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

Abstract

The application discloses a kind of covalent organic framework material solid-phase microextraction fiber and its application in alkaloid metabolism and neurotransmitter analysis.The covalent organic framework material (COFs) includes the repeating structural unit of 4,4',4"-(1,3,5-triazine-2,4,6-triazyl) tri[benzaldehyde] connected by imine bond, ethidium bromide and 2,5-diaminobenzenesulfonic acid.The COFs material is used as the adsorbent of solid-phase microextraction fiber surface coating, and the COFs is coated on the surface of stainless steel fiber by polyacrylonitrile glue.The COFs solid-phase microextraction fiber of the application can be used to extract rhizoma coptidis alkaloids and neurotransmitters in the brain and blood of living rats, and after sampling extraction, it is directly combined with LC-MS / MS for mass spectrometry analysis, with very high sensitivity, and can be used for rapid, accurate and sensitive analysis of trace rhizoma coptidis alkaloid metabolites and neurotransmitters in vivo.

Description

TECHNICAL FIELD

[0001] The present application relates to a covalent organic framework material solid-phase microextraction fiber and its application in drug metabolism analysis, and belongs to the field of solid-phase microextraction. BACKGROUND

[0002] Pharmacokinetics is a discipline for quantitatively studying the absorption, distribution, metabolism and excretion of drugs in the body, and has become an important part of preclinical and clinical studies of drugs. Exploring the pharmacokinetic behavior of drugs in brain tissue can determine whether they can penetrate the blood-brain barrier and reach the effective concentration, which is of great significance in the pharmacodynamics and toxicology studies of drugs for preventing and treating brain diseases. The collection of biological samples is the primary step in pharmacokinetic studies. Traditional sampling methods mainly collect whole blood from animals at specific time points or collect tissues from sacrificed animals for homogenization and subsequent analysis. This method has the disadvantages of requiring a large number of animals, complex pretreatment, non-free drug concentration analysis results, and inability to accurately reflect the real-time changes of components in the body. Therefore, the development of new sampling techniques is of great significance for pharmacokinetic studies.

[0003] Solid-phase microextraction (SPME) is a highly efficient and sensitive sample pretreatment technology that integrates sampling, extraction, concentration and injection into one. It can be combined with high-efficiency analysis and detection methods such as chromatography, electrophoresis and mass spectrometry, and is widely used in the analysis and detection of environmental, food and pharmaceutical fields. SPME technology is a solvent-free or low-solvent extraction technology using a small amount of enrichment medium as the core. Compared with traditional extraction methods, it has the advantages of low cost, easy use and environmental friendliness, and is a new high-efficiency separation and enrichment technology. The SPME sampling device is small in size and minimally invasive, and the coating volume is small and the enrichment efficiency is high, which is very suitable for the separation and enrichment of trace target substances in small-volume biochemical samples, and easy to realize in-situ and micro-injury analysis of living biological samples, giving it unique advantages in in-vivo tracking sampling. Moreover, the non-destructive SPME in-situ sampling method can minimize the interference with the organism, unlike traditional lethal methods, which can ensure the survival of the experimental object during the monitoring period. Long-term in-vivo tracking research on individual biological individuals during the drug administration period can reduce the loss of animal samples in the research and avoid the influence of individual differences to obtain the most authentic metabolic data of experimental animals.

[0004] The core point of the development of SPME technology is the coating. The commercial SPME coating has low specific surface area, high price and poor selectivity for target molecules, which limits its wide application. Therefore, it is the development direction of researchers to develop a SPME coating material with simple preparation, high adsorption performance, good selectivity, regular coating morphology and low cost. Covalent organic frameworks (COFs) are a new class of crystalline porous organic materials, which have 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 coating, many reported solid phase microextraction coatings do not have biocompatibility and protein exclusion effect, and it is difficult to realize the direct extraction of target substances in animal blood and tissue. Therefore, it is of great significance to prepare a coating material with biocompatibility and protein exclusion effect and high extraction efficiency for pharmacokinetic studies.

[0005] According to the ancient book 'Rihua Subao', Huanglian can treat five kinds of diseases and seven kinds of injuries, benefit qi, stop abdominal pain, palpitation, restlessness, moisten the heart and lung; 'Yaoxing Fu' records that Huanglian tastes bitter, is flat, has cold qi and is non-toxic, and can clear heart fire, and eliminate the state of heart fullness. Based on the fact that Huanglian can clear heart fire, since the heart governs the spirit and the fire is in the heart, the heart must be cleared first when clearing the fire, and the fire in the heart will be reduced when the heart fire is reduced. Modern studies have shown that Huanglian Jiedu Decoction can effectively improve the learning and memory function and effectively improve the clinical symptoms of patients with senile dementia, and pharmacological studies have shown that it can improve the symptoms of depression, Alzheimer's disease and other brain diseases by regulating the monoamine neurotransmitters (NE, 5-HT, DA) in the brain. Therefore, the establishment of a new method for the rapid separation and analysis of the effective components of traditional Chinese medicine Huanglian and the neurotransmitter markers in biological samples (blood, tissue, etc.) can provide a basis for the development of new drugs and clinical application of traditional Chinese medicine Huanglian.

[0006] Based on this, the application uses commercial 4,4',4''-(1,3,5-triazine-2,4,6-triazyl) tri[benzaldehyde] (TTB), ethidium bromide (EB) and 2,5-diamino benzenesulfonic acid (DABSA) as monomers to prepare a new EB-TTB-DABSA-COF material with sulfonic acid and quaternary ammonium group ambiphilic ions, large specific surface area and good dispersibility through Schiff base polymerization. SUMMARY

[0007] In view of the long preparation time, high detection cost, inability to extract in situ in real time and inconvenience in tracking a single animal for a long time in the prior art, the main purpose of the present application is to provide a functional covalent organic framework material solid phase microextraction fiber and its application in the detection of various active ingredients of traditional Chinese medicine (Huanglian alkaloids) and neurotransmitters, which can effectively solve the above problems, has the advantages of short detection time, high sensitivity and low detection cost, and can realize the research on the pharmacokinetics of Huanglian alkaloids and the changes of neurotransmitters.

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

[0009] In another aspect, the technical scheme of the present application is to provide a preparation method of a covalent organic framework material, comprising the following steps:

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

[0011] b. adding a solvent, ultrasonic dissolving and mixing uniformly;

[0012] c. adding an aqueous acetic acid solution, mixing thoroughly and reacting;

[0013] d. separating and purifying to obtain a covalent organic framework material.

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

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

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

[0017] Further, the specific method of separating and purifying in step d is: centrifuging to obtain a precipitate, then alternately washing with tetrahydrofuran, ultrapure water and anhydrous ethanol, and vacuum drying.

[0018] In another aspect, the technical scheme of the present application is to provide a covalent organic framework adhesive material, which is prepared by first using polyacrylonitrile and N,N-dimethylformamide to prepare polyacrylonitrile glue, and then adding the covalent organic framework material.

[0019] In another aspect, the technical scheme of the present application is to provide a preparation method of a covalent organic framework adhesive material, comprising the following steps:

[0020] a. mixing polyacrylonitrile and N,N-dimethylformamide, and sealing and reacting to prepare polyacrylonitrile glue;

[0021] b. The covalent organic framework material is added to the polyacrylonitrile glue and mixed to obtain a covalent organic framework adhesive material.

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

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

[0024] In another aspect, the technical scheme of the present application is to provide a covalent organic framework material solid phase microextraction fiber, which is coated with the covalent organic framework adhesive material.

[0025] In another aspect, the technical scheme of the present application is to provide a preparation method of a covalent organic framework material solid phase microextraction fiber, which comprises uniformly pulling and immersing a stainless steel wire to coat the covalent organic framework adhesive material, air-drying and then drying, and repeating the above steps to obtain a solid phase microextraction fiber with uniform coating.

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

[0027] In another aspect, the technical scheme of the present application is to provide the use of the covalent organic framework material, the covalent organic framework adhesive material, and the covalent organic framework material solid phase microextraction fiber in the detection of alkaloids and neurotransmitters in Coptis deltoidea.

[0028] The COFs material of the present application can adsorb alkaloids and neurotransmitters in Coptis deltoidea through hydrophobic interaction, electrostatic interaction, π-π interaction and van der Waals force. The COFs material can be used to prepare a solid phase microextraction fiber, which is combined with LC-MS / MS to study the changes of alkaloids and neurotransmitters in Coptis deltoidea 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 alkaloids and neurotransmitters in Coptis deltoidea in vivo. The extraction process does not require killing the rats, and the extraction and detection processes can be completed within 1 hour, with high sensitivity and low detection cost. At the same time, the loss of experimental animals in the research is effectively reduced, and the influence of individual differences is also avoided, so as to obtain the most real metabolic data of the experimental animals. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Figure 4 is a scanning electron microscope image of the EB-TTB-DABSA-COF fiber.

[0030] Figure 2EDS elemental map of EB-TTB-DABSA-COF material.

[0031] Figure 3 XPS survey spectrum of EB-TTB-DABSA-COF material.

[0032] Figure 4 High resolution spectra of EB-TTB-DABSA-COF material in C 1s, N 1s, O 1s and S 2p channels.

[0033] Figure 5 Protein rejection efficiency plot of EB-TTB-DABSA-COF material and COFs-SPME fiber.

[0034] Figure 6 Comparison of adsorption efficiency of COFs-SPME fiber with 3 commercial material fibers.

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

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

[0037] Figure 9 Optimization of elution time of COFs solid phase microextraction fiber after extraction.

[0038] Figure 10 Optimization of adsorption time of COFs solid phase microextraction fiber in brain surrogate matrix.

[0039] Figure 11 Pharmacokinetic plot of Coptis rhizome in rat blood after gavage (n=5). In the figure, Model represents the model group, and Control represents the control group.

[0040] Figure 12 Neurotransmitter content in rat blood within 10 h after gavage of Coptis rhizome (n=5). In the figure, Model represents the model group, and Control represents the control group.

[0041] Figure 13 Pharmacokinetic plot of Coptis rhizome in rat brain tissue after gavage (n=5). In the figure, Model represents the model group, and Control represents the control group.

[0042] Figure 14 Neurotransmitter content in rat brain tissue within 10 h after gavage of Coptis rhizome (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 application will be further described in detail below with reference to the examples and the accompanying drawings, but the embodiments of the application are not limited to the following ranges. The reagents used in the examples are not specified, and are all commercially available conventional products that can be purchased.

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

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

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

[0047] b. 4 mL of a mixed solvent of mesitylene and 1,4-dioxane (ratio of 2:3, v / v) was added, and the mixture was ultrasonically dissolved and uniformly mixed;

[0048] c. 12 M aqueous acetic acid (molar ratio of TTB to acetic acid was 1:16) was added, and the mixture was thoroughly mixed and reacted at 120°C for 60 h;

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

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

[0051] a. Polyacrylonitrile and N,N-dimethylformamide were mixed (1:100, w / v), and a polyacrylonitrile glue was prepared by sealing and reacting at 90°C for 1 h;

[0052] b. The covalent organic framework material was added to the polyacrylonitrile glue and mixed by oscillation to prepare a covalent organic framework adhesive material; the mass ratio of the covalent organic framework material to the polyacrylonitrile glue was 1:1;

[0053] c. A stainless steel wire with a diameter of 0.2 mm was cut into small pieces with a length of 7 cm, washed with 50% (v / v) methanol aqueous solution, and then dried; the solid phase microextraction fiber with uniform coating was obtained by vertically inserting the stainless steel wire into the covalent organic framework adhesive material by the dip-coating method, and the coating length was 6 mm; after the surface material was air-dried, the coating was fixed by baking at 90°C for 60 s, and the steps of immersion, air-drying, and baking were repeated to make the coating as uniform as possible on the stainless steel wire; finally, the solid phase microextraction fiber (COFs-SPME fiber) with uniform coating was obtained, and the thickness of the coating was 0.02 mm.

[0054] Example 2: Preparation of covalent organic framework material solid-phase microextraction fiber

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

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

[0057] b. 5 mL of mixed solvent of mesitylene and 1,4-dioxane (ratio of 2:3, v / v) was added, ultrasonic dissolution and uniform mixing were carried out;

[0058] c. 12 M aqueous acetic acid solution (molar ratio of TTB and acetic acid was 1:8) was added, mixed thoroughly, and reacted at 100°C for 72 h;

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

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

[0061] a. Polyacrylonitrile and N,N-dimethylformamide were mixed (1:150, w / v), and a polyacrylonitrile glue was prepared by sealing and reacting at 80°C for 2 h;

[0062] b. The covalent organic framework material was added to the polyacrylonitrile glue and mixed by oscillation to prepare a covalent organic framework adhesive material; wherein the mass ratio of the covalent organic framework material to the polyacrylonitrile glue was 1:1.5;

[0063] c. A stainless steel wire with a diameter of 0.2 mm was cut into small pieces with a length of 6 cm, washed with 50% (v / v) methanol aqueous solution and dried; the solid-phase microextraction fiber with uniform coating was obtained by vertical insertion of the stainless steel wire into the covalent organic framework adhesive material by dip coating method, and the coating length was 5 mm; after the surface material was air-dried, the coating was fixed by baking at 60°C for 90 s, and the steps of immersion, air-drying and baking were repeated to make the coating as uniform as possible on the stainless steel wire, and the final coating thickness was 0.06 mm.

[0064] Example 3: Preparation of covalent organic framework material solid-phase microextraction fiber

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

[0066] a. Mix 4,4',4”-(1,3,5-triazine-2,4,6-triyl)tri[benzaldehyde], ethidium bromide and 2,5-diaminobenzenesulfonic acid, with amounts of 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 (in a ratio of 2:3, v / v), sonicate to dissolve and mix thoroughly;

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

[0069] d. Centrifuge to obtain a precipitate, then wash it alternately with tetrahydrofuran, ultrapure water and anhydrous ethanol, and vacuum dry it at 80°C to obtain a covalent organic framework material.

[0070] (2) Preparation of solid-phase microextraction fibers

[0071] a. Polyacrylonitrile and N,N-dimethylformamide were mixed (1:120, w / v) and reacted at 100°C in a sealed environment for 1 h to obtain polyacrylonitrile adhesive;

[0072] b. Add the covalent organic framework material to the polyacrylonitrile adhesive and mix by shaking to obtain the covalent organic framework adhesive material; wherein the mass ratio of the covalent organic framework material to the polyacrylonitrile adhesive is 1:2;

[0073] c. Cut stainless steel wire with a diameter of 0.3 mm into 5 cm segments, clean them with 50% (v / v) methanol aqueous solution and dry them; vertically insert them into the covalent organic framework adhesive material using the dip-coating method, with a coating length of 4 mm. After the surface material is air-dried, bake at 100℃ for 40 seconds to fix the coating. Repeat the dip-coating, 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 and a final coating thickness of 0.08 mm.

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

[0075] First, the morphology and microstructure of COFs-SPME fibers were studied using scanning electron microscopy. For example... Figure 1 As shown in 1a and 1b, the surface of the synthesized EB-TTB-DABSA-COF material exhibits irregularly stacked rod-shaped substances forming a flower-like structure. This unique structure is beneficial for increasing the specific surface area and adsorption capacity of the EB-TTB-DABSA-COF material, thereby achieving its effective adsorption of target substances. Simultaneously, it can be observed that the coated fiber surface is uniformly coated, with consistent shape and good dispersion, and the coating thickness is approximately 20 μm. Figure 1c and 1d) and the surface of the coating is wrapped with a reticular PAN polymer layer, which can effectively exclude macromolecular proteins and other substances while allowing small molecules to enter, and has excellent biocompatibility in the extraction of biological complex matrix samples. Figure 2 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 elements are uniformly distributed.

[0076] In order to further investigate whether the synthesis of EB-TTB-DABSA-COF material is successful, XPS is used to characterize the synthesized material. From the XPS survey spectrum of the synthesized material, as shown in Figure 3 It can be seen that there are five peaks of C1s, N1s, O1s, S2p and Br3d in the wide spectrum. Then, the high-resolution spectra of C1s, N1s, O1s and S2p are further analyzed, and the results are as shown in Figure 4 a-4d, in the C1s spectrum, two obvious peaks appear, corresponding to C-C with a binding energy of 284.3eV and C-O with a binding energy of 286.38eV, indicating that the triazine compound TTB is combined on the COFs. In the high-resolution spectrum of N1s, the peaks with binding energies of 398.3, 399 and 400.63eV correspond to N-H, N-N and N-C respectively; in the high-resolution spectra of O1s and S2p, there are C=O with a binding energy of 531.16eV and R-S-R with a binding energy of 167.35eV respectively, which indicates that DABSA is combined on the COFs material. At the same time, based on Figure 3 the Br3d peak in the XPS survey spectrum and N-H and N-C in the high-resolution spectrum of N1s, it is shown that EB is combined on the COFs. The above results confirm that EB-TTB-DABSA-COF is successfully synthesized.

[0077] Example 5: Evaluation of protein exclusion ability

[0078] There are a large number of protein interferents in vivo, and the protein exclusion ability is an important indicator for evaluating the performance of adsorption materials. Based on this, first, the albumin is used as a probe protein to determine the exclusion ability of the material. The prepared 2mg EB-TTB-DABSA-COF material and COFs-SPME fiber are added into different concentrations of albumin solution (100, 200 and 300μg·mL -1 ) respectively. After oscillation at 500rpm for 2h at room temperature, the absorbance of the supernatant is detected at 280nm wavelength. The calculation formula of protein exclusion rate is: protein exclusion rate = C s / C0×100%, wherein C s is the concentration of residual protein in the solution after adsorption, and C0is the initial concentration of protein in the solution. The results are as shown inFigure 5 As shown, with the increase of albumin concentration, the protein rejection rates of EB-TTB-DABSA-COF material and COFs-SPME fiber were between 90.93-94.59% and 91.50-95.16%, respectively. Meanwhile, in order to further clarify the protein rejection effect, the rejection effect of different proteins (hemoglobin, beta-lactoglobulin and serum actual sample) was also analyzed. The rejection efficiency of EB-TTB-DABSA-COF material and COFs-SPME fiber to different proteins was 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 exhibit excellent rejection effect on different concentrations and different types of proteins. 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 target substances in complex samples.

[0079] Experimental verification

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

[0081] (1) Rat brain striatum extraction: After isoflurane anesthesia of rats, the skin and subcutaneous tissue near the fontanel were separated, and the bone surface was exposed. According to the coordinates (AP: 0.43-0.73 mm, ML: 2.7-3.3 mm), the striatum area was positioned. A hole was drilled on the bone surface with a dental drill, and a cannula was implanted in the striatum area. After fixation, the dustproof cap was tightened. The wound was sutured and roxithromycin was applied to the wound to prevent infection. The rats were raised for 5 days to recover normal physiological state. Before use, the COFs-SPME fiber was activated with 50% (v / v) methanol-water for 20-30 min, and the fiber was inserted into the brain striatum through the brain cannula, and extracted for 2-15 min.

[0082] Rat blood extraction: First, a 7-gauge needle was inserted into the tail vein, and the fiber was inserted into the blood vessel through the needle. The needle was pulled out, and the fiber was extracted in the tail vein for 2-15 min.

[0083] After extraction, the fiber was first washed with ultrapure water for 30-60 s, and then eluted with 0.2% formic acid-methanol: water (1:1, v / v) for 10-60 min.

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

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

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

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

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

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

[0090] Mass spectrometry conditions: model: TSQ Altis high-resolution mass spectrometer; the ion source is in positive ion mode; the scanning mode is SRM; the source area condition parameters are: 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 is used as collision gas and damping gas of C-trap.

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

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

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

[0094] In this experiment, the extraction capacity of the COFs-SPME fiber prepared in Example 1 of the present application and the commercially available WAX, PXC, and C18 material coated solid phase microextraction fiber for alkaloids and neurotransmitters in Coptidis Rhizoma is determined.

[0095] The COFs-SPME fiber prepared in Example 1 of the present application was compared with commercialized WAX, PXC and C18 material coated SPME fibers for extraction of 100 ng·mL -1 various neurotransmitters (NTs) and 10 ng·mL -1 various alkaloids in phosphate buffered saline (PBS, 0.01 M, pH 7.4). 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, the elution condition was 100 μL 0.2% FA-MeOH:ACN:H2O (1:1:1, v / v / v), and the elution time was 30 min. The desorption solution was introduced into the LC-MS / MS instrument for analysis, and the elution of each substance was compared to compare the adsorption capacity of different solid phase microextraction fibers for the analytes, and the results are shown in Figure 6 Figure 6 It can be seen that the COFs-SPME fiber prepared in the present application has better extraction effect than the commercialized material fiber for the extraction of coptis alkaloids including coptisine, demethylated berberine, berberine, epiberberine, jatrorrhizine, columbamine, magnoflorine, oxidized berberine, palmatine and neurotransmitters (glycine, γ-aminobutyric acid, dopamine, 5-hydroxytryptamine and adrenaline), indicating that the COFs-SPME fiber has good extraction performance for alkaloids and neurotransmitter target substances.

[0096] Verification experiment 2: condition optimization of COFs-SPME fiber for extraction of coptis alkaloids and neurotransmitters in blood

[0097] The COFs-SPME fiber prepared in Example 1 of the present application was used for simultaneous spiking of 100 ng·mL -1 various neurotransmitters and 10 ng·mL -1 ​The COFs-SPME fiber was activated with 50% methanol-water before extraction, and then adsorbed 0.5 mL of the spiked serum for 2-15 min; after extraction, the COFs-SPME fiber was washed with ultrapure water for 30 s, and then eluted with 100 μL of 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:H2O, 1:1, v / v), 0.2% formic acid-methanol water (0.2% FA-MeOH:H2O, 6:4, v / v), 0.2% formic acid-acetonitrile water (0.2% FA-ACN:H2O, 1:1, v / v) and 0.2% formic acid-methanol acetonitrile water (0.2% FA-MeOH:ACN:H2O, 1:1:1, v / v / v) for 10-60 min, and the eluent was introduced into the LC-MS / MS instrument for analysis, and the neurotransmitters and alkaloids in the eluent were quantified.

[0098] In order to more intuitively reflect the extraction results, substances with similar adsorption amounts were displayed in the same graph according to the difference in the adsorption amount of the target substance, and therefore were divided into Figure a and Figure b.

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

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

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

[0102] Verification experiment 3: condition optimization of the COFs-SPME fiber for extracting alkaloids and neurotransmitters in brain substitute matrix

[0103] First, the brain substitute matrix was prepared, and agar gel could be used as the interstitial fluid of the organism. The agar gel (2% agar dispersed in PBS buffer, w / v) was mixed with the brain homogenate (sheep brain:PBS buffer = 1:10, w / v) at a ratio of 1:1 (v / w) to prepare the brain substitute matrix of the rat for SPME. The COFs-SPME fiber prepared in Example 1 of the present application was used to simultaneously spike 100 ng·mL -1 a variety of neurotransmitters and 10 ng·mL -1The COFs-SPME fiber was activated with 50% methanol-water and then adsorbed 0.5 mL of the spiked brain substitute for 2-15 min before extraction; after extraction, the COFs-SPME fiber was washed with ultrapure water for 30 s, and then eluted with 100 μL of 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, respectively, and the eluent was introduced into the LC-MS / MS instrument for analysis, and the neurotransmitters and alkaloids in the eluent were quantified.

[0104] The extraction time optimization results are shown in Table 2. Figure 10 As shown in Table 2, the COFs-SPME fiber has the best extraction effect when the extraction time is 4 min.

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

[0106] Verification experiment 4: method verification of COFs-SPME fiber for extraction of alkaloids and neurotransmitters in serum

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

[0108] The COFs-SPME fiber prepared in Example 1 of the present application is used for the extraction of a plurality of neurotransmitters (at concentrations of 1, 25, 50, 75, 100, 150, and 200 ng·mL -1 ) and a plurality of alkaloids (at concentrations of 0.5, 1, 2, 4, 6, 8, and 10 ng·mL -1 ) in blank serum at the same time, a linear good concentration range is selected according to the extraction results, and the detection limit and the quantification limit are calculated in turn, and 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 the neurotransmitters in serum is 1.0-200.0 ng·mL -1 , the detection limit and the quantification limit are 3.3×10 -3 -0.05 ng·mL -1 , and 0.01-0.16 ng·mL -1 , respectively; the linear range of the alkaloids is 0.5-10.0 ng·mL -1 , the detection limit and the quantification limit are 0.10×10 -3 -0.21 ng·mL -1 , and 0.3×10 -3~0.50 ng·mL -1 between 1.0 and 200.0 ng·mL -1 , the detection limit and the limit of quantification were between 4.40 x 10 -3 ~ 0.07 ng·mL -1 and 0.01 ~ 0.24 ng·mL -1 , respectively. This indicates that the COFs-SPME fiber constructed can achieve wide-range and high-sensitivity simultaneous detection of alkaloids and neurotransmitters in serum.

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

[0110]

[0111]

[0112] Verification Experiment 5: Method verification of COFs-SPME fiber for extracting alkaloids and neurotransmitters in brain substitute matrix

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

[0114] The COFs-SPME fiber prepared in Example 1 of the present application was used for the extraction of a plurality of neurotransmitters (concentrations of 1, 25, 50, 75, 100, 150 and 200 ng·mL -1 ) and a plurality of alkaloids (concentrations of 0.5, 1, 2, 4, 6, 8 and 10 ng·mL -1 ) in the brain substitute matrix at the same time, and according to the extraction results, a linearly good concentration range was selected, and the detection limit and the limit of quantification were calculated in turn, and the signal-to-noise ratios of the detection limit and the limit of quantification were selected to be 3 and 10, respectively. The linear range is shown in Table 2, the linear range of the neurotransmitters in the brain substitute matrix is between 1.0 and 200.0 ng·mL -1 , the detection limit and the limit of quantification are between 4.40 x 10 -3 ~ 0.07 ng·mL -1 and 0.01 ~ 0.24 ng·mL -1 , respectively; the linear range of the alkaloids is between 0.5 and 10.0 ng·mL -1 , the detection limit and the limit of quantification are between 0.09 x 10 -3 ~ 0.12 ng·mL -1 and 0.30 x 10 -3 ~ 0.38 ng·mL -1 , respectively. This indicates that the COFs-SPME fiber constructed can achieve wide-range and high-sensitivity detection of alkaloids and neurotransmitters in brain tissue samples.

[0115] Table 2 Linear regression equation of target substances in brain substitute matrix

[0116]

[0117] Verification experiment 6: precision and accuracy of COFs-SPME fiber extraction of alkaloids and neurotransmitters in Coptis deltoidea

[0118] This experiment determines the accuracy and precision of the COFs-SPME fiber prepared in Example 1 of the present application for the analysis method of alkaloids and neurotransmitters in Coptis deltoidea in blood and brain substitute matrix.

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

[0120] Table 3 Precision and accuracy of different concentrations of target substances

[0121]

[0122]

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

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

[0125] The COFs-SPME fiber prepared in Example 1 of the present application is used for the extraction of alkaloid metabolites and neurotransmitters in the blood of PD rats in the tail vein. After fasting for 12 h, the control group (normal rats without treatment) and the PD model group of rats are given Coptis extract 0.48 g·kg -1 . The blood of the rats at each time point (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8 and 10 h) after administration is extracted through the tail vein. The extraction time is 8 min, and after oscillation elution with ultrapure water for 30 s, elution with 0.2% formic acid-methanol water (1:1, v / v) for 10 min, the eluate enters the LC-MS / MS, and the content changes of the pharmacokinetics of alkaloids and neurotransmitters in the blood of PD rats are analyzed. The specific results are shown in Figure 11 and Figure 12 . Figure 11It can be seen that the nine alkaloids basically reach their peak rapidly at 0.5h or 1h, and then gradually decrease. A second peak occurs at 4-6h, suggesting that there may be a 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 that in the control group, indicating that the body improves drug utilization under disease conditions. Figure 12 The study showed the trends in neurotransmitter changes in the PD model group and the control group. Compared with the control group, the neurotransmitter levels in the blood of PD rats showed a slight upward trend, but the difference was not statistically significant. These results indicate that the method established in this invention can effectively track changes in drugs and disease biomarkers in vivo, providing a reference for disease prevention and treatment and the pharmacodynamic material basis of new traditional Chinese medicine formulations.

[0126] Validation Experiment 8: Application of COFs-SPME Fibers in Extracting Coptis chinensis Alkaloids and Neurotransmitters from the Rats Striatum

[0127] This experiment is a pharmacokinetic analysis of berberine alkaloids and changes in neurotransmitters in the striatum of PD rats using COFs-SPME fibers prepared in Example 1 of this invention.

[0128] The COFs-SPME fiber prepared in Example 1 of this invention was used for the extraction of Coptis chinensis alkaloid metabolites and neurotransmitters from the striatum of PD rats. After fasting for 12 hours, rats in the control group (untreated normal rats) and the PD model group were administered Coptis chinensis by gavage at a dose of 0.48 g·kg⁻¹. -1 By stereotactic localization of the brain and using an implanted cannula, berberine and neurotransmitters were extracted from the striatum of PD rats at 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, and 10 hours after gavage. The extraction time was 8 min. After elution with ultrapure water for 30 s, followed by elution with 0.2% formic acid-methanol-water (1:1, v / v) for 10 min, the pharmacokinetics of berberine and changes in neurotransmitter content in the striatum of PD rats and control groups were analyzed using LC-MS / MS. The results are as follows: Figure 13 and Figure 14 As shown. By Figure 13 It was found that the nine berberine alkaloids reached their peak concentration at approximately 1.5 hours, then decreased, with a second absorption peak appearing at 4 hours due to systemic circulation. Unlike in blood, the drug concentration in the brain tissue of the model group animals was significantly higher than that in the control group, indicating that the drug's targeted accumulation was induced in the PD rats under disease conditions, resulting in better efficacy. Simultaneously, from... Figure 14It is shown that the contents of neurotransmitters dopamine, 5-hydroxytryptamine and adrenaline in the model animals increase, which indicates that the pathological indexes of the model animals are effectively recovered by the exertion of the pharmacological effect, and the effectiveness of the alkaloids of Coptis chinensis Franch is verified. The above results show that the method established in the application can realize effective tracking of drugs and disease markers in the target organs of brain tissues, and provides a method for prevention and treatment of brain diseases and pharmacodynamic material basis of new Chinese medicine formula drugs.

Claims

1. A covalent organic framework material characterized in that, The repeating structural unit comprises 4,4',4"-(1,3,5-triazine-2,4,6-triyl)tris[benzaldehyde] connected by imine bond, ethidium bromide and 2,5-diaminobenzenesulfonic acid.

2. A method of preparing the covalent organic framework material of claim 1, wherein, The method comprises the following steps: a. mixing 4,4',4"-(1,3,5-triazine-2,4,6-triyl)tris[benzaldehyde], ethidium bromide and 2,5-diaminobenzenesulfonic acid; b. adding a solvent, ultrasonic dissolving and mixing uniformly; c. adding an aqueous acetic acid solution, mixing thoroughly and reacting; d. separating and purifying to obtain a covalent organic framework material.

3. The method according to claim 2, wherein the molar ratio of 4,4',4"-(1,3,5-triazine-2,4,6-triyl)tris[benzaldehyde], ethidium bromide and 2,5-diaminobenzenesulfonic acid in step a is 4:3-4:3-4; the volume ratio of the mixed solvent of mesitylene and 1,4-dioxane in step b is 2:3; the concentration of the aqueous acetic acid solution in step c is 12M, and the molar ratio of 4,4',4"-(1,3,5-triazine-2,4,6-triyl)tris[benzaldehyde] and acetic acid is 1:8-24; the reaction temperature is 100-150℃, and the reaction time is 48-72h; the specific method of separating and purifying in step d is: centrifuging to obtain a precipitate, then alternately washing with tetrahydrofuran, ultrapure water and anhydrous ethanol, and vacuum drying. The covalent organic framework material of claim 1 is added to the polyacrylonitrile glue prepared from polyacrylonitrile and N,N-dimethylformamide.

4. A covalent organic framework adhesive material characterized in that, The method comprises the following steps:

5. A method of making a covalent organic framework adhesive material, characterized in that, a. mixing polyacrylonitrile and N,N-dimethylformamide, and sealing to react to prepare polyacrylonitrile glue; b. adding the covalent organic framework material of claim 1 to the polyacrylonitrile glue, and oscillating to mix to prepare a covalent organic framework adhesive material.

6. The method according to claim 5, wherein the ratio of polyacrylonitrile and N,N-dimethylformamide in step a is 1:100-150 (w / v); the reaction temperature is 80-100℃, and the reaction time is 1-2h; the mass ratio of the covalent organic framework material and the polyacrylonitrile glue in step b is 1:1-2. The covalent organic framework adhesive material of claim 4 is coated. A stainless steel wire is uniformly pulled and immersed to coat the covalent organic framework adhesive material of claim 4, and after air drying, the above steps are repeated to obtain a solid phase microextraction fiber with uniform coating.

7. A covalent organic framework material solid phase microextraction fiber, characterized in that, The diameter of the stainless steel wire is 0.2-0.3mm, the length is 5-7cm, and the coating thickness is 0.02-0.08mm.

8. A method of preparing a covalent organic framework material solid phase microextraction fiber, characterized in that, 10. Use of the covalent organic framework material of claim 1, the covalent organic framework adhesive material of claim 4, and the covalent organic framework material solid phase microextraction fiber of claim 7 in detection of alkaloids in Coptis and neurotransmitters.

9. The production method according to claim 8, characterized by, ​ ​

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