Solid-phase microextraction probe as well as preparation method and application thereof

Abstract

The invention relates to a solid-phase microextraction probe as well as a preparation method and application thereof. The solid phase microextraction probe comprises a probe carrier and an extraction coating coated on the probe carrier, the extraction coating is a COF-DT material, and the COF-DT material is prepared by connecting 2, 5-dihydroxy terephthalaldehyde (Dha) and 1, 3, 5-tris (4-aminophenyl) benzene (Tab) through an imine bond. The probe disclosed by the invention shows excellent effect and repeatability in the aspect of adsorbing the neurotransmitter, and can be combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) equipment for use, so that the analysis of the neurotransmitter in a wide concentration range is realized.

Description

Technical Field

[0001] The invention relates to the technical field of solid phase microextraction, and in particular to a solid phase microextraction probe and a preparation method and application thereof. Background Art

[0002] Neurotransmitters (NTs) are a class of endogenous chemicals that, as key signaling molecules in the human body, play a vital role in the balance and function of brain tissue. They play a central role in maintaining the normal functioning of the nervous system, regulating emotions, and influencing cognitive functions. Unfortunately, a variety of central nervous system diseases, such as Parkinson's disease, Alzheimer's disease, and Huntington's disease, are often closely related to abnormal neurotransmitter levels. Such abnormalities not only lead to dysfunction of the nervous system, but may also trigger a series of complex pathological changes. Therefore, accurate neurotransmitter detection in brain tissue is not only crucial for understanding the pathological mechanisms of these diseases, but also provides key information for clinical treatment. However, due to the wide variety of neurotransmitters and their low concentrations, coupled with the complexity of brain tissue, their accurate detection faces great challenges. Therefore, the development of efficient and sensitive detection methods is of great significance for early diagnosis, disease monitoring, and the formulation of treatment strategies.

[0003] When analyzing neurotransmitters in brain tissue, the key challenge lies in dealing with the complexity of the sample, especially the multiple compounds contained in brain tissue. Although traditional sample pretreatment methods such as liquid-liquid extraction (LLE), solid phase extraction (SPE), and dispersive liquid-liquid microextraction (DLLME) are effective in some cases, they have limitations when dealing with brain tissue samples with limited volume. In contrast, solid phase microextraction (SPME) technology, with its advantages of rapid sampling and low sample demand, significantly simplifies the sample preparation process and is suitable for the extraction of endogenous compounds in complex biological matrices. However, the challenge of SPME technology is that the existing coatings have limited protein exclusion capabilities, which may hinder adsorption efficiency. Therefore, the development of SPME coatings with higher selectivity and effective reduction of protein interference is crucial to ensure accurate and reliable neurotransmitter analysis.

[0004] Covalent organic frameworks (COFs), as an innovative class of porous crystalline materials, have attracted much attention due to their huge specific surface area, high permanent porosity, good thermochemical stability, and multifunctional modification potential. The unique structure of COFs, such as hydrogen bonding, π-π stacking, and hydrophilic-hydrophobic balance, endows them with excellent interaction capabilities with target analytes, especially in adsorption and separation applications. In recent years, COFs have been significantly applied as solid phase microextraction (SPME) coating materials in the field of bioseparation, especially their ordered and tunable mesoporous structures have shown great potential in reducing macromolecular interference in complex biological samples. However, there are currently few studies that combine COFs with nanopores with SPME technology for direct application in the analysis of neurotransmitters in biological brain tissue samples. Summary of the invention

[0005] The present invention uses specially designed COFs materials combined with SPME technology to enhance the selective adsorption capacity of neurotransmitters and improve the accuracy and stability of detection. This combination not only improves the efficiency of neurotransmitter detection, but also provides a new way to accurately identify and separate complex substances in biological samples. In addition, this innovative method has shown significant creativity and application value in the field of neurotransmitter detection in biological brain tissue samples, opening up new research and application prospects. This groundbreaking combination not only proves the great potential of COFs in biological sample analysis, but also provides new directions and inspiration for future scientific research.

[0006] The present invention provides a solid phase microextraction probe and a preparation method and use thereof. The present invention utilizes the nanoscale pore size of COF-DT material combined with solid phase microextraction technology to achieve direct and effective analysis of mouse brain tissue homogenate. The probe has the ability to efficiently and accurately detect trace neurotransmitters in brain tissue samples, providing an important analytical tool for neuroscience research. This innovation not only improves the efficiency of sample processing, but also ensures the accuracy of the analysis results.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] On the one hand, the present invention provides a solid phase microextraction probe, which includes a probe carrier and an extraction coating coated on the probe carrier, wherein the extraction coating is a COF-DT material, and the COF-DT material is made of 2,5-dihydroxyterephthalaldehyde (Dha) and 1,3,5-tri(4-aminophenyl)benzene (Tab) connected by an imine bond.

[0009] Preferably, the pore size of the COF-DT material is 4.7 nanometers. The unique composition of the probe of the present invention enables it to exhibit excellent performance in the detection of neurotransmitters at trace levels, greatly improving the sensitivity and accuracy of the detection.

[0010] Preferably, the coating has a thickness of 100 microns.

[0011] Preferably, the length of the coating is 1 cm. The present invention adopts a specific coating thickness and coating length to ensure efficient sample adsorption and release.

[0012] Preferably, the probe carrier is a stainless steel fiber. The stainless steel fiber of the present invention has excellent mechanical strength and chemical stability, ensuring the uniformity and durability of the coating.

[0013] On the other hand, the present invention provides a method for preparing the above-mentioned solid phase microextraction probe, which comprises: first coating a layer of adhesive solution on the surface of the probe carrier, and then adhering the powder of COF-DT material to the end of the probe carrier coated with the adhesive solution, and drying.

[0014] Preferably, the end of the probe carrier coated with the adhesive solution is dipped with the powder of the COF-DT material to a length of 1 cm.

[0015] Preferably, the adhesive solution is a cyclohexane solution of neutral silicone glue or a polyacrylonitrile (PAN) solution with N-dimethylformamide (DMF) as a dispersant, with a concentration of 200 mg / g.

[0016] Preferably, the drying is oven drying at a temperature of 120 degrees for 40 minutes.

[0017] Preferably, the preparation method can be repeated multiple times, for example, repeated twice.

[0018] Preferably, the preparation method also includes a step of pre-treating the probe carrier, and the pre-treatment method is as follows: the probe carrier is ultrasonically cleaned using superacetone, deionized water and methanol in sequence; then, it is immersed in aqua regia solution for surface roughening treatment to enhance its adhesion to the adhesive and COF-DT material; then, it is thoroughly rinsed with deionized water until the pH value is neutral, and dried under a nitrogen flow.

[0019] Preferably, the preparation method further comprises the step of subjecting the powder of the COF-DT material to high temperature aging of the extracted coating after drying.

[0020] In another aspect, the present invention provides a use of the above-mentioned solid phase microextraction probe in detecting neurotransmitter compounds. The probe of the present invention can effectively adsorb and separate multiple key neurotransmitters, including but not limited to acetylcholine, dopamine, etc., through its unique COF-DT material coating, thereby achieving rapid and accurate detection of neurotransmitter compounds in mouse brain tissue, and is particularly suitable for applications in neuroscience and biomedical research.

[0021] Preferably, the neurotransmitter compounds include acetylcholine (Ach), L-3,4-dihydroxyphenylalanine (DOPA), dopamine (DA), norepinephrine (NE), epinephrine (E), 3-methoxytyramine hydrochloride (3-MT), 5-hydroxytryptamine (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), glutamate (Glu), γ-aminobutyric acid (GABA), and the structure thereof is as follows: Figure 1 shown.

[0022] On the other hand, the present invention provides a method for detecting neurotransmitter compounds using the above-mentioned solid phase microextraction probe, wherein the extraction conditions adopted during the detection are: extraction time is 20 minutes, desorption time is 15 minutes, eluent is 0.1% formic acid / water (V / V), and vortex speed is 1500rpm.

[0023] Preferably, the neurotransmitter compounds include acetylcholine (Ach), L-3,4-dihydroxyphenylalanine (DOPA), dopamine (DA), norepinephrine (NE), epinephrine (E), 3-methoxytyramine hydrochloride (3-MT), 5-hydroxytryptamine (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), glutamate (Glu), and γ-aminobutyric acid (GABA).

[0024] In some specific embodiments, the solid phase microextraction probe of the present invention includes a probe carrier and an extraction coating coated on the surface of the probe carrier, wherein the extraction coating is a COF-DT material, and the COF-DT material is made by connecting 2,5-dihydroxyterephthalaldehyde (Dha) and 1,3,5-tris(4-aminophenyl)benzene (Tab) through an imine bond.

[0025] The probe carrier is made of stainless steel fiber. The solid phase microextraction coating (COF-D coating) is coated on one end surface of the stainless steel fiber. The length of the stainless steel fiber is about 3 cm and the diameter is 500 μm. The coating length of the solid phase microextraction coating on the stainless steel fiber is about 1 cm and the thickness is about 100 μm.

[0026] The method for preparing the solid phase microextraction probe comprises the following steps: coating an adhesive solution on the etched carrier surface, then adhering powder of COF-DT material on the surface of the carrier to which the adhesive solution is attached, and obtaining a solid phase microextraction coating after drying to obtain the solid phase microextraction probe. Specifically, the adhesive solution is a cyclohexane solution of neutral silicone glue. As a further preferred embodiment, the adhesive solution is prepared according to a ratio of 0.5 g of PAN dissolved in each milliliter of DMF.

[0027] The solid phase microextraction probe of the present invention is particularly suitable for analyzing neurotransmitters in brain tissue samples.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The core of the present invention is an extraction coating composed of COF-DT material, which is coated on the surface of the probe carrier; the COF-DT material gives the probe excellent hydrophilicity and macromolecular exclusion ability, while maintaining good thermal stability, thereby effectively enriching a variety of neurotransmitters in brain tissue; the best extraction effect can be achieved by optimizing parameters such as extraction time, desorption time and analytical solvent; this probe shows excellent effect and repeatability in adsorbing neurotransmitters, and can be used in combination with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) equipment to achieve neurotransmitter analysis within a wide concentration range.

[0030] The coating of the present invention has a coverage length of about 1 cm and a thickness of about 100 microns. Such coating dimensions are carefully selected to ensure optimal extraction effects, because the adsorption amount is closely related to the length and thickness of the coating.

[0031] The method for preparing the coating of the present invention is simple and easy, and does not require complicated equipment. The prepared coating not only has good thermal stability, but also shows excellent adsorption effect and good reproducibility for a variety of neurotransmitters.

[0032] The solid phase microextraction coating prepared by the present invention is used for the analysis of neurotransmitters in brain tissue homogenate, and has the advantages of good linear range, low detection limit, high recovery rate, etc. The analytical method extracts multiple neurotransmitters in brain tissue homogenate through COF-DT material, and can be used in conjunction with UPHLC-MS / MS to detect neurotransmitters, and the signal intensity generated by the neurotransmitter has a good linear relationship with its content, which can realize the direct extraction of neurotransmitters in brain tissue homogenate, as well as the analysis and detection of trace and large concentration range neurotransmitters. Compared with the prior art, its operation is simpler and the detection ability is greatly improved, which has practical application value.

[0033] In the solid phase microextraction probe, the adsorbent used in the solid phase microextraction coating is COF-DT material, which has the advantages of being rich in hydroxyl groups, having a macromolecular exclusion effect, and having good thermal stability; the solid phase microextraction coating prepared with it as a raw material has good heat resistance and good reproducibility, and has excellent adsorption effects on a variety of neurotransmitters. The present invention also provides the solid phase microextraction method, which provides a more direct and simpler NT extraction process compared to the traditional brain NT analysis method, without the need for complex protein precipitation and derivatization steps. This innovative method not only saves time, but also improves the accuracy of the analysis, making it a powerful tool for analyzing complex biological samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the structure of COF-DT solid phase microextraction coating used to enrich the target;

[0035] Figure 2 is the scanning electron microscope image of COF-DT coating;

[0036] Figure 3 Optimize the adsorption time of COF-DT solid phase microextraction coating;

[0037] Figure 4 Solvent optimization for COF-DT solid phase microextraction coating analysis;

[0038] Figure 5 Optimize the analysis time of COF-DT solid phase microextraction coating;

[0039] Figure 6 Optimization of vortex intensity for COF-DT solid phase microextraction coating analysis;

[0040] Figure 7 Total ion current of COF-DT solid phase microextraction coating for the detection of neurotransmitters in mouse brain tissue samples. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with specific examples and accompanying drawings, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

[0042] Example 1: Preparation of COF-DT materials

[0043] First, 68 mg of 1,3,5-tris(4-aminophenyl)benzene and 44 mg of 2,5-dihydroxyterephthalaldehyde were added to a glass ampoule, followed by 2 mL of 1,2-dichlorobenzene and 2 mL of n-butanol, and 0.4 mL of 8 M acetic acid aqueous solution. The mixture was sonicated for 10 minutes to achieve uniform dispersion, then rapidly frozen to 77 K in a liquid nitrogen bath, and subjected to three freeze-pump-thaw cycles to remove gas. Afterwards, the sealed ampoule was heated at 120 ° C for 3 days. The resulting precipitate was separated by centrifugation and washed with tetrahydrofuran, water and ethanol to remove any unreacted components, and finally dried at 120 ° C overnight under vacuum conditions to obtain COF-DT material (yield 90%).

[0044] Example 2: Preparation of the solid phase microextraction probe of the present invention

[0045] The preparation method of the solid phase microextraction probe of this embodiment is carried out according to the following steps:

[0046] 1) Treatment of the carrier: The stainless steel fiber was thoroughly cleaned of impurities by ultrasonic cleaning in acetone, water and methanol in sequence. Then, the cleaned fiber was etched in aqua regia at 50°C for 1 minute to produce a rough surface. After etching, the fiber was rinsed with deionized water until the pH value reached 7, and then dried in a nitrogen atmosphere at room temperature for use;

[0047] 2) Preparation of SPME coating: The stainless steel fiber treated in step 1) was immersed in a polyacrylonitrile (PAN) adhesive to a depth of about 1.0 cm, quickly removed, and then rolled in the ground COF-DT (obtained from Example 1) powder to achieve a uniform coating of about 1.0 cm in length. Subsequently, the COF-DT coated SPME fiber was cured at 120°C for 40 minutes. To ensure uniform coating, the application and curing steps of the adhesive were repeated twice, and the obtained coating thickness was uniform. The SPME coating was stored under nitrogen for future use.

[0048] 3) The microstructure of the prepared COF-DT coating was characterized by scanning electron microscopy. Figure 2 SEM images shown.

[0049] Example 3: Optimization of solid phase microextraction coating operating conditions

[0050] In this embodiment, the operating conditions of the solid phase microextraction coating made of COF-DT material when used for solid phase microextraction are optimized through experiments.

[0051] 1) Instrument information: An Agilent 1290 Infinity II UHPLC system was used, coupled to an Agilent 6495 triple quadrupole mass spectrometer equipped with a dual jet stream electrospray ionization (ESI) source.

[0052] 2) Chromatographic conditions: Separation of neurotransmitters was achieved by using an ACQUITY UPLC○R HSS PFP column (2.1 mm × 100 mm, 1.8 μm, Waters, USA) at 30°C with an injection volume of 5 μl. The mobile phase consisted of (A) water containing 0.1% (volume ratio) formic acid and (B) methanol containing 0.1% (volume ratio) formic acid, with a flow rate of 0.3 ml / min. The gradient elution program was as follows: 0-2 min, 0% B; 2-4 min, 0%-10% B; 4-6 min, 10%-12% B; 6-9 min, 12%-90% B; 9.0-10.0 min, 90% B; 10.0-10.1 min, 90%-0% B, 10.1-11 min, 0% B, and the post-treatment time was set to 2 min.

[0053] 3) Mass spectrometry conditions: capillary voltage was 3.5 kV, gas temperature and flow rate were 250 °C and 15.0 L / min, respectively, and sheath gas temperature and flow rate were 350 °C and 10.0 L / min, respectively. Nitrogen was used as nebulizer and collision gas. Positive ion mode was used in data acquisition through multiple reaction monitoring (MRM). For neurotransmitters, Table 1 provides detailed MRM parameters, including retention time, precursor ion, product ion, and collision energy.

[0054] Table 1. UHPLC–MS / MS parameters for monitoring neurotransmitters and their internal standards (ISs)

[0055]

[0056]

[0057] *: The ion with the largest response is used for quantification of the analyte.

[0058] 4) Optimization of optimal extraction conditions

[0059] The efficiency of neurotransmitter extraction using solid phase microextraction based on COF-DT coating may be affected by various parameters, such as extraction and desorption time, desorption solvent, and vortex speed. This study investigated the effects of different extraction times (5, 10, 20, 30, and 40 minutes), different desorption times (2, 5, 10, 15, 20, and 25 minutes), different desorption solvents (water, acetonitrile (ACN), methanol (MeOH), and water containing 1 v / v% formic acid (FA), 1 v / v% formic acid (FA) in acetonitrile, and 1 v / v% formic acid (FA) in methanol) and different vortex speeds (500, 1000, 1500, 2000, and 2500 rpm). The optimization experiment involved single factor investigation and optimization using the controlled variable method. The results are shown in Figure 2. Figure 3-6 shown.

[0060] SPME aims to achieve a dynamic equilibrium of the target analyte between the SPME coating and the sample solution. The extraction performance is closely related to the extraction time, and the adsorption capacity gradually increases with time to reach equilibrium. Extraction times ranging from 5 to 40 minutes have been studied, such as Figure 3 The intensities of the 10 neurotransmitters increased as the extraction time increased from 5 to 20 minutes, and 20 minutes was determined to be the time required for most neurotransmitters to reach equilibrium.

[0061] Poor desorption conditions during SPME may affect analytical accuracy because the analytes may remain on the coating. Therefore, it is crucial to choose a suitable desorption solvent to prevent the adverse effects of the residues in subsequent experiments. H2O, ACN, MeOH and its 1% formic acid solution were used for desorption, such as Figure 4As shown in Figure 3, except for Gln, GABA and 5-HIAA, the other seven NTs were better eluted in acidified solution, which may be related to their pKa values. Therefore, H2O containing 1% FA was selected as the appropriate desorption solvent.

[0062] Selecting optimal conditions is extremely important when considering the appropriate desorption time to ensure complete release of analytes from the SPME coating while avoiding cross-contamination in subsequent experiments. Figure 5 As shown in the figure, the desorption amount of NTs increased from 2 min to 15 min, and then stabilized or slightly decreased, indicating that 15 min desorption time was sufficient to release NTs. Therefore, 20 min was selected as the optimal extraction time and 15 min as the ideal desorption time.

[0063] The vortex rate is very important for the extraction efficiency and directly affects the mass diffusion of the analyte between the sample solution and the fiber. An appropriate vortex rate helps the analyte to diffuse, and a too high rate may affect the interaction between the analyte and the coating. The vortex rates of 500 to 2500 rpm were studied, and it was found that the intensity of NTs increased with the increase of the vortex rate, reaching a peak at 1500 rpm. Therefore, 1500 rpm was determined to be the optimal vortex rate for SPME.

[0064] The prepared COF-DT coated SPME fiber was directly immersed in a dilute solution containing 100 μl of brain sample and 900 μl of deionized water. The extraction was performed at room temperature for 20 min ( Figure 3 After extraction, the SPME fiber was quickly rinsed with deionized water for 1 second and then transferred to a desorption solvent containing 100 μL of 0.1% formic acid in water (as shown). Figure 4 The desorption process was promoted by spinning at 1500 rpm for 15 minutes (as shown in Figure 5 and Figure 6 In addition, the COF-based SPME fiber can be reused after washing with methanol and vortexing.

[0065] Example 4: Performance Characterization of Solid Phase Microextraction Coating

[0066] This example characterizes the performance of the solid phase microextraction coating prepared from the COF-DT material of Example 1 in Example 2.

[0067] 1) Preparation of alternative matrix: 2% agar gel and phosphate buffered saline (PBS) containing 5% bovine serum albumin (BSA) were used as alternative matrices to effectively simulate the viscosity of brain interstitial fluid and avoid the interference of endogenous neurotransmitters.

[0068] 2) Solution preparation: Quantification of neurotransmitters was performed based on the signal of the analyte in the surrogate matrix calibration curve. Standard stock solutions of neurotransmitters and internal standards were prepared at a concentration of 1 mg / mL and dissolved in different solvents based on solubility and stability. These stock solutions were then mixed and diluted with the surrogate matrix to make the calibration curve. The working solution of the internal standard was prepared by diluting the stock solution, and all stock and standard solutions were stored at -20°C to ensure stability and returned to room temperature before use.

[0069] 3) Linear analysis: Under the optimal extraction conditions, the COF-DT coating was used to extract a series of freshly prepared neurotransmitter solutions, with the concentration range for Glu and GABA ranging from 1 to 200 μg / mL, and the concentration range for NE, DOPA, E, DA, Ach, 3-MT, 5-HT and 5-HIAA ranging from 0.1 to 200 ng / mL. Three groups were tested in parallel for each test condition and analyzed by UHPLC-MS / MS to obtain the linearity of the COF-DT coating test. As shown in Table 2, the correlation coefficients (R 2 ) were all over 0.99, the detection limits of Glu and GABA were 0.5 μg / mL, the detection limits of Ach and 3-MT were 0.2 ng / mL, and the detection limits of NE, DOPA, E, DA, 5-HT and 5-HIAA were 0.05 ng / mL. Under the optimized chromatographic and extraction conditions, the total ion chromatograms of neurotransmitter analytes were obtained, providing symmetrical peak shapes, good separation and high resolution (see Figure 7 ).

[0070] Table 2. Linear range and detection limit of COF-DT coating

[0071]

[0072]

[0073] Example 5: Suitability of Solid Phase Microextraction Coatings for Determination of Neurotransmitters in Mouse Brain Tissue Samples

[0074] In this example, the solid phase microextraction coating (Example 2) made of the COF-DT material of Example 1 was used for suitability analysis in neurotransmitter analysis in mouse brain tissue samples.

[0075] 1) Sample collection and processing: 6 male C57BL6 mice (6-8 weeks old) were purchased from Jiangsu Jinfa Pharmaceutical Technology Co., Ltd. The mice were sacrificed for subsequent experiments, and the brain tissue was immediately removed and collected and weighed. The brain tissue sample was then mixed with a cold aqueous solution of 0.1% FA at a ratio of 1:5 (mg / μL) in a 2mL centrifuge tube. The mixture was homogenized on ice for 1 minute using a tissue homogenizer to prepare QC samples, which were then stored at -80°C until further testing.

[0076] 2) Spiking treatment: QC samples with three different concentrations were pretreated and analyzed under optimal extraction and detection conditions to evaluate the accuracy and precision of the developed method. QC samples were spiked at three concentration levels: NE, DOPA, E, DA, Ach, 3-MT, 5-HT and 5-HIAA at 0.5 ng / mL, 5 ng / mL and 50 ng / mL, respectively, while Glu and GABA at 0.5 μg / mL, 5 μg / mL and 50 μg / mL, respectively. The recovery was calculated as the percentage of the concentration of NT added relative to the measured concentration, and the precision of the method was evaluated by the RSD value of five repeated measurements at each level.

[0077] 3) Recovery and matrix effects: As shown in Table 3, all recoveries ranged from 80.9% to 117.5%, with RSDs less than 9.5%, indicating that the method was both reliable and reproducible. The matrix effects of all NTs were found to be acceptable, ranging from 84.3% to 117.3%, indicating that the matrix effects were negligible compared with the use of alternative brain tissue as a matrix.

[0078] Table 3. Recovery, precision and matrix effect of the proposed method (n=5)

[0079]

[0080]

[0081] Example 6: Solid Phase Microextraction Coating for Determination of Neurotransmitter Compounds in Mouse Brain Tissue

[0082] In this example, the solid phase microextraction coating of Example 2 made from the COF-DT material of Example 1 was used for the analysis of neurotransmitters in mouse brain tissue samples.

[0083] 1) Sample collection and processing: Male C57BL6 mice (6-8 weeks old) were purchased from Jiangsu Jinfa Pharmaceutical Technology Co., Ltd. The mice were sacrificed for subsequent experiments, and the brain tissue of the striatum was immediately removed and collected and weighed. The brain tissue sample was then mixed with a cold aqueous solution of 0.1% FA at a ratio of 1:5 (mg / μL) in a 2 mL centrifuge tube. The mixture was homogenized on ice using a tissue homogenizer for 1 minute and then stored at -80°C until further testing.

[0084] 2) Sample solid phase microextraction: The prepared COF-DT coated solid phase microextraction fiber was directly immersed in a dilute solution containing 100 μl of brain sample with internal standard and 900 μl of deionized water. The extraction was performed at room temperature for 20 minutes. After extraction, the SPME fiber was quickly rinsed with deionized water for 1 second and then transferred to a desorption solvent in 100 μl of water containing 0.1% formic acid. The desorption process was promoted by spinning at 1500 rpm for 15 minutes.

[0085] 3) Quantitative analysis of neurotransmitters in the striatum of mice: The present invention was combined with UPHLC-MS / MS instrument analysis to analyze the contents of ten neurotransmitters in the striatum of healthy mice, and the test results are shown in Table 4. The data reflect the concentration levels of various neurotransmitters in the striatum samples of control mice, among which the concentrations of Glu and GABA are relatively high, while the concentrations of other neurotransmitters are low. The standard deviation indicates the fluctuation range of the measured value, and a smaller standard deviation means that the measurement results are more consistent.

[0086] Table 4. Contents of ten neurotransmitters in the striatum of mice (ng / mg)

[0087]

[0088]

[0089] The developed prepared COF-DT-coated SPME fiber coupled with UHPLC-MS / MS method presents a new strategy for the selective extraction, separation, and detection of trace analytes in complex biological matrices, which not only deepens the understanding of the effects of nicotine addiction on neurotransmitter balance but also serves as a powerful tool for exploring the role of neurotransmitters in neurological diseases and psychiatric disorders.

Claims

1. A solid phase microextraction probe, comprising a probe carrier and an extraction coating coated on the probe carrier, wherein: The extraction coating is a COF-DT material, and the COF-DT material is made by connecting 2,5-dihydroxyterephthalaldehyde (Dha) and 1,3,5-tris(4-aminophenyl)benzene (Tab) through an imine bond.

2. The solid phase microextraction probe according to claim 1, wherein: The pore size of the COF-DT material is 4.7 nanometers.

3. The solid phase microextraction probe according to claim 1 or 2, wherein: The thickness of the coating is 100 microns; Preferably, the length of the coating is 1 cm.

4. The solid phase microextraction probe according to any one of claims 1 to 3, wherein: The probe carrier is stainless steel fiber.

5. A method for preparing the solid phase microextraction probe according to any one of claims 1 to 4, the method comprising: A layer of adhesive solution is first coated on the surface of the probe carrier, and then the end of the probe carrier coated with the adhesive solution is dipped with powder of COF-DT material and dried to obtain; Preferably, the end of the probe carrier coated with the adhesive solution is dipped with the powder of the COF-DT material to a length of 1 cm.

6. The method according to claim 5, wherein: The adhesive solution is a cyclohexane solution of neutral silicone glue or a polyacrylonitrile (PAN) solution with N-dimethylformamide (DMF) as a dispersant, with a concentration of 200 mg / g; Preferably, the drying is oven drying at a temperature of 120 degrees for 40 minutes.

7. The method according to claim 5 or 6, wherein: The preparation method also includes the step of pre-treating the probe carrier, and the pre-treatment method is as follows: the probe carrier is ultrasonically cleaned using superacetone, deionized water and methanol in sequence; then, it is immersed in aqua regia solution for surface roughening treatment to enhance its adhesion to the adhesive and COF-DT material; then, it is thoroughly rinsed with deionized water until the pH value is neutral, and dried under a nitrogen flow.

8. The method according to any one of claims 5 to 7, wherein: The preparation method further comprises the step of subjecting the powder of the COF-DT material to high temperature aging of the extracted coating after drying.

9. Use of the solid phase microextraction probe according to any one of claims 1 to 4 in detecting neurotransmitter compounds; Preferably, the neurotransmitter compounds include acetylcholine (Ach), L-3,4-dihydroxyphenylalanine (DOPA), dopamine (DA), norepinephrine (NE), epinephrine (E), 3-methoxytyramine hydrochloride (3-MT), 5-hydroxytryptamine (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), glutamate (Glu), and γ-aminobutyric acid (GABA).

10. A method for detecting neurotransmitter compounds using the solid phase microextraction probe according to any one of claims 1 to 4, wherein: The extraction conditions adopted during the detection were: extraction time of 20 min, desorption time of 15 min, eluent of 0.1% formic acid / water (V / V), and vortex speed of 1500 rpm; Preferably, the neurotransmitter compounds include acetylcholine (Ach), L-3,4-dihydroxyphenylalanine (DOPA), dopamine (DA), norepinephrine (NE), epinephrine (E), 3-methoxytyramine hydrochloride (3-MT), 5-hydroxytryptamine (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA), glutamate (Glu), and γ-aminobutyric acid (GABA).

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