Magnetic carboxylated covalent organic framework as well as preparation method and application thereof

Abstract

The invention relates to the technical field of drug detection, in particular to a magnetic carboxylated covalent organic framework as well as a preparation method and application thereof. The magnetic carboxylated covalent organic framework comprises a Fe3O4 (at) SiO2 magnetic core, and a covalent organic framework generated by a reaction of a monomer 2, 4, 6-triformyl phloroglucinol and a dissolved amine monomer is used as a shell layer; the dissolved amine monomer is selected from one of p-phenylenediamine, benzidine and 4, 4 '-diamino-p-terphenyl; wherein the shell layer is modified by carboxylation. The magnetic carboxylated covalent organic framework can accurately regulate and control the aperture, is used for chemical detection based on a magnetic solid-phase extraction technology, is simple and rapid, has low toxicity, uses less organic reagents, has high protein exclusion rate, can exclude complex matrixes such as proteins in a whole blood sample in one step, and can enrich three immunosuppressants at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and particularly relates to a magnetic carboxylated covalent organic framework, a preparation method thereof, and an application thereof. Background Art

[0002] Solid organ transplantation refers to the transplantation of the heart, lung, kidney, pancreas or liver. After solid organ transplantation, immunosuppressants can suppress the immune system to ensure the short-term and long-term survival of organ transplantation. Taking the correct dose of immunosuppressants at the correct time is the key to post-transplant care. Immunosuppressants have the characteristics of a wide variety, complex action mechanisms, extensive action targets, long-term medication, large individual differences, and the rapid development of targeted immunosuppressants. While playing an immunosuppressive role, there is also a high risk of adverse reactions. Due to its narrow therapeutic window, large variability in individual pharmacokinetics and pharmacodynamics, the adverse reactions of drugs and drug interactions are also closely related to the blood drug concentration of immunosuppressants. Therefore, transplant recipients must regularly detect the blood drug concentration and related biochemical indicators of immunosuppressants to avoid the increased risk of infection and adverse reactions caused by excessive immunosuppressants, as well as the rejection reaction caused by insufficient immunosuppression.

[0003] Therapeutic drug monitoring (TDM) is a clinical pharmacy branch discipline that conducts individualized research and application of drug therapy through the integration of multiple disciplines; according to the theories of clinical pharmacology, biopharmaceutics, and pharmacotherapeutics, combined with the principles of pharmaceutical analysis, molecular biology, and epidemiology, modern testing means are used to measure the concentrations of drugs and their metabolites in biological samples, determine the effective concentration range, apply pharmacokinetic-pharmacodynamic principles to adjust the dosing regimen, and improve the therapeutic effect. And because serum contains a large amount of protein and the concentration of immunosuppressants in the blood is generally relatively low, and patients often use other drugs in combination, which may cause certain interference to the analysis. Therefore, the pretreatment method must have high selectivity and high sensitivity to accurately qualitatively and quantitatively analyze the subsequent analysis.

[0004] In the prior art, the commonly used analytical methods for detecting immunosuppressants are pretreatment techniques combined with liquid chromatography-tandem mass spectrometry. The pretreatment methods for determining immunosuppressants in whole blood mainly include liquid-liquid extraction (LLE), protein precipitation (PP), supercritical fluid extraction (SFE), and solid-phase extraction (SPE). At present, the commonly used liquid-liquid extraction method has problems such as excessive use of organic reagents and low extraction rate; protein precipitation has defects such as low protein precipitation efficiency, easy clogging of high-performance liquid chromatography, damage to instrument performance, and interference from other drugs in the analysis; supercritical fluid extraction has problems such as complex equipment and large investment consumption; solid-phase extraction has defects such as long extraction time, cumbersome steps (protein removal is required before extraction), easy clogging of extraction columns, and the need for special equipment. Summary of the Invention

[0005] To solve at least one of the problems of the prior art, the present invention provides a magnetic carboxylated covalent organic framework with precisely adjustable pore size, which can be used for chemical detection based on magnetic solid-phase extraction (M-SPE) technology. This detection method has the advantages of simple and rapid operation, low toxicity, less use of organic reagents, high protein exclusion rate, and the ability to exclude complex matrices such as proteins in whole blood samples in one step while enriching three immunosuppressants.

[0006] A magnetic carboxylated covalent organic framework, having a core-shell structure, includes: a magnetic core, which is Fe 3 O 4 @SiO 2 ; a shell layer, which is a covalent organic framework (COFs) formed by the reaction of monomer 2,4,6-triformylphloroglucinol (Tp) with a dissolved amine monomer; the dissolved amine monomer is selected from one of p-phenylenediamine (Pa), benzidine (Bd), and 4,4”-diamino-p-terphenyl (Dt); wherein, the shell layer is carboxylated (-COOH) modified.

[0007] Preferably, sodium chloroacetate is used for the carboxylation modification.

[0008] Preferably, the pore size of the magnetic carboxylated covalent organic framework is 2-4 nm.

[0009] Preferably, the pore size of the magnetic carboxylated covalent organic framework is 2.5-3.9 nm.

[0010] In some specific embodiments, the pore size of the magnetic carboxylated covalent organic framework is 2.57 nm, 3.02 nm or 3.89 nm. Among them, when the pore size is 2.57 nm, the exclusion effect on proteins is the best, and when the pore size is 3.02 nm, the adsorption effect on immunosuppressants is the best.

[0011] The magnetic carboxylated covalent organic framework within the above pore size range can efficiently extract immunosuppressants in whole blood samples and has the advantage of high protein exclusion rate. In particular, when the pore size of the magnetic carboxylated covalent organic framework is 2.9-3.2 nm, the enrichment effect on immunosuppressants and the exclusion effect on proteins are better.

[0012] It is found in the research of the present invention that by selecting amine monomers with different numbers of benzene rings, namely p-phenylenediamine, benzidine and 4,4”-diamino-p-terphenyl, to prepare covalent organic frameworks (COFs), the pore size thereof can be precisely regulated; in particular, after carboxylation modification with sodium chloroacetate, when used for extracting immunosuppressants (such as cyclosporine A, tacrolimus and rapamycin) in whole blood samples, the enrichment efficiency can be significantly improved, and proteins in the biological sample system can be excluded, thus omitting the protein precipitation step, reducing the detection error caused by protein interference, simplifying the detection process, and saving the time and cost of pretreatment.

[0013] The present invention also provides a preparation method of the above magnetic carboxylated covalent organic framework, including:

[0014] Providing magnetic Fe 3 O 4 ;

[0015] Coating the surface of the magnetic Fe 2 with silicon dioxide (SiO 3 O 4 ) to obtain Fe 3 O 4 @SiO 2 ;

[0016] Dispersing the Fe 3 O 4 @SiO 2 in a solvent, and then adding the monomer 2,4,6-triformylphloroglucinol (Tp) and the dissolved amine monomer, and reacting to generate a magnetic covalent organic framework MCOFs;

[0017] Reacting the magnetic covalent organic framework MCOFs with sodium chloroacetate to obtain a magnetic carboxylated covalent organic framework (A-MCOFs).

[0018] In the present invention, the magnetic Fe 3 O 4 can be prepared by existing conventional methods.

[0019] In some embodiments, the magnetic Fe 3 O 4 is prepared from ferric chloride hexahydrate, anhydrous sodium acetate and ethylene glycol as raw materials, and the mass ratio of the three is (1-2):(2-3):(10-20).

[0020] In some specific embodiments, the magnetic Fe 3 O 4The preparation method includes: adding ferric chloride hexahydrate and anhydrous sodium acetate into ethylene glycol and stirring vigorously; then heating at 180 - 220 °C (such as 200 °C) for 6 - 10 hours (such as 8 hours), cooling to room temperature after the reaction ends, and washing with ethanol and ultrapure water; the dried black powdery product is magnetic Fe 3 O 4 。

[0021] In the present invention, the Fe 3 O 4 @SiO 2 can be prepared by existing conventional methods. For example, the classic tetraethyl orthosilicate hydrolysis method is used to coat the surface of Fe 2 ) with silicon dioxide (SiO 3 O 4 .

[0022] In some specific embodiments, the preparation method of the Fe 3 O 4 @SiO 2 includes: adding magnetic Fe 3 O 4 to dilute hydrochloric acid (0.1 mol L -1 ), then dispersing Fe 3 O 4 in a mixed solution of ultrapure water, ethanol, and ammonia water (concentration 25 - 28%), and performing ultrasonic treatment. Then tetraethyl orthosilicate (TEOS) is added and mechanically stirred; the obtained product is collected with a magnet, washed alternately with ultrapure water and ethanol, and then the product is dried under vacuum at 60 °C (such as 12 hours), and the powdery product collected is Fe 3 O 4 @SiO 2 . Specifically, the mass ratio of the ultrapure water, ethanol, and ammonia water is 16:65:1.

[0023] Preferably, the solvent for preparing magnetic covalent organic frameworks MCOFs is 1,4 - dioxane.

[0024] Preferably, when preparing magnetic covalent organic frameworks (MCOFs), the molar ratio of monomer 2,4,6 - trimethoxybenzoylphloroglucinol (Tp) to the dissolved amine monomer is 2:(2.5 - 3.5), preferably 2:3.

[0025] Preferably, when preparing magnetic covalent organic frameworks (MCOFs), acetic acid is also added as a catalyst.

[0026] Preferably, when preparing magnetic covalent organic frameworks (MCOFs), the reaction temperature is 15 - 30 °C, such as 25 °C. The reaction time can be 1 - 5 days, such as 3 days.

[0027] In some specific embodiments, the method for preparing the magnetic covalent organic framework (MCOFs) includes: dispersing the Fe 3 O 4 @SiO 2 in 1,4-dioxane, and then adding a 1,4-dioxane suspension of the monomer 2,4,6-triformylphloroglucinol (Tp) and a 1,4-dioxane suspension of the dissolved amine monomer; subjecting the materials to ultrasonic treatment to ensure sufficient contact; adding the catalyst acetic acid; reacting at 15 - 30 °C for 1 - 5 days; performing magnetic separation; washing the separated product with N,N'-dimethylformamide and ethanol, and drying in vacuum (60 °C, 48 h) to obtain the magnetic covalent organic framework (MCOFs).

[0028] Preferably, the mass ratio of the magnetic covalent organic framework MCOFs to sodium chloroacetate is 1:(45 - 55), for example, 1:50.

[0029] In some specific embodiments, the method for reacting the magnetic covalent organic framework MCOFs with sodium chloroacetate includes: mixing the magnetic covalent organic framework MCOFs, sodium chloroacetate, and an aqueous sodium hydroxide solution (e.g., 1.5 M) (with vigorous shaking), reacting; performing magnetic separation on the obtained product, washing with distilled water and ethanol to remove unreacted sodium chloroacetate and sodium hydroxide, and drying in vacuum (24 h at 60 °C) to obtain the magnetic carboxylated covalent organic framework (A-MCOFs).

[0030] The present invention also includes the magnetic carboxylated covalent organic framework prepared by the above method.

[0031] The present invention also includes the application of the above magnetic carboxylated covalent organic framework in detecting whole blood samples.

[0032] Specifically, the above magnetic carboxylated covalent organic framework is used to detect immunosuppressants in whole blood samples, such as cyclosporine A, tacrolimus, and rapamycin.

[0033] The present invention also provides a method for detecting immunosuppressants in whole blood samples, including:

[0034] S1: Sample pretreatment;

[0035] Performing magnetic solid-phase extraction (M-SPE) on the whole blood sample using the above magnetic carboxylated covalent organic framework to obtain a pretreated solution;

[0036] S2: Performing LC-MS / MS (liquid chromatography tandem mass spectrometry) detection on the pretreated solution to obtain the content of immunosuppressants in the whole blood sample.

[0037] Preferably, in step S1: during sample pretreatment, 2.5 - 15.0 mg of the above-mentioned magnetic carboxylated covalent organic framework is used for every 50 μL of whole blood sample, and more preferably 12.5 mg.

[0038] Preferably, in step S1: during sample pretreatment, in the presence of a buffer solution, the magnetic carboxylated covalent organic framework is used for magnetic solid-phase extraction of the whole blood sample; the pH of the buffer solution is 3 - 11, and preferably the pH is 9.

[0039] Preferably, the buffer solution is 0.85 - 0.9% physiological saline.

[0040] Preferably, the volume ratio of the buffer solution to the whole blood sample is (10 - 20):1, and more preferably 15:1.

[0041] Preferably, in step S1: during sample pretreatment, the time for magnetic solid-phase extraction is 2 - 18 min, more preferably 2 - 10 min, and further preferably 10 min.

[0042] The whole blood sample described in the present invention includes whole blood and serum.

[0043] In some embodiments, the detection method of the immunosuppressant in the whole blood sample includes:

[0044] S1: Sample pretreatment;

[0045] Mix the magnetic carboxylated covalent organic framework, the whole blood sample and the buffer solution, vortex to make the magnetic carboxylated covalent organic framework uniformly dispersed in the whole blood sample; the time for magnetic solid-phase extraction is 2 - 18 min; use an external magnet for magnetic separation, discard the supernatant, and obtain a precipitate;

[0046] Elute the precipitate with an elution solvent (such as vortex for 10 - 15 min), use an external magnet for magnetic separation, blow-dry the supernatant with liquid nitrogen, re-dissolve with a reconstitution solution, and disperse evenly to obtain a pretreated solution;

[0047] S2: Perform LC-MS / MS detection on the pretreated solution to obtain the content of the immunosuppressant in the whole blood sample.

[0048] Preferably, the elution solvent is methanol, acetonitrile, a methanol solution with 2% acetic acid by volume, a methanol solution with 4% acetic acid by volume, a methanol solution with 2% ammonia water by volume, or a methanol solution with 4% ammonia water by volume.

[0049] Preferably, the reconstitution solution is an aqueous methanol solution, where the volume ratio of methanol to water is 7:3.

[0050] Preferably, the detection conditions for liquid chromatography:

[0051] Use a 100 mm × 2.1 mm, 5 μm Waters X bridge C18 column; the injection volume for auto - sampler injection: 10 μL.

[0052] Mobile phase A is an aqueous solution containing 0.1% (v / v) formic acid and 0.1% (v / v) 1 mM ammonium acetate; mobile phase B is a methanol solution containing 0.1% (v / v) formic acid.

[0053] Specifically, the mobile phase A is prepared from 998 mL of ultrapure water, 1 mL of 1 M ammonium acetate solution and 1 mL of formic acid.

[0054] Specifically, the mobile phase B is prepared from 999 mL of methanol + 1 mL of formic acid.

[0055] The flow rate is 0.4 mL / min.

[0056] Gradient elution:

[0057] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 35-45 55-65 0.6 35-45 55-65 1.3 0-5 95-100 3.0 0-5 95-100 3.5 35-45 55-65 4.0 35-45 55-65

[0058] Preferably, the gradient elution is as follows:

[0059] Time (min) Mobile Phase A (%) Mobile Phase B (%) 0 40 60 0.6 40 60 1.3 0 100 3.0 0 100 3.5 40 60 4.0 40 60

[0060] Detection under the above - optimized chromatographic conditions can significantly shorten the detection time, reduce the sample detection amount and improve the recovery rate.

[0061] Mass spectrometry parameters: nebulizing gas (GS1): 50 L / min; heating gas (GS2): 50 L / min; source temperature (TEM): 400 °C; ion spray voltage (IS): 5500; curtain gas (CUR): 20 L / min; collision gas (CAD): 8 L / min; scanning mode: multiple reaction monitoring MRM; ion source: ESI, positive ion.

[0062] While enriching immunosuppressants, the magnetic carboxylated covalent organic framework of the present invention can exclude proteins in whole - blood samples in one step, saving the protein - removing step. The present invention uses magnetic carboxylated covalent organic framework materials as magnetic solid - phase extraction materials, and adopts high - performance liquid chromatography - tandem mass spectrometry to establish an analytical method for three immunosuppressants in whole - blood samples. This method is simple to operate, saves pretreatment time and cost, has good extraction effect, is green and environmentally friendly. The raw materials used in the present invention are cheap and easily available, the application environment is friendly, and the market prospect is broad. The materials of the present invention are expected to become special materials for biological sample extraction. Brief Description of the Drawings

[0063] Figure 1 It is the chromatogram of three immunosuppressants in Example 4.

[0064] Figure 2 Scanning electron microscope images of the magnetic carboxylated covalent organic frameworks, magnetic Fe 3 O 4 and Fe 3 O 4 @SiO 2 prepared in Examples 1 - 3.

[0065] Figure 3 Infrared spectra of the magnetic carboxylated covalent organic frameworks, magnetic Fe 3 O 4 and Fe 3 O 4 @SiO 2 prepared in Examples 1 - 3.

[0066] Figure 4 Pore size distribution diagrams of the magnetic carboxylated covalent organic frameworks prepared in Examples 1 - 3.

[0067] Figure 5 Comparison diagrams of the adsorption and exclusion effects of magnetic carboxylated covalent organic frameworks with different pore sizes prepared in Examples 1 - 3.

[0068] Figure 6 Diagram showing the effect of the amount of the magnetic carboxylated covalent organic framework prepared in the examples of the present invention on the extraction effect.

[0069] Figure 7 Diagram showing the effect of the buffer pH on the extraction effect in Experiment 4.

[0070] Figure 8 Diagram showing the effect of the extraction time on the extraction effect in Experiment 5.

[0071] Figure 9 Diagram showing the effect of the type of elution solvent on the extraction effect in Experiment 6. Detailed implementation manners

[0072] The following examples will further illustrate the present invention, but do not limit the present invention accordingly.

[0073] Example 1

[0074] This example provides a magnetic carboxylated covalent organic framework, and its preparation method is as follows:

[0075] 1) First, add 5.4 g of ferric chloride hexahydrate and 11.5 g of anhydrous sodium acetate to 100 mL of ethylene glycol and stir vigorously for 60 minutes. Then transfer the well - stirred solution to the inner liner of a high - pressure reaction kettle and heat at 200 °C for 8 hours. After the reaction is completed and cooled to room temperature, wash 5 times each with ethanol and ultrapure water. Dry in vacuum at 60 °C for 12 hours, and the dried black powdery product is magnetic Fe 3 O4 .

[0076] 2) According to the hydrolysis method of ethyl orthosilicate, silicon dioxide (SiO 2 ) for Fe 3 O 4 The surface coating method is as follows: take the magnetic Fe synthesized in the previous step 3 O 4 , added to 200 mL of dilute hydrochloric acid (0.1 mol L -1 ), and then rinsed with ultrapure water to remove HCl on the surface of magnetic nanoparticles. 3 O 4 Dispersed in ultrapure water, ethanol, and ammonia (28%) (the mass ratio of ultrapure water, ethanol, and ammonia is 16:65:1), and ultrasonically treated. Then 1 mL of tetraethyl orthosilicate (TEOS) was added and mechanically stirred for 12 hours. The obtained product was collected with a magnet, washed alternately with ultrapure water and ethanol for 5 times, and then dried under vacuum at 60°C for 12 hours. The collected powdery product was Fe 3 O 4 @SiO 2 .

[0077] 3) Then, take the synthesized Fe 3 O 4 @SiO 2 Dispersed in 1,4-dioxane, 2 mol of aldehyde monomer 2,4,6-triformyl phloroglucinol (Tp) was dispersed in 1,4-dioxane, and then ultrasonically obtained a uniform suspension. Secondly, 3 mol of amine monomer p-phenylenediamine (Pa) was dispersed in 1,4-dioxane solution and added to the previously prepared suspension, and ultrasonically made it fully contact. Then 0.1 mL of acetic acid was slowly added as a catalyst, sealed at 25°C for 3 days without stirring, and powder was obtained by magnetic separation, washed with N,N'-diaminoformamide and ethanol to remove unreacted monomers, and finally dried in vacuum at 60°C for 48 hours to obtain magnetic covalent organic frameworks MCOFs.

[0078] 4) Finally, the prepared MCOFs and sodium chloroacetate were placed in a 50 mL centrifuge tube, and a 1.5 M sodium hydroxide aqueous solution was slowly added, and the mixture was mixed vigorously. The obtained material was magnetically separated, and the unreacted sodium chloroacetate and sodium hydroxide were removed with distilled water and ethanol, and vacuum dried at 60 ° C for 24 h. The magnetic carboxylated covalent organic framework was numbered A-MCOF-1. The mass ratio of MCOFs to sodium chloroacetate was 1:50.

[0079] Example 2

[0080] This example provides a magnetic carboxylated covalent organic framework. The difference in its preparation method from that of Example 1 is only that: the diamine monomer p-phenylenediamine (Pa) in Example 1 is replaced by benzidine (Bd).

[0081] The magnetic carboxylated covalent organic framework prepared in this example is numbered A-MCOF-2.

[0082] Example 3

[0083] This example provides a magnetic carboxylated covalent organic framework. The difference in its preparation method from that of Example 1 is only that: the diamine monomer p-phenylenediamine (Pa) in Example 1 is replaced by 4,4”-diamino-p-terphenyl (Dt).

[0084] The magnetic carboxylated covalent organic framework prepared in this example is numbered A-MCOF-3.

[0085] Example 4

[0086] In this example, the magnetic carboxylated covalent organic frameworks (A-MCOF) prepared in Examples 1-3 are used for magnetic solid-phase extraction.

[0087] Using A-MCOFs for magnetic solid-phase extraction, combined with liquid chromatography-tandem mass spectrometry to analyze three immunosuppressants in biological samples. The specific steps are as follows:

[0088] Take 12.5 mg of A-MCOFs in a 1.5 mL centrifuge tube. Add 750 μL of 0.9% saline buffer (pH = 9) and 50 μL of serum to the centrifuge tube (the concentrations of the three immunosuppressant drugs, cyclosporine A (CsA), tacrolimus (FK-506), and rapamycin (RPM), in the total system are all 200 ng / ml, which is the known spiked amount). Vortex for 10 min to evenly disperse the carboxylated MCOF in the sample. Use an external magnet to separate, pour out the supernatant, and reserve the precipitate.

[0089] Add 1 mL of methanol (elution solvent) to the above precipitate. Vortex for 15 min. After thorough washing, use an external magnet to separate. Pour the supernatant into a new centrifuge tube. After nitrogen blowing to dryness, re-dissolve with 1 mL of methanol: ultrapure water (70:30), disperse evenly, and inject the prepared reaction system into liquid chromatography-tandem mass spectrometry for detection. The liquid chromatography-tandem mass spectrometry conditions are as follows:

[0090] The detection conditions of liquid chromatography: Use Waters Xbridge C 18The chromatographic column is 100 mm×2.1 mm, 5 μm; the injection volume by the auto-sampler is 10 μL. Mobile phase A is composed of 998 mL of ultrapure water + 1 mL of 1 M ammonium acetate solution + 1 mL of formic acid, and mobile phase B is composed of 999 mL of methanol + 1 mL of formic acid. The elution program is shown in Table 1 below:

[0091] Table 1

[0092]

[0093] Mobile phase A is prepared from 998 mL of ultrapure water, 1 mL of 1 M ammonium acetate solution and 1 mL of formic acid. Mobile phase B is prepared from 999 mL of methanol + 1 mL of formic acid.

[0094] The mass spectrometry parameters are as follows: nebulizing gas (GS1): 50 L / min; heating gas (GS2): 50 L / min; source temperature (TEM): 400 °C; ion spray voltage (IS): 5500; curtain gas (CUR): 20 L / min; collision gas (CAD): 8 L / min; scan mode: multiple reaction monitoring MRM; ion source: ESI, positive ion. See Table 2 below for details.

[0095] Table 2

[0096]

[0097] The results detected in this example are shown in Table 3 below.

[0098] Table 3

[0099]

[0100] The chromatograms of 3 immunosuppressants (cyclosporine A, tacrolimus, rapamycin) are shown in Figure 1 .

[0101] Experiment 1

[0102] The scanning electron microscope images of the magnetic carboxylated covalent organic frameworks, magnetic Fe 3 O 4 and Fe 3 O 4 @SiO 2 prepared in Examples 1-3 are shown in Figure 2 , and the infrared images are shown in Figure 3 . Figure 3 In, the abscissa Wavenuber represents the wave number, and the ordinate Transmittance represents the transmittance.

[0103] Experiment 2

[0104] Test the exclusion effect of A-MCOFs on proteins.

[0105] Prepare a 1 mg / mL bovine serum albumin solution to simulate the serum environment. Weigh 12.5 mg of the magnetic carboxylated covalent organic frameworks prepared in Examples 1-3 into 1.5 mL centrifuge tubes respectively, add 1 mL of the bovine serum albumin solution, vortex for 10 min, separate using an external magnet, and take the supernatant into a new centrifuge tube.

[0106] Use the Folin-phenol method to determine the protein in the supernatant. Take 0.25 mL of the solution in the supernatant after enrichment centrifugation and place it in a test tube, add 0.25 mL of ultrapure water to dilute it by one time, then add 2.5 mL of Folin-phenol reagent A, mix well, let it stand at room temperature for 10 min, then add 0.25 mL of Folin-phenol reagent B, mix immediately, and let it stand at room temperature for 30 min. Then measure the absorbance value at a wavelength of 500 nm, and calculate the protein concentration in the enriched sample system by referring to the standard curve.

[0107] The protein removal rate of A-MCOFs is calculated using the following formula:

[0108] BSA rejection(%)=C 0 / C f ×100

[0109] BSA rejection represents the protein removal rate of A-MCOFs; C 0 represents the supernatant concentration after magnetic solid-phase extraction (MSPE) enrichment centrifugation, and C f represents the initial concentration of the added protein.

[0110] The measured protein rejection rate of A-MCOF-1 is 99.7%; the protein rejection rate of A-MCOF-2 is 99.3%; the protein rejection rate of A-MCOF-3 is 97.4%.

[0111] It shows that the carboxylated MCOF in the present invention has a good protein rejection effect, excluding proteins in the serum sample system outside the material while enriching drugs, eliminating the step of precipitating proteins with organic solvents in the traditional solid-phase extraction method.

[0112] Figure 4 is the pore size distribution of the magnetic carboxylated covalent organic frameworks prepared in Examples 1-3. Figure 4 In it, the abscissa PoreWidth represents the pore size, and the ordinate represents the pore volume. It can be seen from Figure 4 that the pore sizes (Pore Size) of the three magnetic carboxylated covalent organic framework materials A-MCOF-1, A-MCOF-2, and A-MCOF-3 synthesized from amine monomers with different numbers of benzene rings are 2.57 nm, 3.02 nm, and 3.89 nm respectively.

[0113] Figure 5 Comparison chart of the adsorption and exclusion effects of magnetic carboxylated covalent organic frameworks with different pore sizes prepared in Examples 1-3. Figure 5 In the figure, the abscissa Adsorbent represents the adsorbent, the ordinate Recovery represents the recovery rate, and BSA rejection represents the protein removal rate of A-MCOFs. Figure 5 It can be seen that as the pore size increases, the protein exclusion efficiency gradually decreases. Among them, both A-MCOF-1 and A-MCOF-2 show good exclusion effects. A-MCOF-1 has the best exclusion effect, and the pore size of A-MCOF-2 is sufficient to meet the required requirements.

[0114] Experiment 3

[0115] Effect of the dosage of the adsorbent on the drug recovery rate.

[0116] Weigh 2.5, 5.0, 7.5, 10.0, 12.5, and 15.0 mg of A-MCOF-2 (prepared in Example 2) into 50 μL of whole blood samples, and the concentrations of 3 immunosuppressants (cyclosporine A, tacrolimus, and rapamycin) in the samples are all 200 ng / mL.

[0117] Detect according to the same method as in Example 4. The detection results are shown in Figure 6 . From Figure 6 the results, it can be seen that when the dosage of A-MCOF-2 increases from 1 mg to 12.5 mg, the recovery rates of cyclosporine A, tacrolimus, and rapamycin increase with the increase of the adsorbent dosage, indicating that as the dosage of A-MCOF-2 increases, the number of adsorption sites increases and the adsorption capacity increases accordingly; but when the dosage of the adsorbent is greater than 12.5 mg, the recovery rate reaches equilibrium and remains basically unchanged. Considering various factors, the optimal dosage of the adsorbent magnetic carboxylated covalent organic framework relative to the 50 μL sample volume in the present invention is 12.5 mg.

[0118] Experiment 4

[0119] Effect of the pH value of the buffer solution on the drug recovery rate:

[0120] This experiment studied the effect of the pH of 0.9% normal saline (buffer solution) on the activity of the surface adsorption sites of the adsorbent.

[0121] Use the magnetic carboxylated covalent organic framework (A-MCOF-2) prepared in Example 2 and detect according to the same method as in Example 4. The extraction recovery results under the conditions of buffer solution pH values of 3, 5, 7, 9, and 11 were investigated respectively. The detection results are shown in Figure 7 . From Figure 7As can be seen from the results, from pH 3 to 9, the recovery rates of the three immunosuppressants (cyclosporine A, tacrolimus, rapamycin) gradually increase and reach equilibrium at pH 9. Since A-MCOF-2 is protonated under alkaline conditions and has electrostatic interaction with the drugs, the optimal pH of the extraction buffer is 9.

[0122] Experiment 5

[0123] Effect of extraction time on drug recovery rate:

[0124] In this example, the effect of extraction time on drug recovery rate was studied. The extraction time will affect the adsorption equilibrium between the drug to be measured and the modified MOF material, and thus affect the drug extraction recovery rate.

[0125] Using the magnetic carboxylated covalent organic framework (A-MCOF-2) prepared in Example 2, the detection was carried out according to the same method as in Example 4.

[0126] The recovery rates of the three immunosuppressants were investigated when the extraction times were 2, 6, 10, 14, and 18 min respectively. The detection results are shown in Figure 8 . From Figure 8 the results, it can be seen that when the extraction time increases from 2 min to 10 min, the recovery rates of all drugs increase. After continuing to extend the extraction time, the adsorption has reached equilibrium. Therefore, the optimal extraction time is 10 min.

[0127] Experiment 6

[0128] Investigation of the type of elution solvent on drug recovery rate.

[0129] This experiment studied the effect of different elution solvents on the elution efficiency of the drugs to be measured adsorbed on the carboxylated MCOF.

[0130] Using the magnetic carboxylated covalent organic framework (A-MCOF-2) prepared in Example 2, referring to the method in Example 4, the effects of replacing the elution solvent methanol with acetonitrile, methanol solution with 2% acetic acid by volume, methanol solution with 4% acetic acid by volume, methanol solution with 2% ammonia water by volume, and methanol solution with 4% ammonia water by volume on the extraction recovery rate were investigated. The detection results are shown in Figure 9 . Figure 9 In, Acetonitrile is acetonitrile, Methanol is methanol, Methanol + 2% HCOOH is methanol + 2% formic acid, Methanol + 4% HCOOH is methanol + 4% formic acid, Methanol + 2% NH 3 ·H 2 O is methanol + 2% ammonia water, Methanot + 4% NH 3 .H 2 O is methanol + 4% ammonia water.

[0131] As can be seen from Figure 9 the results, methanol has the best elution efficiency for the three immunosuppressants. Therefore, methanol is used as the best elution solvent.

[0132] Experiment 7

[0133] Methodological analysis performance.

[0134] Using the magnetic carboxylated covalent organic framework (A-MCOF-2) prepared in Example 2, referring to the method of Example 4, the linear range, fitting coefficient, detection limit, quantification limit and relative standard deviation of this method for detecting three immunosuppressants were studied in this experiment. The results are shown in Table 4.

[0135] Table 4 Methodological analysis performance

[0136]

[0137] As can be seen from the results in Table 4, tacrolimus and rapamycin showed good linearity in the range of 0.5 - 100 ng / mL -1 (cyclosporine A was 5.0 - 1000.0 ng / mL -1 ), and the correlation coefficient R 2 values of the standard curves all exceeded 0.99. The detection limits (LOD, S / N = 3) and quantification limits (LOQ, S / N = 10) of cyclosporine A, tacrolimus and rapamycin were 0.136 - 0.249 ng / mL -1 and 0.367 - 0.861 ng / mL -1 . The intra-day (n = 3) and inter-day (n = 3) precisions (RSD, %) of the spiked levels of immunosuppressants were 0.8 - 1.7% and 0.85 - 3.89%, respectively. The results indicate that the M-SPE-HPLC-MS / MS method based on A-MCOF-2 can be used to detect trace levels of immunosuppressants.

[0138] Experiment 8

[0139] This example was compared with other methods, focusing on the analyte, sample type, sample volume, sample pretreatment method, analysis time, recovery rate, precision and other factors. The results are shown in Table 5.

[0140] Table 5 Comparison with other detection methods

[0141]

[0142] CsA: Cyclosporine A; FK-506: Tacrolimus; RPM: Rapamycin; aDSPE-HPLC-MS / MS: Adsorptive dispersive solid-phase extraction-high performance liquid chromatography-tandem mass spectrometry; bPP: Protein precipitation; cLLE: Liquid-liquid extraction;

[0143] As can be seen from the results in Table 5, the method of Example 4 does not require the addition of organic reagents for protein precipitation, which is more environmentally friendly. In addition, compared with other methods, the method of the present invention has a shorter analysis time, simpler sample treatment, and comparable or better recovery and precision. The results show that the method of the present invention is an efficient pretreatment technology and a sensitive analytical method for detecting residual immunosuppressants in whole blood.

[0144] Example 10

[0145] Using the magnetic carboxylated covalent organic framework (A-MCOF-2) prepared in Example 2, the whole blood samples of 10 patients were analyzed and detected according to the method of Example 4, and the results were compared with the currently most recognized method (PP-UPLC-MS / MS).

[0146] The results are shown in Table 6. The relative standard deviation ranges from 0.7% to 12.5%, and the results between the two methods are basically the same. The accuracy and reliability of the MSPE-HPLC-MS / MS method developed by the present invention are confirmed, making it suitable for the analysis and identification of immunosuppressants in biological samples.

[0147] Table 6 Analysis and comparison of real samples

[0148]

[0149] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A magnetic carboxylated covalent organic framework, characterized in that: It is a core-shell structure, including: The magnetic core is Fe3O4@SiO2; The shell layer is a covalent organic framework formed by the reaction of the monomer 2,4,6-triformylphloroglucinol and a dissolving amine monomer; the dissolving amine monomer is selected from one of p-phenylenediamine, benzidine and 4,4"-diamino-p-terphenyl; Wherein, the shell layer is carboxyl-modified.

2. The magnetic carboxylated covalent organic framework according to claim 1, characterized in that The pore size of the magnetic carboxylated covalent organic framework is 2-4 nm; Preferably, the pore size of the magnetic carboxylated covalent organic framework is 2.5-3.9 nm; More preferably, the pore size of the magnetic carboxylated covalent organic framework is 2.57 nm, 3.02 nm or 3.89 nm.

3. The magnetic carboxylated covalent organic framework according to claim 1 or 2, characterized in that: The carboxylation modification was carried out using sodium chloroacetate.

4. The method for preparing the magnetic carboxylated covalent organic framework according to any one of claims 1 to 3, characterized in that: include: Provides magnetic Fe3O4; The surface of the magnetic Fe3O4 is coated with silicon dioxide to obtain Fe3O4@SiO2; The Fe3O4@SiO2 is dispersed in a solvent, and then a monomer 2,4,6-triformyl phloroglucinol and the dissolved amine monomer are added to react to generate a magnetic covalent organic framework MCOFs; The magnetic covalent organic framework MCOFs is reacted with sodium chloroacetate to obtain a magnetic carboxylation covalent organic framework.

5. The preparation method according to claim 4, characterized in that: When preparing the magnetic covalent organic framework, the molar ratio of the monomer 2,4,6-triformylphloroglucinol to the dissolving amine monomer is 2:(2.5-3.5), preferably 2:

3.

6. The preparation method according to claim 4 or 5, characterized in that: The mass ratio of the magnetic covalent organic framework MCOFs to sodium chloroacetate is 1:(45-55), preferably 1:50; Preferably, the method of reacting the magnetic covalent organic framework MCOFs with sodium chloroacetate comprises: mixing the magnetic covalent organic framework MCOFs, sodium chloroacetate and sodium hydroxide aqueous solution, reacting; magnetically separating the obtained product, washing with distilled water and ethanol to remove unreacted sodium chloroacetate and sodium hydroxide, and vacuum drying to obtain a magnetic carboxylated covalent organic framework.

7. Use of the magnetic carboxylated covalent organic framework according to any one of claims 1 to 3 in detecting whole blood samples; Preferably, the whole blood sample includes whole blood and serum; Preferably, the magnetic carboxylated covalent organic framework is used to detect immunosuppressants in whole blood samples, including cyclosporine A, tacrolimus, and rapamycin.

8. A method for detecting immunosuppressants in whole blood samples, characterized in that: include: S1: sample preparation; Performing magnetic solid phase extraction on a whole blood sample using the magnetic carboxylated covalent organic framework according to any one of claims 1 to 3 to obtain a pretreatment solution; S2: Performing LC-MS / MS detection on the pretreatment solution to obtain the content of immunosuppressants in the whole blood sample.

9. The detection method according to claim 8, characterized in that: Step S1: During sample pretreatment, 2.5-15.0 mg of the magnetic carboxylated covalent organic framework is used per 50 μL of whole blood sample, more preferably 12.5 mg; and / or, Step S1: During sample pretreatment, the whole blood sample is subjected to magnetic solid phase extraction using the magnetic carboxylated covalent organic framework in the presence of a buffer solution; the pH of the buffer solution is 3-11, more preferably pH 9; preferably, the buffer solution is 0.85-0.9% physiological saline; The volume ratio of the buffer to the whole blood sample is (10-20):1, preferably 15:1; and / or, Step S1: During sample pretreatment, the time of magnetic solid phase extraction is 2-18 min, more preferably 2-10 min, and further preferably 10 min.

10. The detection method according to claim 8 or 9, characterized in that: The method for detecting the immunosuppressant in the whole blood sample comprises: S1: sample preparation; The magnetic carboxylated covalent organic framework, the whole blood sample and the buffer solution are mixed and vortexed to make the magnetic carboxylated covalent organic framework evenly dispersed in the whole blood sample; the magnetic solid phase extraction time is 2-18 minutes; an external magnet is used for magnetic separation, and the supernatant is discarded to obtain a precipitate; The precipitate is eluted with an elution solvent, magnetically separated using an external magnet, the supernatant is blown dry with nitrogen, and redissolved with a redissolving solution to be evenly dispersed to obtain a pretreatment solution; S2: performing LC-MS / MS detection on the pretreatment solution to obtain the content of immunosuppressants in the whole blood sample; Preferably, the elution solvent is methanol, acetonitrile, a methanol solution with a volume ratio of 2% acetic acid, a methanol solution with a volume ratio of 4% acetic acid, a methanol solution with a volume ratio of 2% ammonia water, or a methanol solution with a volume ratio of 4% ammonia water; Preferably, the reconstituted solution is a methanol-water solution, wherein the volume ratio of methanol to water is 7:3; Preferably, the detection conditions of liquid chromatography are as follows: The chromatographic column is Waters X bridge C18 100mm×2.1mm Mobile phase A was an aqueous solution containing 0.1% (v / v) formic acid and 0.1% (v / v) 1 mM ammonium acetate; mobile phase B was a methanol solution containing 0.1% (v / v) formic acid; Gradient elution: Preferably, the gradient elution is as follows: 。

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