Solid-phase microextraction probe based on hollow covalent organic framework material and application of solid-phase microextraction probe in lipidomics analysis

Abstract

The invention discloses a solid-phase microextraction probe based on a hollow covalent organic framework material and application of the solid-phase microextraction probe in lipidomics analysis, and relates to the technical field of lipidomics analysis. The preparation method of the hollow covalent organic framework material comprises the following steps: carrying out a Schiff base reaction on 1, 3, 5-tri (4-aminophenyl) benzene and trimesic aldehyde at 20-30 DEG C to obtain a covalent organic polymer; and dispersing the covalent organic polymer in an organic solvent, adding glacial acetic acid, and reacting at 65-75 DEG C to obtain the hollow covalent organic framework material. The hollow covalent organic framework material disclosed by the invention is high in matrix interference resistance and high in lipid extraction capacity, and can realize efficient enrichment of micromolecular lipid metabolites. The prepared SPME probe can be applied to in-vivo lipid sampling analysis and lipidomics research, and a more accurate and effective method is provided for real-time capture of in-vivo lipid abundance.

Description

Technical Field

[0001] The present invention relates to the technical field of lipidomics analysis, and particularly to a solid-phase microextraction probe based on hollow covalent organic framework materials and its application in lipidomics analysis. Background Art

[0002] Lipidomics is an important branch of metabolomics. With its high sensitivity to physiological processes such as cell membrane components, energy metabolism, and membrane signal transduction, it has been widely used in the study of the impact of lipid homeostasis regulation under pollutant exposure. By systematically analyzing the abundance changes of lipid classes and molecular species in organisms under pollutant stress, key regulatory nodes related to the lipid metabolic pathway can be revealed, thereby providing a theoretical basis for identifying biotoxic effects and screening potential biomarkers. In addition, lipids are an important nutrient. Through lipidomics, we can also study the impact of pollutant exposure on the nutritional components of aquatic products, providing a reliable basis for food safety and nutritional health assessment.

[0003] However, most of the existing methods for lipid extraction are limited to ex vivo sampling, which may not reflect the true state of organisms. Moreover, this sampling method has defects such as cumbersome operation procedures and large consumption of organic solvents, which do not meet the requirements of current green analytical chemistry. In contrast, the solid-phase microextraction (SPME) in vivo sampling technique, as a sample pretreatment technique with simple operation and no need for a large amount of solvents, can provide a more accurate and effective method for the real-time capture of in vivo lipid abundance.

[0004] Due to the complex composition of biological matrices, sufficient lipid sampling amounts usually need to be ensured in lipidomics analysis; however, the existing SPME probes have low lipid extraction capacities and weak anti-matrix interference abilities, which cannot meet the analysis requirements of lipidomics. There is an urgent need to develop a solid-phase microextraction coating material with strong anti-matrix interference ability and high adsorption capacity for lipids to promote the development and application of SPME in vivo sampling in lipidomics analysis.

[0005] A solid-phase microextraction probe for detecting perfluorinated compounds, its preparation method and application in Patent CN110204670A uses monomers with fluoroalkyl bonds and hydroxyl groups to prepare covalent organic framework materials through the condensation reaction of amino groups and aldehyde groups, having good adsorption performance and being able to efficiently enrich perfluorinated compounds in milk and aquatic animals, with an enrichment efficiency reaching 95%. -F is modified in this material, and specific adsorption of perfluoro and polyfluoro compounds can be achieved through fluorine-fluorine bond and hydrogen bond interactions, but omics research often requires probes with broad-spectrum adsorption, which limits its application in the field of lipidomics analysis. Summary of the Invention

[0006] The object of the present invention is to overcome the defects and deficiencies of existing SPME probes, and to provide a solid-phase microextraction probe based on a hollow covalent organic framework material, which has strong anti-matrix interference ability, can adsorb lipids in a broad spectrum, and has a high lipid extraction capacity.

[0007] Another object of the present invention is to provide a preparation method of a solid-phase microextraction probe based on a hollow covalent organic framework material.

[0008] Another object of the present invention is to provide an application of a solid-phase microextraction probe based on a hollow covalent organic framework material in lipidomics analysis.

[0009] In some embodiments, the hollow covalent organic framework material presents a hollow microsphere structure in the TEM microscopic morphology characterization.

[0010] Provide a solid-phase microextraction probe based on a hollow covalent organic framework material, including a matrix and a solid-phase microextraction coating covering the matrix, and the solid-phase microextraction coating is made of a hollow covalent organic framework material; The preparation method of the hollow covalent organic framework material includes the following steps: S1, performing a Schiff base reaction on 1,3,5-tris(4-aminophenyl)benzene and benzene-1,3,5-tricarbaldehyde at 20-30 °C to obtain a covalent organic polymer; S2, dispersing the covalent organic polymer in an organic solvent, adding glacial acetic acid and reacting under the condition of 65-75 °C to obtain the hollow covalent organic framework material.

[0011] For the solid-phase microextraction probe based on the hollow covalent organic framework material of the present invention, its solid-phase microextraction coating is a covalent organic framework material prepared by using a specific reaction monomer. Its pore size of the structure is small, which can avoid large biomolecules from entering the pores and affecting the extraction of lipids, and improve the anti-matrix interference ability; in addition, during the preparation process, the reaction monomers are rapidly condensed at a specific temperature to obtain an amorphous covalent organic polymer, and then at a specific temperature, glacial acetic acid is used as a catalyst to catalyze the induced rearrangement reaction to form a covalent organic framework material with a hollow structure. This material has a specific hollow structure that is beneficial to providing more adsorption sites and space, thereby effectively improving the adsorption capacity of lipids.

[0012] In some embodiments, in step S1, the molar ratio of 1,3,5-tris(4-aminophenyl)benzene to benzene-1,3,5-tricarbaldehyde is 1:(0.8-1.2).

[0013] In some embodiments, in the Schiff base reaction of step S1: the catalyst is glacial acetic acid (HAc), and the concentration of the glacial acetic acid in the reaction system is 5-15 vol%.

[0014] In some of these embodiments, in the Schiff base reaction of step S1, the solvent is acetonitrile (ACN).

[0015] Preferably, the reaction temperature of step S1 is 22 - 27 °C.

[0016] In some of these embodiments, in the Schiff base reaction of step S1, the reaction time is 5 - 7 h.

[0017] In some of these embodiments, the covalent organic polymer in step S1 is washed with absolute ethanol and / or 1,4 - dioxane, and dried at 55 - 65 °C.

[0018] In some of these embodiments, in step S2, the organic solvent is 1,4 - dioxane and mesitylene with a volume ratio of (3 - 5):1.

[0019] In some of these embodiments, in step S2, the concentration in the glacial acetic acid reaction system is 5 - 15 vol%.

[0020] Preferably, in step S2, the reaction temperature is 68 - 72 °C, and the reaction time is 46 - 52 h.

[0021] In some of these embodiments, the hollow covalent organic framework material in step S2 is washed with tetrahydrofuran and / or ethanol, and dried under vacuum.

[0022] In some of these embodiments, it includes a basic unit structure as shown in formula (Ⅰ): Formula (Ⅰ).

[0023] In some of these embodiments, in the hollow covalent organic framework material, the repeating structural unit constructed with the basic unit is as shown in formula (Ⅱ): Formula (Ⅱ).

[0024] Provided is a method for preparing a solid - phase microextraction probe based on a hollow covalent organic framework material, which is characterized by including the following steps: (1) Using a stainless - steel wire as a substrate, ultrasonically cleaning it in water, methanol, and acetone in sequence, and drying; (2) Vertically placing the dried stainless - steel wire into a silicone rubber - cyclohexane solution, rotating it at least one circle to make the adhesive evenly distributed on the surface of the stainless - steel wire; then inserting the wire coated with the adhesive into the prepared hollow covalent organic framework material and rotating it at least one circle to make the target material evenly adhered to the surface of the stainless - steel wire, and drying to obtain a solid - phase microextraction probe based on the hollow covalent organic framework material.

[0025] Provided is an application of a solid-phase microextraction probe based on a hollow covalent organic framework material in lipidomics analysis.

[0026] In some of these embodiments, it includes the following steps: using the solid-phase microextraction probe based on the hollow covalent organic framework material to perform in vivo sampling on an organism, and detecting by liquid chromatography-mass spectrometry and performing lipidomics analysis.

[0027] Compared with the prior art, it has the following beneficial effects: The present invention provides a solid-phase microextraction probe based on a hollow covalent organic framework material. The solid-phase microextraction coating is a covalent organic framework material prepared by using a specific reaction monomer. Its structure has a smaller pore size, which can prevent larger biomolecules from entering the pores, and has strong anti-matrix interference ability; at the same time, the prepared covalent organic framework material has a hollow microsphere structure in its microscopic morphology, which is beneficial to providing more adsorption sites and space, thereby effectively improving the adsorption capacity of lipids. Description of the Drawings

[0028] Figure 1 HCOF of Example 1 of the present invention TAPB-BTCA Material and COF of Comparative Example 1 TAPB-BTCA Characterization result diagrams of the materials. Among them, Figure 1 a is the SEM diagram of the HCOF TAPB-BTCA material, Figure 1 b is the TEM diagram of the HCOF TAPB-BTCA material, Figure 1 c is the SEM diagram of the COF TAPB-BTCA material, Figure 1 d is the TEM diagram of the COF TAPB-BTCA material.

[0029] Figure 2 COP of Example 1 of the present invention TAPB-BTCA and HCOF TAPB-BTCA materials and XRD diagrams of the COF TAPB-BTCA materials of Comparative Example 1.

[0030] Figure 3 SEM diagram of the SPME probe of Example 1 of the present invention.

[0031] Figure 4 COP of Example 1 of the present invention TAPB-BTCA and HCOF TAPB-BTCA Fourier transform infrared spectroscopy (FT-IR) diagrams of the materials.

[0032] Figure 5 COP of Example 1 of the present invention TAPB-BTCA and HCOF TAPB-BTCA Nitrogen isothermal adsorption and desorption diagrams of the materials.

[0033] Figure 6 COP for Example 1 of the present invention TAPB-BTCA and HCOF TAPB-BTCA Water contact angle diagrams of the materials Figure 6 where a is the water contact angle diagram of the COP TAPB-BTCA material, Figure 6 and b is the water contact angle diagram of the HCOF TAPB-BTCA material

[0034] Figure 7 HCOF for Example 1 of the present invention TAPB-BTCA Comparison diagram of the probe with two commercial products for lipid extraction effect

[0035] Figure 8 PCA diagram after 24 h of exposure in lipidomics analysis for Example 2 of the present invention. Among them, Figure 8 a to Figure 8 c are the PCA diagrams of the Pys group, PS-NPs group, and PNPs after 24 h of exposure in the positive ion mode; Figure 8 d to Figure 8 f are the PCA diagrams of the Pys group, PS-NPs group, and PNPs after 24 h of exposure in the negative ion mode

[0036] Figure 9 PCA diagram after 7 days of exposure in lipidomics analysis for Example 2 of the present invention. Among them, Figure 9 a to Figure 9 c are the PCA diagrams of the Pys group, PS-NPs group, and PNPs after 7 days of exposure in the positive ion mode; Figure 9 d to Figure 9 f are the PCA diagrams of the Pys group, PS-NPs group, and PNPs after 7 days of exposure in the negative ion mode Detailed implementation manners

[0037] The present invention will be further described below in conjunction with the detailed implementation manners, but the embodiments do not limit the present invention in any form

[0038] Example 1 A preparation method of a solid-phase microextraction probe based on a hollow covalent organic framework material, comprising the following steps: S1, preparing a covalent organic polymer (COP TAPB-BTCA ); Specifically: Weigh 1,3,5-tris(4-aminophenyl)benzene (0.04 mmol, 14.1 mg) and benzene-1,3,5-tricarbaldehyde (0.04 mmol, 6.49 mg) into a 10 mL sample bottle respectively, add 5 mL of acetonitrile and dissolve them by ultrasound. Then add 0.5 mL of glacial acetic acid and react at room temperature for 6 h. Collect the yellow precipitate formed by the reaction, wash it 3 times with absolute ethanol and 1,4-dioxane, and dry it at 60 °C to obtain a covalent organic polymer; S2. Prepare hollow covalent organic framework materials (HCOF TAPB-BTCA ); Specifically: Take 10 mg of the COP obtained in step S1 TAPB-BTCA , disperse it in 3 mL of 1,4-dioxane / toluene solution (4:1, v / v), add 0.3 mL of glacial acetic acid, and react at 70 °C for 48 h; After the reaction, collect the product and wash it 3 times with tetrahydrofuran and ethanol, and dry it under vacuum to obtain hollow covalent organic framework materials.

[0039] S3. Prepare a solid-phase microextraction probe; Specifically: Cut a stainless steel wire (diameter 400 μm) into 8 cm lengths, then ultrasonically treat it in water, methanol and acetone for 30 min in sequence, and dry it. Then vertically place the stainless steel wire into a 0.5 g / mL silicone rubber-cyclohexane solution with a depth of 1.0 cm, rotate it once to make the adhesive silicone rubber evenly distributed on the surface of the stainless steel wire. Subsequently, quickly insert the wire coated with glue into the above-prepared HCOF TAPB-BTCA and rotate it once to ensure that the target material is evenly adhered to the surface of the stainless steel wire. Finally, dry it in an oven at 80 °C for 30 min, and repeat the above operation 3 times to ensure uniform thickness, and obtain an SPME probe coated with HCOF TAPB-BTCA .

[0040] Comparative Example 1 A preparation method of a covalent organic framework material (COF TAPB-BTCA ), including the following steps: Weigh TAPB (0.04 mmol, 14.1 mg) and BTCA (0.04 mmol, 6.49 mg) into a 25 mL beaker respectively, add 5 mL of ACN and ultrasonically treat the mixture until the two monomers are completely dissolved. Then add 1 mL of HAc to the mixture and stir at room temperature until the solvent is almost completely evaporated. Collect the precipitate, wash it 3 times with ethanol, and dry it under vacuum to obtain a covalent organic framework material. Performance test 1. Microscopic morphology and XRD characterization The HCOF of Example 1 TAPB-BTCAMaterials and COF of Comparative Example 1 TAPB-BTCA Scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used for characterization respectively, and SEM characterization was performed on the SPME probe. The results are as Figure 1-3 shown.

[0041] From Figure 1 the SEM image of a, it can be seen that the HCOF TAPB-BTCA material is spherical particles with a particle size of about 270 nm.

[0042] From Figure 1 the TEM image of b, it can be seen that the prepared HCOF TAPB-BTCA material has a hollow spherical structure in its microscopic morphology, which can provide more space for the attachment of lipids.

[0043] From Figure 1 c and Figure 1 d, it can be seen that the COF TAPB-BTCA material has a solid spherical structure.

[0044] From Figure 2 the XRD pattern, it can be seen that the HCOF TAPB-BTCA material shows a sharp diffraction peak at 5.7°, and weak diffraction peaks are observed at 9.9° and 11.5°, indicating that the prepared HCOF TAPB-BTCA has the same crystal form as COF TAPB-BTCA , which shows that the HCOF TAPB-BTCA obtained by the rearrangement reaction in the present invention has the same high crystallinity as COF TAPB-BTCA . The high crystallinity can provide more active sites for lipid attachment.

[0045] From Figure 3 the SEM image, it can be seen that the HCOF TAPB-BTCA material in the SPME probe is uniformly coated on the surface of the stainless steel wire, and the HCOF TAPB-BTCA coating thickness is 50 μm.

[0046] 2. Fourier Transform Infrared Spectroscopy (FT-IR) Characterization The COP TAPB-BTCA and HCOF TAPB-BTCA of Example 1 were respectively subjected to FT-IR characterization; The results are as Figure 4 shown, in which the characteristic stretching vibration (1581 cm -1 ) of imino group (C=N) exists, and at the same time, the aldehyde C-H stretching vibration peak of BTCA (2810 cm -1 ) and the N-H stretching vibration peak of TAPB (3419 cm -1), all disappeared, indicating that the imine bond (C=N) has formed in COP TAPB-BTCA It has been formed in the

[0047] 3. Nitrogen isothermal adsorption and desorption experiment The COP of Example 1 TAPB-BTCA and HCOF TAPB-BTCA were respectively subjected to nitrogen adsorption and desorption experiments.

[0048] The results are as Figure 6 shown. The adsorption isotherms of COP TAPB-BTCA and HCOF TAPB-BTCA are of type IV, indicating that the materials have good adsorption force; the specific surface area of COP TAPB-BTCA is 77.8 m 2 / g, while the specific surface area of HCOF TAPB-BTCA reaches 575.4 m 2 / g.

[0049] 4. Contact angle measurement The COP of Example 1 TAPB-BTCA and HCOF TAPB-BTCA were respectively subjected to contact angle measurement.

[0050] The results are as Figure 6 shown. The contact angle of COP TAPB-BTCA is 84.5°, showing hydrophilicity; while the contact angle of HCOF TAPB-BTCA is 99.6°, showing hydrophobicity.

[0051] Example 2 Application of an HCOF TAPB-BTCA coated SPME probe in lipidomics analysis In this example, an SPME in vivo sampling was carried out using the HCOF TAPB-BTCA coated SPME probe prepared in Example 1 and applied to the lipidomics study of tilapia under the combined pollution exposure of polystyrene nanoplastics (PS-NPs) and pyrethroid pesticides (PYs). The specific operations are as follows: 1. Pretreatment: Before the experiment, tilapia fry were cultured in the laboratory for 8 months (the culture water was dechlorinated tap water, disinfected by ultraviolet light, fully aerated, and filtered through activated carbon for the water source).

[0052] Before preparing the formal experiment, tilapia were divided into four groups (6 fish per group), namely: blank group (Blank), polystyrene nanoplastics group (PS-NPs, particle size 100-1000 nm), pyrethroid group (PYs), and combined exposure group of PYs and PS-NPs (PNPs); they were placed in a glass water tank containing 50 L of dechlorinated tap water for adaptive feeding for one week to ensure that the fish adapted to the experimental environment.

[0053] 2. SPME in vivo sampling: Tilapia in each group were exposed in the corresponding environment for 7 days (168 h); among them, the PS-NPs group: 500 μg / L PS-NPs; the PYs group: 2.5 μg / L PYs; the combined exposure group of PYs and PS-NPs: 2.5 μg / L PYs + 500 μg / L PS-NPs. The exposure time was 168 h.

[0054] The SPME in vivo sampling technique was used to continuously monitor the lipid changes in the fish body for a total duration of 168 h. The sampling points were set at 0 h, 24 h, 36 h, 48 h, 72 h, 120 h, and 168 h after the fish were exposed to the target spiked pollutants.

[0055] The in vivo sampling process was as follows: Insert the HCOF TAPB-BTCA probe under the guidance of a stainless-steel needle, and then withdraw the needle to expose the HCOF TAPB-BTCA coating in the fish muscle. After extraction for 40 min, carefully insert the needle back and then remove the HCOF TAPB-BTCA probe together. Then rinse the surface of the probe coating with deionized water and dry the moisture with lint-free paper. Subsequently, desorb the lipids on the surface of the probe coating in 70 μL of acetonitrile / water (v / v = 8:2) at 600 rpm for 20 min.

[0056] 3. Mass spectrometry and liquid chromatography detection: The eluate was detected by a combination of ultra-high performance liquid chromatography (UPLC, Waters) and Synapt G2-Si quadrupole time-of-flight high-resolution mass spectrometry (MS, Waters) to complete lipidomics analysis.

[0057] Detection conditions for ultra-high performance liquid chromatography: Use a CSH C18 chromatographic column (2.1×100 mm 2, with a particle size of 1.7 μm, was separated using a Waters chromatographic column. Mobile phase A was an acetonitrile / water (volume ratio 6:4) solution containing 10 mM ammonium formate (AmAc), and mobile phase B was an isopropanol / acetonitrile (volume ratio 9:1) solution containing 10 mM ammonium formate. The flow rate was 0.3 mL / min, and the column oven temperature was 60 °C. The injection volume was 5 μL, and scanning analysis was performed in both positive and negative ion modes. The mobile phase gradient program was as follows: 0 min, 32% B; 2 min, 32% B; 4 min, 60% B; 12 min, 97% B; 16 min, 97% B; 16.1 min, 32% B; 18 min, 32% B.

[0058] Mass spectrometry detection conditions: The mass spectrometry scanning range was 50 - 1500 m / z, and the scanning time was 100 ms. The ion source temperature and desolvation gas temperature were 150 °C and 600 °C, respectively. The desolvation gas flow rate was 1100 L / h, and the curtain gas flow rate was 150 L / h. Except for the collision gas being argon, all other gases were nitrogen. In the positive ion mode, the capillary voltage was 1 kV; in the negative ion mode, the capillary voltage was 2 kV.

[0059] A quality control sample (QC) was injected after every 5 sample analyses. This sample was made from an equal mixture of all samples and was used to monitor system stability and performance. All samples were analyzed in a random order. Mass calibration was performed by continuously injecting the standard calibration substance leucine enkephalin, which is widely used in mass spectrometry analysis.

[0060] The number of lipids detected by the liquid chromatography - mass spectrometry detection method was counted. The group with the largest number of detected lipids was set as 100, and other groups were normalized to obtain the coverage rate of each probe. The results are as Figure 7 shown.

[0061] The raw data was imported into Progenesis QI (Nonlinear Dynamics Waters, UK) software for pre - processing, including baseline correction, peak extraction, peak alignment, deconvolution, and data normalization, etc. Subsequently, the pre - processed data was subjected to multivariate statistical analysis using EZinfo for Waters (Umetrics, Sweden) software, mainly principal component analysis (PCA), to preliminarily explore the metabolic characteristic differences between different experimental groups and lay a foundation for the screening of potential biomarkers. The principal component analysis (PCA) plot is shown in Figures 8 - 9.

[0062] To further explore the data and verify the differential metabolites in the experimental data, the preprocessed data mentioned above was imported into the MetaboAnalyst data processing platform. ANOVA was performed on the data, and the fold change of metabolites in different groups was calculated. Differential metabolites were screened with the thresholds of p < 0.05, FC > 2 or FC < 0.5. The results are shown in Table 1.

[0063] From Figure 7 it can be seen that compared with the two commercial probes of DVB / PDMS and PDMS, the HCOF TAPB-BTCA probe can extract more types of lipid molecules, indicating that the HCOF TAPB-BTCA probe has significant advantages in lipidomics research.

[0064] It can be seen from the principal component analysis (PCA) that after 24 h of exposure to different pollutants, whether it is a single or combined exposure group, an obvious clustering trend was shown ( Figure 8 ), and the metabolism of tilapia was significantly disturbed. After seven days of exposure, the PS-NPs group and the PYs group no longer showed an obvious clustering trend ( Figure 9 ), indicating that as the exposure time prolongs, the fish may gradually regulate its own metabolism through metabolic adaptation mechanisms to cope with the physiological stress caused by the continuous presence of PS-NPs and PYs.

[0065] Table 1

[0066] It can be seen from Table 1 that in the positive ion mode, the sorted differential expression (up-regulated + down-regulated) lipid metabolites screened in each group are: PYs group (3531) > PS-NPs group (2962) > PNPs group (2918); while in the negative ion mode, a total of 2180 differential expression metabolites were monitored, and the inter-group difference trend was highly consistent with that in the positive ion mode.

[0067] The above experimental results show that the HCOF TAPB-BTCA probe can achieve efficient extraction of lipids in fish, and can continuously track and monitor the dynamic changes of fish metabolism under continuous pollutant exposure, and has great application prospects in in vivo sampling analysis of lipidomics.

[0068] The above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limiting the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A solid-phase microextraction probe based on a hollow covalent organic framework material, characterized in that, It includes a substrate and a solid-phase microextraction coating covering the substrate, and the solid-phase microextraction coating is made of a hollow covalent organic framework material; The preparation method of the hollow covalent organic framework material includes the following steps: S1. Carry out a Schiff base reaction on 1,3,5-tris(4-aminophenyl)benzene and trimesic aldehyde at 20 - 30 °C to obtain a covalent organic polymer; S2. Disperse the covalent organic polymer in an organic solvent, add glacial acetic acid and react under the condition of 65 - 75 °C to obtain the hollow covalent organic framework material.

2. The solid-phase microextraction probe based on a hollow covalent organic framework material according to claim 1, characterized in that, In step S1, the molar ratio of 1,3,5-tris(4-aminophenyl)benzene to trimesic aldehyde is 1:(0.8 - 1.2).

3. The solid-phase microextraction probe based on the hollow covalent organic framework material according to claim 1, characterized in that, In the Schiff base reaction of step S1: The catalyst is glacial acetic acid, and the concentration of the glacial acetic acid is 5 - 15 vol%.

4. The solid-phase microextraction probe based on the hollow covalent organic framework material according to claim 1, wherein In the Schiff base reaction of step S1, the solvent is acetonitrile.

5. The solid-phase microextraction probe based on the hollow covalent organic framework material according to claim 1, wherein In step S2, the organic solvent is 1,4-dioxane and mesitylene with a volume ratio of (3 - 5):

1.

6. The solid-phase microextraction probe based on the hollow covalent organic framework material according to claim 1, wherein In step S2, the concentration of the glacial acetic acid is 5 - 15 vol%.

7. The solid-phase microextraction probe based on the hollow covalent organic framework material according to claim 1, wherein, The hollow covalent organic framework material includes a basic unit structure shown in formula (Ⅰ): Formula (I).

8. A method for preparing the solid-phase microextraction probe based on the hollow covalent organic framework material according to any one of claims 1-7, characterized in that, It includes the following steps: (1) Use a stainless steel wire as the substrate, and ultrasonically clean it in water, methanol and acetone in sequence, and then dry it; (2) Vertically place the dried stainless steel wire into a silicone rubber - cyclohexane solution, rotate it at least one circle to make the adhesive evenly distributed on the surface of the stainless steel wire; then insert the stainless steel wire coated with the adhesive into the prepared hollow covalent organic framework material and rotate it at least one circle to make the target material evenly adhered to the surface of the stainless steel wire, and dry it to obtain a solid-phase microextraction probe based on the hollow covalent organic framework material.

9. The application of the solid-phase microextraction probe based on the hollow covalent organic framework material according to any one of claims 1 - 7 in lipidomics analysis.

10. The application according to claim 9, characterized in that, It includes the following steps: Use the solid-phase microextraction probe based on the hollow covalent organic framework material to perform in-vivo sampling on an organism, and detect and perform lipidomics analysis through mass spectrometry combined with liquid chromatography.

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