Application of zwitterionic covalent organic framework solid-phase microextraction fiber in neurotransmitter analysis

Abstract

The application discloses application of a zwitterionic covalent organic framework solid-phase microextraction fiber in neurotransmitter analysis, wherein the zwitterionic covalent organic framework material is prepared by Schiff base reaction condensation with ethidium bromide, 2,5-diaminobenzoic acid and triformylphloroglucin as functional monomers and acetic acid as a catalyst to obtain the zwitterionic covalent organic framework material. The coating of the zwitterionic covalent organic framework solid-phase microextraction fiber prepared in the application has good hydrophilicity, and the structure is rich in functional groups such as hydroxyl groups, carboxyl groups, phenyl groups and quaternary ammonium groups, which can interact with target analytes through ion exchange, pi-pi stacking, hydrogen bonding and hydrophilic-hydrophobic interaction, thereby showing strong specific adsorption and high enrichment capacity for neurotransmitters. The zwitterionic covalent organic framework solid-phase microextraction fiber prepared in the application realizes efficient enrichment and extraction analysis of neurotransmitters with different polarities in complex biological sample matrices.

Description

Technical Field

[0001] This invention relates to the fields of chemical synthesis, chemical analysis and detection, and sample pretreatment technology, specifically to a method for preparing and applying an ionic covalent organic framework solid-phase microextraction fiber coating. Background Technology

[0002] Neurotransmitters, as a class of endogenous chemical substances, are widely distributed in the central nervous system, brain tissue, and body fluids of mammals. They play a crucial role in the physiological processes of intercellular communication and signal transduction. Abnormal changes in the levels of neurotransmitters in the body are also closely related to diseases such as cancer, immune responses, and inflammation, and they hold promise as new targets for the diagnosis and treatment of diseases like cancer, as well as potential biomarkers for these diseases. Therefore, the detection of trace neurotransmitters in biological fluids is of great significance for promoting disease detection and the development of new therapeutic drugs.

[0003] Currently, various sample preparation techniques, such as liquid-liquid extraction, solid-phase extraction, dispersive liquid-liquid microextraction, magnetic solid-phase extraction, and solid-phase microextraction, are used for the enrichment and detection of neurotransmitters in various biological matrices. However, traditional pretreatment methods, such as solid-phase extraction (SPE) and liquid-liquid extraction (LLE), require large sample volumes and consume organic solvents, which often limits their application in routine analyses, such as the analysis of solid tissue samples. Solid-phase microextraction (SPME) technology not only offers advantages such as fast sampling speed, small sample volume requirements, no need for large amounts of organic solvents, and low invasiveness, minimally invasiveness, low cost, and high throughput, but has also been widely used for the extraction and enrichment of endogenous compounds and metabolites in complex biological matrices such as biofluids and solid tissues. Covalent organic frameworks (COFs) are a class of innovative porous crystalline materials that primarily connect organic monomers together through covalent bonds to form 2D or 3D networks. Covalent organic frameworks (COFs) possess large specific surface areas, high porosity, and excellent thermal and chemical stability, attracting widespread attention due to their immense application potential in sensing, separation, catalysis, optoelectronics, and biomedicine. Currently, there are reports on the use of covalent organic frameworks for neurotransmitter extraction; however, existing framework materials can only be used for small amounts of single neurotransmitters (such as catecholamines, or basic or acidic neurotransmitters). Therefore, there is a need to develop functionalized materials capable of simultaneously extracting multiple neurotransmitters. Furthermore, existing covalent organic frameworks suffer from low enrichment efficiency and low sensitivity. Therefore, how to prepare a covalent organic framework solid-phase microextraction fiber to improve the selectivity and enrichment efficiency of analytical methods for neurotransmitters is a pressing and challenging problem in this field. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide an ionic covalent organic framework solid-phase microextraction fiber, its preparation method, and its applications, thereby solving the problems of poor selectivity, low enrichment efficiency, low sensitivity, high detection limits, and poor extraction effect of target analytes in existing technologies for neurotransmitters. The ionic covalent organic framework solid-phase microextraction fiber prepared by this invention achieves efficient enrichment and extraction analysis of neurotransmitters of different polarities in complex biological sample matrices.

[0005] To achieve the above objectives, one aspect of the technical solution of the present invention is to provide an application of an ionic covalent organic framework material in the preparation of an adsorbent for sample pretreatment. The ionic covalent organic framework material is obtained by condensation of ethidium bromide, 2,5-diaminobenzoic acid and trialdehyde-resorcinol as functional monomers, with acetic acid as a catalyst, through a Schiff base reaction.

[0006] Furthermore, the specific preparation method of the ionic covalent organic framework material is as follows: Ethidium bromide, 2,5-diaminobenzoic acid, and trialdehyde phloroglucinol are accurately weighed, mixed, and then a reaction solvent is added, followed by the addition of acetic acid solution, and ultrasonic dispersion is performed. Subsequently, the mixture is degassed and sealed using a three-stage refrigeration pump-thawing cycle, and the reaction is continued. After the reaction is completed, the mixture is cooled to room temperature, centrifuged to collect the precipitate, and washed alternately with tetrahydrofuran, deionized water, and acetone before drying to obtain deep red EB-Da-COF.

[0007] Furthermore, the molar ratio of ethidium bromide, 2,5-diaminobenzoic acid and trialdehyde phloroglucinol is 0.8–1.2:0.8–1.2:1.2–1.8.

[0008] Furthermore, the acetic acid solution has a concentration of 6M, and its ratio with trialdehyde phloroglucinol is 0.5mL:0.45mmol / L.

[0009] Furthermore, the reaction temperature is 110–120°C, the reaction time is 60–72 hours, and the reaction solvent is a mixed solution of 1,3,5-trimethylbenzene and 1,4-dioxane in a volume ratio of 2:3.

[0010] Furthermore, sample pretreatment includes extraction analysis of neurotransmitters in serum samples and brain tissue.

[0011] On the other hand, the technical solution of the present invention provides an application of ionic covalent organic framework solid-phase microextraction fiber as an adsorbent in sample pretreatment. The fiber is based on nickel-titanium fiber filaments, which are self-assembled layer by layer using a sol-gel coating method to coat the ionic covalent organic framework material onto the fiber filaments, thus obtaining the ionic covalent organic framework solid-phase microextraction fiber. Specifically, the following steps are included:

[0012] (1) Preparation of PAN adhesive: Weigh 4.5-5.0g of PAN and 72.5mL of N,N-dimethylformamide, mix them, stir evenly, and seal and place at 90℃ for 1h;

[0013] (2) Weigh 300.0-325.0 mg of the ionic covalent organic framework material, then add 2.5-3.1 g of PAN glue, then add 0.15-0.2 g of glycerol, vortex, and then stir overnight to obtain the ionic covalent organic framework solid-phase microextraction fiber coating.

[0014] (3) Coating the ionic covalent organic framework solid-phase microextraction fiber with an ionic covalent organic framework solid-phase microextraction fiber to obtain the ionic covalent organic framework solid-phase microextraction fiber.

[0015] Furthermore, the coating length of the ionic covalent organic framework solid-phase microextraction fiber is 4.5±2 mm, and the thickness is 10±5 μm. The concentration of the mixed adhesive can be slightly adjusted according to the coating effect.

[0016] Furthermore, sample pretreatment is used for the extraction and analysis of neurotransmitters in serum samples and brain tissue.

[0017] Compared with the prior art, the present invention has the following superior effects:

[0018] (1) The ionic covalent organic framework material prepared by the Schiff base reaction of the present invention has a simple preparation step and the obtained material has good mechanical stability and thermal stability. It is connected by relatively stable imine bonds, which improves the service life of the extraction fiber.

[0019] (2) In this invention, 2,5-diaminobenzoic acid with cation exchange function, ethidium bromide with anion exchange function, and trialdehyde phloroglucinol are used as functional monomers to prepare an ionic covalent organic framework, and further prepare a solid phase microextraction fiber coating. The prepared fiber coating has a uniform surface distribution and thickness, and has a dense honeycomb porous structure, which greatly improves its adsorption and extraction capacity.

[0020] (3) The ionic covalent organic framework prepared by the present invention using 2,5-diaminobenzoic acid with cation exchange activity, ethidium bromide with anion exchange activity, and trialdehyde phloroglucinol as functional monomers has a large specific surface area (183.26 m²). 2 The ionic covalent organic framework solid-phase microextraction fiber coating prepared in this invention has good biocompatibility and protein exclusion ability, and is characterized by a narrow particle size distribution (average pore size of 2.27 nm), resulting in a high specific surface area, mesoporous structure and protein exclusion effect. This allows it to be directly used for the extraction of target substances from biological samples without the need for a protein precipitation step.

[0021] (4) In addition to good hydrophilicity, the ionic covalent organic framework solid-phase microextraction fiber coating prepared by the present invention has strong specific adsorption and high efficiency enrichment of neurotransmitters due to the interaction between the functional groups such as hydroxyl, carboxyl, phenyl and quaternary ammonium in its structure and the target analytes through ion exchange, π-π stacking, hydrogen bonding and hydrophilic-hydrophobic interaction.

[0022] (5) The three monomers of this invention (2,5-diaminobenzoic acid, ethidium bromide and trialdehyde phloroglucinol) are inexpensive and readily available, have short synthesis steps, simple operation, mild reaction conditions, and high synthesis yield, making them suitable for large-scale preparation and promotion.

[0023] (6) The ionic covalent organic framework solid-phase microextraction fiber coating prepared by the present invention has good stability and reusability, which can ensure the high efficiency and reliability of the extraction process, and shows significant advantages in saving resources, reducing costs and protecting the environment. Attached Figure Description

[0024] Figure 1 The images show scanning electron microscope (SEM) images of the zwitterionic covalent organic framework (A), a portion of the surface of the zwitterionic covalent organic framework solid-phase microextraction fiber (B and C), and a cross-section (D) of the zwitterionic covalent organic framework solid-phase microextraction fiber coating prepared in Example 1 of this invention.

[0025] Figure 2 This is the overall EDS elemental distribution map of the ionic covalent organic framework prepared in Example 1 of the present invention.

[0026] Figure 3 This is the Fourier transform infrared spectrum of the ionic covalent organic framework prepared in Example 1 of the present invention.

[0027] Figure 4 The above are the overall XPS spectrum (A) and fine spectra of C (B), N (C) and O (D) of the ionic covalent organic framework prepared in Example 1 of this invention.

[0028] Figure 5 This is a water contact angle test diagram of the ionic covalent organic framework prepared in Example 1 of the present invention.

[0029] Figure 6 This is the N2 adsorption-desorption isotherm of the ionic covalent organic framework prepared in Example 1 of this invention.

[0030] Figure 7 A comparison of the extraction efficiency of target analytes by ionic covalent organic framework fiber coating materials prepared for different monomers and commercial coatings.

[0031] Figure 8This is a diagram showing the macromolecular exclusion effect of the ionic covalent organic framework and solid-phase microextraction fiber coating prepared in Example 1 of the present invention.

[0032] Figure 9 This is a graph showing the effect of repeated use of the ionic covalent organic framework solid-phase microextraction fiber coating prepared in Example 1 of the present invention. Detailed Implementation

[0033] The present invention will be further described below with reference to embodiments. These embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0034] The present invention will now be described in detail with reference to specific embodiments.

[0035] Selection of UPLC-MS / MS conditions

[0036] Chromatographic conditions: A Thermo Ultimate 3000 ultra-high performance liquid chromatograph (Thermo Fisher Scientific) and a Phenomenex Kinetex C18 (100×3 mm, 2.6 μm) column were used for chromatographic separation. The column temperature was 40 °C. The mobile phases were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), with the following gradients: 0–2 min, 2% B; 5–7 min, 2%–95% B; 7–7.5 min, 95%–2% B; 7.5–11 min, 2% B. The flow rate was 0.2 mL / min, and the injection volume was 5 μL.

[0037] Mass spectrometry conditions: TSQ Altis high-resolution mass spectrometer (Thermo Fisher Scientific), H-ESI ion source, MRM scanning mode, source region parameters: positive ion source voltage 3500V, negative ion source voltage 2800V, capillary temperature 325℃, heating temperature 325℃; sheath gas 30Arb, auxiliary gas 10Arb, backflush gas 0, nitrogen was used as the collision gas and the damping gas for the C-trap. Optimized parameters for the MRM scan are shown in Table 1.

[0038] Table 1. Basic chemical parameters of the target neurotransmitter, such as LogP and PKa, and optimization conditions of MRM.

[0039]

[0040]

[0041] Example 1: Preparation of SPME Fibers

[0042] (1) Preparation of zwitterionic covalent organic framework (EB-Da-COF) materials

[0043] Accurately weigh ethidium bromide (0.3 mmol), 2,5-diaminobenzoic acid (0.3 mmol), and trialdehyde phloroglucinol (0.45 mmol), mix them, and then add 5 mL of a mixed solution of 1,3,5-trimethylbenzene and 1,4-dioxane (2:3, v / v), followed by 0.5 mL of 6M acetic acid solution. The mixture is then sonicated for 10 min to achieve uniform dispersion. Subsequently, the mixture is degassed using a three-stage refrigeration pump-thawing cycle and sealed, and reacted at 120 °C for 72 h. After the reaction, the mixture is cooled to room temperature, centrifuged to collect the precipitate, and washed alternately with tetrahydrofuran, deionized water, and acetone, repeated three times. Finally, it is dried in a vacuum drying oven at 80 °C for 12 h to obtain a deep red zwitterionic covalent organic framework material (yield approximately 90%).

[0044] In addition, the preparation steps of Da-COF are as follows: accurately weigh 2,5-diaminobenzoic acid (0.6 mmol) and trialdehyde phloroglucinol (0.45 mmol), mix them, and the subsequent preparation method is the same as that of EB-Da-COF.

[0045] The preparation steps of EB-COF are as follows: accurately weigh ethidium bromide (0.6 mmol) and trialdehyde phloroglucinol (0.45 mmol), mix them, and the subsequent preparation method is the same as that of EB-Da-COF.

[0046] (2) Preparation of fiber filaments

[0047] At room temperature, 0.3 mm diameter nickel-titanium fibers were cut into 6.5 cm long metal filaments. The cut ends of the filaments were then polished with a grinding wheel to ensure a smooth finish, which is beneficial for subsequent coating with adsorbent materials. After cutting and polishing, the filaments were repeatedly washed three times with a mixed solution of methanol, isopropanol, and water in a volume ratio of 3:3:4 to remove surface grease and stains. After washing, the filaments were dried in a 60°C oven for later use.

[0048] (3) Preparation of polyacrylonitrile (PAN) adhesive

[0049] Add 5.0g of PAN to a 100mL wide-mouth bottle with a cap, add 72.5mL of N,N-dimethylformamide, and then stir with a glass rod to disperse the PAN evenly. Cover and shake to mix thoroughly. Seal and place in a 90℃ oven for 1 hour. Release the gas every 15 minutes.

[0050] (4) Preparation of SPME fibers

[0051] Weigh 325.0 mg of ionic covalent organic framework material into a 5 mL capped glass bottle, add a magnetic stir bar, add 3.1 g of PAN adhesive and 0.2 g of glycerol, cap and vortex for 10 min, then magnetically stir overnight. SPME fibers were prepared using a sol-gel coating method, repeatedly coating the fibers with a dip-coating machine until the desired coating thickness was achieved (4.5 mm length, 10 μm thickness). The prepared SPME fibers were sealed and stored for later use. Before use, the fibers were activated with a 1:1 methanol-water mixture for 30 min.

[0052] Example 2: Preparation of SPME Fibers

[0053] (1) Preparation of zwitterionic covalent organic framework material (EB-Da-COF)

[0054] Accurately weigh ethidium bromide (0.3 mmol), 2,5-diaminobenzoic acid (0.3 mmol), and trialdehyde phloroglucinol (0.3 mmol), mix them, then add 3.5 mL of a mixed solution of 1,3,5-trimethylbenzene and 1,4-dioxane (2:3, v / v), followed by 0.5 mL of 6M acetic acid solution. The mixture is then sonicated for 10 min to achieve uniform dispersion. Subsequently, the mixture is degassed using a three-stage refrigeration pump-thawing cycle and sealed, and reacted at 110 °C for 60 h. After the reaction is complete, the precipitate is cooled to room temperature, collected by centrifugation, and washed alternately with tetrahydrofuran, deionized water, and acetone, repeated three times. Finally, it is dried in a vacuum drying oven at 80 °C for 12 h to obtain deep red EB-Da-COF (yield approximately 90%).

[0055] (2) Preparation of fiber filaments

[0056] At room temperature, 0.3 mm diameter nickel-titanium fibers were cut into 6.5 cm long metal filaments. The cut ends of the filaments were then polished with a grinding wheel to ensure a smooth finish, which is beneficial for subsequent coating with adsorbent materials. After cutting and polishing, the filaments were repeatedly washed three times with a mixed solution of methanol, isopropanol, and water in a volume ratio of 3:3:4 to remove surface grease and stains. After washing, the filaments were dried in a 60°C oven for later use.

[0057] (3) Preparation of polyacrylonitrile (PAN) adhesive

[0058] Add 4.5g of PAN to a 100mL wide-mouth bottle with a cap, add 72.5mL of N,N-dimethylformamide, and then stir with a glass rod to evenly disperse the PAN. Cover and shake to mix thoroughly. Seal and place in a 90℃ oven for 1 hour. Release the gas every 15 minutes.

[0059] (4) Preparation of SPME fibers

[0060] Weigh 300.0 mg of ionic covalent organic framework material into a 5 mL capped glass bottle, add a magnetic stir bar, add 2.5 g of PAN adhesive and 0.15 g of glycerol, cap and vortex for 10 min, then magnetically stir overnight. SPME fibers were prepared using a sol-gel coating method, repeatedly coating the fibers with a dip-coating machine until the desired coating thickness was achieved (3.5 mm in length and 15 μm in thickness). The prepared SPME fibers were then sealed and stored for later use. Before use, the fibers were activated with a 1:1 methanol-water mixture for 30 min.

[0061] Example 3: Preparation of SPME Fibers

[0062] (1) Preparation of zwitterionic covalent organic framework material (EB-Da-COF)

[0063] Accurately weigh ethidium bromide (0.3 mmol), 2,5-diaminobenzoic acid (0.3 mmol), and trialdehyde phloroglucinol (0.45 mmol), mix them, and then add 5 mL of a mixed solution of 1,3,5-trimethylbenzene and 1,4-dioxane (2:3, v / v). Next, add 0.5 mL of 6M acetic acid solution and sonicate the mixture for 10 min to achieve uniform dispersion. Subsequently, degas the mixture using a three-stage refrigeration pump-thawing cycle and seal it. React at 120 °C for 72 h. After the reaction is complete, cool the precipitate to room temperature, collect it by centrifugation, and wash it alternately with tetrahydrofuran, deionized water, and acetone, repeating this process three times. Finally, dry the precipitate in a vacuum drying oven at 80 °C for 12 h to obtain a deep red EB-Da-COF (yield approximately 90%).

[0064] (2) Preparation of fiber filaments

[0065] At room temperature, 0.3 mm diameter nickel-titanium fibers were cut into 6.5 cm long metal filaments. The cut ends of the filaments were then polished with a grinding wheel to ensure a smooth finish, which is beneficial for subsequent coating with adsorbent materials. After cutting and polishing, the filaments were repeatedly washed three times with a mixed solution of methanol, isopropanol, and water in a volume ratio of 3:3:4 to remove surface grease and stains. After washing, the filaments were dried in a 60°C oven for later use.

[0066] (3) Preparation of polyacrylonitrile (PAN) adhesive

[0067] Add 5.0g of PAN to a 100mL wide-mouth bottle with a cap, add 72.5mL of N,N-dimethylformamide, and then stir with a glass rod to evenly disperse the PAN. Cover and shake to mix thoroughly. Seal and place in a 90℃ oven for 1 hour. Release the gas every 15 minutes.

[0068] (4) Preparation of SPME fibers

[0069] Weigh 310.0 mg of ionic covalent organic framework material into a 5 mL capped glass bottle, add a magnetic stir bar, add 3.0 g of PAN adhesive and 0.2 g of glycerol, cap and vortex for 10 min, then magnetically stir overnight. SPME fibers were prepared using the sol-gel coating method, repeatedly coating the fibers with a dip-coating machine until the desired coating thickness was achieved (6.5 mm in length and 5 μm in thickness). The prepared SPME fibers were then sealed and stored for later use. Before use, the fibers were activated with a 1:1 methanol-water mixture for 30 min.

[0070] Example 4: Characterization of zwitterionic covalent organic framework solid-phase microextraction fiber coating

[0071] The morphology of the zwitterionic covalent organic framework solid-phase microextraction fiber coating prepared in Example 1 was characterized using scanning electron microscopy. Figure 1 As shown, these are zwitterionic covalent organic framework materials ( Figure 1 A), Local surface observation of the prepared zwitterionic covalent organic framework solid-phase microextraction fiber ( Figure 1 Cross-sectional view of B and 1C) and zwitterionic covalent organic framework solid-phase microextraction fiber coating ( Figure 1 D). From Figure 1 As can be seen from A, the zwitterionic covalent organic framework is composed of numerous loose, linear villous structures, whose loose structural characteristics provide it with a large specific surface area and abundant porosity. From Figure 1 As can be seen from B and 1C, the prepared extraction fiber has a uniformly rough surface, and the coating is evenly distributed on the surface, exhibiting a porous structure of varying sizes. Figure 1 D indicates that the coating thickness is uniform, which is beneficial for increasing the specific surface area of ​​the coating to form more adsorption sites. It also facilitates the internal mass transfer between the target analyte and the coating material, and helps the rapid adsorption and desorption of neurotransmitter molecules.

[0072] Elemental analysis was performed on the C, N, and O elements in the zwitterionic covalent organic framework material. The results are as follows: Figure 2 As shown, C, N, and O elements are uniformly distributed in the covalent organic framework material, with contents of 74.64%, 10.87%, and 14.49%, respectively, indicating that the covalent organic framework material was successfully prepared.

[0073] The functional groups of EB-Da-COF were analyzed using Fourier transform infrared spectroscopy (FT-IR). The results are as follows: Figure 3 As shown, a position at 3295 cm⁻¹ was observed in the infrared spectra of both 2,5-diaminobenzoic acid (Da) and ethidium bromide (EB). -1and 3158cm -1 The characteristic absorption peak of -NH2 near the wavelength is different; in contrast, a peak at 3387 cm⁻¹ appears in the infrared spectrum of 2,5-diaminobenzoic acid. -1 An absorption peak appears at wavelength due to the stretching vibration of the OH group in -COOH, and at 1148 cm⁻¹. -1 The CO stretching vibration absorption peak near the wavelength and located at 671 cm⁻¹ -1 The absorption peak of the C=O bending vibration at a specific wavelength appears in the infrared spectrum of ethidium bromide at 1404 cm⁻¹. -1 The characteristic peak of quaternary ammonium at wavelength and 1256 cm⁻¹ -1 CN absorption characteristic peak at 1075 cm⁻¹ and at 1075 cm⁻¹ -1 Absorption peaks from the methyl bending vibration of ethidium bromide near the wavelength. In the infrared spectrum of trialdehyde phloroglucinol (TP), at 2895 cm⁻¹. -1 The characteristic absorption peak of HC=O in the aldehyde group appears near the wavelength and at 1645 cm⁻¹. -1 The C=O absorption peak is located near the wavelength. Unlike ethidium bromide, 2,5-diaminobenzoic acid, and trialdehyde phloroglucinol, it is located at 3295 cm⁻¹ in the EB-Da-COF infrared spectrum. -1 and 3158cm -1 The characteristic absorption peak of -NH2 near the wavelength and at 1645 cm⁻¹ -1 The characteristic peak of the C=O absorption peak near the wavelength disappears, and at 1577 cm⁻¹... -1 A new C=N absorption peak appeared near the wavelength, indicating the formation of an imine bond. Furthermore, a peak was observed at 1256 cm⁻¹. -1 CN absorption characteristic peak at 1075 cm⁻¹ and at 1075 cm⁻¹ -1 The absorption peak of the methyl bending vibration near the wavelength and the peak formed by the C=O bending vibration of the carboxyl group at 671 cm⁻¹ -1 The weak peak indicates that EB-Da-COF was successfully synthesized via a Schiff base reaction between 2,5-diaminobenzoic acid, ethidium bromide, and trialdehyde phloroglucinol.

[0074] The elemental composition of EB-Da-COF was analyzed using XPS. The results are as follows: Figure 4 As shown in Figure A, the ionic covalent organic framework contains C, N, and O elements. (C1s spectrum) Figure 4 In spectrum B), characteristic peaks for CN, CO, C=N, and CC bonds appeared at 287.63 eV, 285.79 eV, 284.77 eV, and 284.12 eV, indicating the presence of imine bonds and a Schiff base reaction between monomers. In the N1s spectrum (… Figure 4In C), characteristic peaks of C=N bonds also appear at 399.43 eV and 399.03 eV, and a quaternary ammonium group (-NR4) appears at 400.92 eV. + The characteristic peaks indicate the presence of ethidium bromide in EB-Da-COF. In the O1s spectrum ( Figure 4 In D), a significant peak appears at 531.89 eV, formed by the carbonyl C=O / OC=O bond in -COOH, further demonstrating the successful introduction of 2,5-diaminobenzoic acid into EB-Da-COF. In summary, the FT-IR results further confirm the successful synthesis of the ionic covalent organic framework.

[0075] Since EB-Da-COF has abundant hydrophilic groups such as -OH and -COOH, it is speculated that it may have a certain degree of hydrophilicity. Figure 5 The contact angle results are shown; the water contact angle of EB-Da-COF is 50.0°, indicating that EB-Da-COF has good hydrophilicity. This good hydrophilicity makes EB-Da-COF beneficial for the extraction and enrichment of highly polar neurotransmitters (NTs).

[0076] The specific surface area and pore properties of EB-Da-COF were determined using the Brunauer-Emmett-Teller (BET) gas-phase adsorption method. The results are as follows: Figure 6 As shown, the N2 adsorption isotherm of EB-Da-COF is basically a type IV curve, indicating that EB-Da-COF mainly has a mesoporous structure with a pore size ranging from 2 to 50 nm. According to the pore size distribution measured by the BJH method, its average pore size is 2.27 nm, indicating a relatively narrow particle size distribution. Furthermore, the specific surface area and pore volume of EB-Da-COF are 183.26 m² / m³. 2 / g and 0.36cm 3 The high specific surface area and mesoporous structure of EB-Da-COF give it good adsorption performance and protein exclusion effect.

[0077] Example 5: Investigation on the extraction and enrichment effect of SPME fibers on neurotransmitters of different polarities

[0078] To investigate the extraction efficiency of the prepared EB-COF, Da-COF, and EB-Da-COF with three commercially available adsorbents (HLB, PXC, and C18) SPME fibers for target neurotransmitters, the extraction conditions were as follows: the prepared SPME fibers were immersed in PBS buffer containing a certain concentration of neurotransmitters (γ-aminobutyric acid GABA, glutamine Gln, glutamate Glu, choline Cho, glycine Gly, tyrosine Tyr, threonine Thr, ornithine Orn, serine Ser, tryptophan Try, methionine Met, arginine Arg, serotonin 5-HT, dopamine DA, thyroid-stimulating hormone TSH, histidine His, adrenaline E, norepinephrine NE, and melatonin 5-MT), and extracted with shaking for 30 min, followed by desorption for 30 min. UPLC-MS / MS data were then collected and analyzed, with each fiber sample repeated three times. The extraction efficiency of different coatings was evaluated by analyzing the amount and number of analytes extracted by the fibers using UPLC-MS / MS data. The results were normalized to the optimal extraction coating for each analyte and are expressed as a percentage. Results are as follows: Figure 7 As shown in Figure A, the prepared ion-exchange EB-Da-COF exhibits significantly higher extraction efficiency and a wider extraction coverage compared to EB-COF and Da-COF developed with other monomer combinations; for example... Figure 7 As shown in B, compared with fibers coated with commercial adsorbents (HLB, PXC and C18), the prepared EB-Da-COF fibers have higher adsorption efficiency and coverage for these polar compounds, which is related to its multiple adsorption forces such as anion exchange, cation exchange, π-π stacking, hydrogen bonding and hydrophilic-hydrophobic interactions.

[0079] Example 6: Investigation of Macromolecular Size Exclusion Properties

[0080] The protein exclusion properties of the ionic covalent organic framework material and the ionic covalent organic framework solid-phase microextraction fiber prepared in Example 1 were studied. A specific case was as follows: 2 mg of EB-Da-COF material and 5 SPME fibers were added to or immersed in mouse serum containing a selected protein at a concentration of 100 μg / mL and diluted with PBS buffer. The mixture was then shaken at 500 rpm for 2 h at room temperature. Finally, the EB-Da-COF material and SPME fibers were separated. The concentration change of macromolecular proteins in the supernatant was analyzed at 280 nm using a UV spectrophotometer. The protein exclusion rate was calculated as: Protein exclusion rate = Cs / C0 × 100%, where Cs is the concentration of the remaining macromolecules in the solution after adsorption by the EB-Da-COF material, and C0 is the initial concentration in the solution. First, albumin, which accounts for about 50% of total plasma protein, was selected as the exclusion target. The exclusion rates of EB-Da-COF and SPME fibers were investigated when the albumin concentration was 100 μg / mL, 200 μg / mL, and 300 μg / mL.

[0081] The results are as follows Figure 8 As shown in Figure A, the protein exclusion rates of EB-Da-COF and SPME fibers remained above 87% even with increasing albumin concentration. Furthermore, considering the complexity of the serum matrix, the exclusion rates of EB-Da-COF and SPME fibers were also investigated using hemoglobin, β-lactoglobulin, and mouse serum at concentrations of 100 μg / mL. The results are as follows... Figure 8 As shown in Figure B, the exclusion efficiency of EB-Da-COF and SPME fibers for the two selected proteins and serum samples remained above 90%. These results indicate that EB-Da-COF and SPME fibers can significantly reduce matrix interference from macromolecules such as proteins, and can be directly used for the extraction and analysis of NTs in complex biological samples.

[0082] Example 7: Evaluation of the reusability of SPME fibers

[0083] The repeatability and practicality of the zwitterionic covalent organic framework solid-phase microextraction fiber coating prepared in Example 1 are investigated, with specific examples as follows:

[0084] SPME fibers were subjected to repeated adsorption-desorption cycles targeting Tyr, Glu, Cho, DA, 5-MT, and His. After each extraction cycle, the SPME fibers were washed sequentially with 1% FA+H2O / MeOH / CAN (40:30:30, v / v / v), MeOH (methanol), CAN (acetonitrile), and water. The results are as follows: Figure 9As shown, after 7-8 cycles, a significant decrease in the strength of the target compounds was observed. The relative standard deviations of the relative strengths of the six target compounds after 8 cycles were 3.29% (Tyr), 0.14% (Glu), 0.18% (Cho), 1.09% (DA), 1.30% (5-MT), and 6.15% (His), respectively. This indicates that the adsorption efficiency of SPME fibers gradually decreases with increasing usage, suggesting that SPME fibers can be reused within 8 cycles. Furthermore, with increasing usage, PAN swelling due to repeated contact with organic solvents was observed in the SPME fibers. These results indicate that the reusability of SPME is inseparable from the stability of the EB-Da-COF structure.

[0085] Example 8: Validation of SPME method for detecting and analyzing neurotransmitters in serum samples

[0086] The following is a specific example of applying the ionic covalent organic framework solid-phase microextraction fiber from Example 1 to the determination of neurotransmitters in mouse serum samples.

[0087] 200 μL of mouse serum sample was diluted to 1 mL with PBS buffer and placed in a vial. The coated end of the prepared SPME fiber was then immersed in the diluted serum sample and extracted by shaking for 30 min. The SPME fiber was then washed with ultrapure water for 10 s to remove surface inorganic salts and insoluble contaminants. Subsequently, the neurotransmitters adsorbed on the SPME fiber were desorbed by shaking with 100 μL of a mixed solvent of 1% FA (formic acid) + H2O / ACN / MeOH (4:3:3, v / v / v). After desorption for 10 min, the SPME fiber was removed, and the eluent was analyzed by UPLC-MS / MS.

[0088] The established detection and analysis method was validated through the above operations. The results are shown in Tables 2 and 3. This method has low limits of detection and quantitation for 19 NTs and exhibits good linear correlation (r). 2 All values ​​were greater than 0.99. The inter-day and intra-day RSDs ranged from 2.29% to 16.38%, and the matrix effect ranged from 0.16% to 19.93%. At all three spiking concentration levels, the precision was less than 20%, and the accuracy ranged from 80% to 120%. The data results show that the SPME fiber coating combined with ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) of this invention exhibits good accuracy and precision in the extraction and detection of neurotransmitters in serum. This demonstrates the application of the ion-type covalent organic framework solid-phase microextraction fiber-ultra-high performance liquid chromatography-tandem mass spectrometry method in the detection of neurotransmitters of different polarities in serum, and it shows good application results.

[0089] Table 2. Methodological parameters for the detection of serum NTs by ionic covalent organic framework solid-phase microextraction-ultra-high performance liquid chromatography-tandem mass spectrometry.

[0090]

[0091]

[0092] "*" and "#" represent the concentration units of μg / mL and ng / mL, respectively. "x" and "y" represent the concentration of the target substance and the detected peak area, respectively.

[0093] Table 3. Precision and accuracy of ionic covalent organic framework solid-phase microextraction-ultra-high performance liquid chromatography-tandem mass spectrometry for NTs in serum.

[0094]

[0095] "*" represents the low, medium, and high spiking concentrations of 1, 25, and 50 μg / mL, respectively; "#" represents the low, medium, and high spiking concentrations of 5, 25, and 100 ng / mL, respectively.

[0096] Example 9: SPME fibers for in-situ collection and analysis of neurotransmitters in mouse brains

[0097] The following is a specific example of applying the ionic covalent organic framework solid-phase microextraction fiber from Example 1 to in-situ sampling and analysis of neurotransmitters in the mouse brain:

[0098] Method validation in brain alternative matrix: First, agar was dissolved in PBS buffer to prepare an agar gel (2% agar, w / v). Then, sheep brain was thawed, and an appropriate amount of sheep brain was placed in a 2mL grinding tube. Clean stainless steel beads were added, and the sheep brain sample was heated to 50Hz using TissueMaster. TM Brain homogenate was obtained by homogenizing in a high-throughput tissue homogenizer for 2 minutes. Agarose gel was then mixed with the sheep brain homogenate at a 1:1 (w / v) ratio to obtain a brain substitute matrix. Methodological validation was performed by adding different concentrations of target neurotransmitters to 500 μL of the substitute matrix.

[0099] The established detection and analysis method was validated through the above operations. Tables 4 and 5 show the methodological parameters, precision, and accuracy results of SPME fibers for neurotransmitter analysis in mouse brains, respectively. The data (Table 4) show that all analytes exhibit good linearity (r0.05) in the replacement brain matrix. 2The method exhibits a high RSD (>0.97) and a low limit of quantitation. By analyzing six samples of the same concentration consecutively within a single day, the RSD% ranged from 6.10% to 14.87%. The matrix effect was examined by extracting and analyzing blank substitute brain matrix supplemented with analytes, and comparing the signals of each analyte at three concentration levels in a solvent (1% FA+H2O / ACN / MeOH (4:3:3, v / v / v)). The matrix effect fluctuated between 80.20% and 119.48%. This indicates that the method can be applied to in-situ sampling analysis of neurotransmitters and can be used in the determination of actual samples. The precision of the method was assessed by repeated measurements at three concentration levels using RSD, and the results are shown in Table 5. The precision ranged from 2.51% to 19.68%, and the accuracy ranged from 80.37% to 122.07%. The above results indicate that fiber coating combined with ultra-high performance liquid chromatography-mass spectrometry has good accuracy and precision for the extraction and detection of neurotransmitters in brain tissue, suggesting that this method is applicable to the in-situ extraction and analysis of NTs in brain tissue.

[0100] Table 4. Methodological parameters for the detection of neurotransmitters in brain tissue using solid-phase microextraction-ultra-high performance liquid chromatography-tandem mass spectrometry with ionic covalent organic frameworks.

[0101]

[0102]

[0103] "*" and "#" represent the concentration units of μg / mL and ng / mL, respectively. "x" and "y" represent the concentration of the target substance and the detected peak area, respectively.

[0104] Table 5. Precision and accuracy of solid-phase microextraction-ultra-high performance liquid chromatography-tandem mass spectrometry for the detection of neurotransmitters in brain tissue using ionic covalent organic frameworks.

[0105]

[0106] "*" represents the low, medium, and high spiked concentrations of 50, 500, and 1000 μg / mL, respectively; "#" represents the low, medium, and high spiked concentrations of 50, 500, and 1000 ng / mL, respectively.

[0107] Example 10: In-situ extraction and analysis of neurotransmitters in different brain regions of mice using SPME combined with HPLC-MS / MS.

[0108] SPME in situ sampling analysis: Using the numbers marked on the coronal sections of the mouse brain on a slide mold (brain) (coronal section, suitable for mouse brain, 1 mm thickness), and in conjunction with mouse brain atlases, the regions of the mouse brain were determined. The intersection of the inferior sulcus (number 4) and the midsagittal line was used as the location of the anterior fontanelle, dividing the mouse brain into seven regions. After fixing the mouse brain in the mold, SPME in situ static sampling analysis was performed on the prefrontal cortex region (3-4), hippocampal region (6-7), and cerebellar region (9-11). While the mouse brain was not completely thawed, the solid-phase microextraction fiber coating was inserted into the positioned mouse brain tissue, with the depth adjusted appropriately. Static extraction was performed for 40 min, and the fibers were removed. Brain tissue and other debris on the surface of the solid-phase microextraction fibers were wiped away with lint-free paper, and then the fibers were rinsed with mass spectrometry-grade water for 10 s. Neurotransmitters adsorbed on SPME fibers were desorbed by shaking using 100 μL of 1% FA+H2O / ACN / MeOH (4:3:3, v / v / v) mixed solvent. After desorption for 10 min, the SPME fibers were removed, and the elution solution was analyzed by UPLC-MS / MS.

[0109] Using the established method, the levels of various neurotransmitters in the prefrontal cortex, hippocampus, and cerebellum of normal mice and mice with a depression model were tested. The specific results are shown in Table 6. Compared with the normal control group, the concentration levels of neurotransmitters in different brain regions of the depression model group were mostly decreased. Analysis of the concentrations of neurotransmitters in the prefrontal cortex, hippocampus, and cerebellum showed that most of these neurotransmitters were concentrated around the hippocampus, followed by the prefrontal cortex of the mouse brain.

[0110] Table 6. Results of neurotransmitter analysis in different brain regions of mice.

[0111]

[0112] Example 11: Analysis of neurotransmitters in actual mouse serum samples using SPME combined with UPLC-MS / MS

[0113] 200 μL of mouse serum sample was diluted to 1 mL with PBS buffer and placed in a vial. The coated end of the prepared SPME fiber was then immersed in the diluted serum sample and extracted by shaking for 30 min. The SPME fiber was then washed with ultrapure water for 10 s to remove surface inorganic salts and insoluble contaminants. Subsequently, the neurotransmitters adsorbed on the SPME fiber were desorbed by shaking with 100 μL of a 1% FA+H2O / ACN / MeOH (4:3:3, v / v / v) mixed solvent for 10 min. The SPME fiber was then removed, and the eluent was analyzed by UPLC-MS / MS. Using the established method, the levels of various neurotransmitters in the serum of normal mice and mice with a depression model were tested. The specific results are shown in Table 7. As shown in Table 7, when comparing the NTs in the serum of mice in each group, the concentration levels of 19 neurotransmitters in the serum of the depression model mice were significantly lower than those in the normal group, except for a few neurotransmitters whose levels were abnormal due to individual differences among mice. This was especially true for monoamine neurotransmitters, which is consistent with the monoamine deficiency hypothesis associated with depression.

[0114] Table 7. Results of neurotransmitter analysis in actual mouse serum samples.

[0115]

[0116] The foregoing has illustrated and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. All such changes and modifications fall within the scope of the claimed invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

[0117] The above description is merely a preferred embodiment of the present invention. All equivalent variations and modifications made in accordance with the claims of this invention should fall within the scope of this invention.

Claims

1. Use of an ionic covalent organic framework material in the preparation of a sorbent for sample pre-treatment, characterized in that, The sample pre-treatment includes extraction analysis of neurotransmitters in serum samples and brain tissues; the ionic covalent organic framework material is prepared by Schiff base reaction condensation with ethiodide, 2,5-diaminobenzoic acid and tricarboxyphloroglucinol as functional monomers, and acetic acid as a catalyst.

2. Use according to claim 1, characterized in that, The specific preparation method of the ionic covalent organic framework material is as follows: accurately weighing ethiodide, 2,5-diaminobenzoic acid and tricarboxyphloroglucinol, mixing, then adding a reaction solvent, and then adding an acetic acid solution and ultrasonic dispersion; then, degassing and sealing through a freeze-pump-thaw cycle, and then reacting; after the reaction is completed, cooling to room temperature, centrifugal collection of the precipitate, and drying after washing.

3. Use according to claim 2, characterized in that, The molar ratio of the ethiodide, 2,5-diaminobenzoic acid and tricarboxyphloroglucinol is 0.8-1.2:0.8-1.2:1.2-1.

8.

4. Use according to claim 2, characterized in that, The concentration of the acetic acid solution is 6 M, and the ratio of the acetic acid solution to tricarboxyphloroglucinol is 0.5 mL:0.45 mmoL.

5. Use according to claim 2, characterized in that, The reaction temperature is 110-120 DEG C, the time is 60-72 hours, and the reaction solvent is a mixed solution of mesitylene and 1,4-dioxane in a volume ratio of 2:

3.

6. Use of an ionizable covalent organic framework solid-phase microextraction fiber as a sorbent in sample pre-treatment, characterized in that, The sample pre-treatment includes extraction analysis of neurotransmitters in serum samples and brain tissues; the fiber is prepared by layer-by-layer self-assembly through a sol-gel coating method, and the ionic covalent organic framework material is coated on the fiber to obtain an ionic covalent organic framework solid-phase microextraction fiber.

7. Use according to claim 6, characterized in that, The preparation of the ionic covalent organic framework solid-phase microextraction fiber specifically includes the following steps: (1) preparation of PAN glue: 4.5-5.0 g of PAN and 72.5 mL of N,N-dimethylformamide are mixed, stirred uniformly, and sealed and placed; (2) 300.0-325.0 mg of the ionic covalent organic framework material is weighed, then 2.5-3.1 g of PAN glue and 0.15-0.2 g of glycerol are added, vortexed, and then stirred overnight to obtain an ionic covalent organic framework solid-phase microextraction fiber coating; (3) the ionic covalent organic framework solid-phase microextraction fiber coating is coated on the fiber to obtain an ionic covalent organic framework solid-phase microextraction fiber.

8. Use according to claim 7, characterized in that, In step (1), the temperature for sealing and placing is 90 DEG C, and the time is 1 h.

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