polyacrylonitrile and N,N-dimethylformamide are used to prepare polyacrylonitrile glue, and then the covalent organic framework material according to claim 1 is added.

5. A method for preparing a covalent organic framework bonding material, characterized in that: The following steps are involved: a. Mix polyacrylonitrile and N,N-dimethylformamide, and react them in a closed manner to obtain polyacrylonitrile glue; b. Add the covalent organic framework material according to claim 1 to polyacrylonitrile glue and shake and mix to obtain a covalent organic framework bonding material.

6. The preparation method according to claim 5, characterized in that: In step a, the ratio of polyacrylonitrile to N,N-dimethylformamide is 1:100-150 (w / v); the reaction temperature is 80-100° C., and the reaction time is 1-2 hours; In step b, the mass ratio of the covalent organic framework material to the polyacrylonitrile glue is 1:1-2.

7. A covalent organic framework material solid phase microextraction fiber, characterized in that: Coating the covalent organic framework bonding material according to claim 4.

8. A method for preparing a covalent organic framework material solid phase microextraction fiber, characterized in that: The stainless steel wire is pulled at a uniform speed and dipped into the covalent organic framework bonding material according to claim 4, air-dried and then dried, and the above steps are repeated to obtain a solid phase microextraction fiber with a uniform coating.

9. The preparation method according to claim 8, characterized in that: The diameter of the stainless steel wire is 0.2-0.3 mm, the length is 5-7 cm, and the coating thickness is 0.02-0.08 mm.

10. Use of the covalent organic framework material according to claim 1, the covalent organic framework adhesive material according to claim 4, and the covalent organic framework material solid phase microextraction fiber according to claim 7 in the detection of Coptis chinensis alkaloid metabolism and neurotransmitters.

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