Cationic cof material and its application in detecting angiotensin and aldosterone

Abstract

This invention belongs to the field of bioassay technology, specifically relating to a cationic COF material and its application in the detection of angiotensin and aldosterone. Specifically, this invention successfully synthesizes a cationic covalent organic framework material with high specific surface area, abundant porous structure, and good thermal / chemical stability. Its surface quaternary ammonium salt cationic functional groups can selectively adsorb negatively charged angiotensin via electrostatic interactions and adsorb aldosterone via π-π conjugation and hydrophobic interactions. It exhibits excellent enrichment capacity and selectivity for four substances: Ang I, Ang II, Ang III, and Aldo, and can be recycled multiple times, effectively reducing the detection cost of large batches of clinical samples. By coupling the prepared SPE material with LC-MS / MS, a method for the simultaneous detection of Ang I, Ang II, Ang III, and Aldo in plasma was established, demonstrating good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to a cationic COF material and its application in the detection of angiotensin and aldosterone. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Angiotensin I (Ang I), angiotensin II (Ang II), angiotensin III (Ang III), and aldosterone (Aldo) are key components of the renin-angiotensin-aldosterone system (RAAS). They play indispensable roles in maintaining normal blood pressure, cardiovascular homeostasis, and electrolyte balance. As the core effector molecules of this system, angiotensin I, II, III, and aldosterone are of significant physiological importance in the body's response to changes in blood volume and in maintaining homeostasis.

[0004] Currently, the main methods for detecting angiotensin and aldosterone in plasma include radioimmunoassay, chemiluminescent immunoassay, and mass spectrometry. Among these, liquid chromatography-mass spectrometry (LC-MS / MS) combines the high separation efficiency of liquid chromatography with the high sensitivity and specificity of mass spectrometry. It enables simultaneous qualitative and quantitative analysis of multiple components, overcoming the shortcomings of single-component determination and cross-reactivity in immunoassays, making it the preferred technique for detecting trace substances in complex biological samples. However, the blood matrix is ​​complex, and angiotensin and aldosterone are mostly present at trace levels in plasma. Therefore, efficient sample pretreatment techniques are necessary to remove matrix interference and achieve the separation and enrichment of target analytes; otherwise, the accuracy and reproducibility of LC-MS / MS detection will be severely affected.

[0005] Solid-phase extraction (SPE) is a commonly used technique for biological sample pretreatment. Its core component is the adsorbent material, and the performance of the adsorbent material directly determines the SPE efficiency. Currently, SPE materials used for extracting angiotensin and aldosterone from plasma are mainly polymer packing materials such as Strata-X, Oasis MAX, and Oasis HLB. However, the inventors have discovered that some packing materials can only achieve the extraction of a single component or a few components, and cannot simultaneously enrich Ang I, Ang II, Ang III, and Aldo. Furthermore, general-purpose packing materials such as Oasis HLB have limited selectivity and enrichment capabilities, resulting in poor matrix removal. Imported SPE products are also expensive, leading to high costs for large-scale clinical testing. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a cationic COF material and its application in the detection of angiotensin and aldosterone. Specifically, this invention designs and synthesizes a quaternary ammonium salt cationic COF material based on the physicochemical properties of angiotensin and aldosterone molecules and the functionalization of ionic COFs. This material serves as a solid-phase extraction adsorbent for the selective separation and enrichment of Ang I, Ang II, Ang III, and Aldo in plasma. Simultaneously, LC-MS / MS analysis technology is used to establish a combined detection method for Ang I, Ang II, Ang III, and Aldo. This achieves efficient, rapid, and accurate combined detection of trace amounts of Ang I, Ang II, Ang III, and Aldo in plasma. Furthermore, the material is recyclable, reducing detection costs. Based on the above research results, this invention is thus completed.

[0007] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a cationic COF material, which is Py-BPy. 2+ -COF was prepared as follows: Py-TA and 2,2'-BPy-DCA were added to a first mixed solution containing mesitylene, 1,4-dioxane, and acetic acid. After degassing, the solution was purified by a single heating reaction to obtain Py-BPy-COF material. The Py-BPy-COF material was then dispersed in a second mixed solution of nitrobenzene and dibromoethane, and purified by a second heating reaction to obtain Py-BPy. 2+ -COF material.

[0008] In a second aspect, the present invention provides a solid-phase extraction adsorbent comprising at least the above-described cationic COF material.

[0009] A third aspect of the invention provides the application of the above-described cationic COF material or solid-phase extraction adsorbent in the enrichment and / or detection of angiotensin and aldosterone. The angiotensin includes Ang I, Ang II, and Ang III.

[0010] The aforementioned SPE material effectively overcomes the limitations of single or few components, and can simultaneously enrich the four core components of the RAAS system: Ang I, Ang II, Ang III, and aldosterone. At the same time, the material's targeted adsorption characteristics can effectively remove matrix interferences such as proteins and lipids in plasma, improve the purity of the target analytes, and reduce matrix effects for subsequent LC-MS / MS detection.

[0011] A fourth aspect of the present invention provides a method for the combined detection of angiotensin and aldosterone, the method comprising: performing solid-phase extraction on a pretreated plasma sample using the aforementioned cationic COF material or solid-phase extraction adsorbent, and detecting the sample based on a liquid chromatography-mass spectrometry method.

[0012] The beneficial technical effects of one or more of the above technical solutions are as follows: The above-mentioned technical solution successfully synthesized a cationic covalent organic framework material, Py-BPy². + -COF, a material with high specific surface area, abundant porous structure, and good thermal / chemical stability, exhibits excellent selective adsorption of negatively charged angiotensin via electrostatic interactions and adsorption of aldosterone via π-π conjugation and hydrophobic interactions, making it an ideal SPE adsorbent. It demonstrates excellent enrichment capacity and selectivity for Ang I, Ang II, Ang III, and Aldo, and can be recycled multiple times, effectively reducing the detection cost of large batches of clinical samples. A novel method for the simultaneous detection of Ang I, Ang II, Ang III, and Aldo in plasma was established by coupling the prepared SPE material with LC-MS / MS. Method validation results show a wide linear range (e.g., Ang I 0.25-25 ng / mL). -1 The detection limit is low (as low as 0.005 ng / mL). -1 It has high recovery rate (80.1%-94.9%) and good precision (RSD<10%), which can fully meet the needs of clinical trace detection and has good practical application value and social benefits. Attached Figure Description

[0013] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0014] Figure 1 In embodiment (a) of the present invention, Py-BPy 2+ TEM image of Py-BPy-COF; (b) TEM image of Py-BPy-COF; (c) TEM image of Py-BPy-COF. 2+ SEM image of Py-BPy-COF; (d) SEM image of Py-BPy-COF; (ei) SEM image of Py-BPy 2+- EDS surface scan elemental distribution of COF; EDS surface scan elemental distribution of (jm)Py-BPy-COF.

[0015] Figure 2 In the embodiments of the present invention, (a) Py-BPy-COF and Py-BPy 2+ (a) FTIR plot of Py-BPy-COF; (b) Py-BPy-COF and Py-BPy 2+ -COF XRD pattern; (c)Py-BPy 2+ -COF BET plot; (d)Py-BPy 2+(e) Pore size distribution of Py-BPy-COF; (f) Pore size distribution of Py-BPy-COF; (g) Py-BPy-COF 2+ (h) TG plot of Py-BPy-COF; (i) TG plot of Py-BPy-COF and Py-BPy 2+ Zeta potential diagram of -COF.

[0016] Figure 3 Py-BPy in this embodiment of the invention 2+ XPS plots of Py-BPy-COF and Py-BPy-COF: (a) Py-BPy 2+ -COF total XPS plot; (b)Py-BPy 2+ -COF C 1s plot; (c)Py-BPy 2+ -COF N 1s plot; (d) Py-BPy 2+ -COF O 1s plot; (e) Py-BPy 2+ (f) Br plot of Py-BPy-COF; (g) C 1s plot of Py-BPy-COF; (h) N 1s plot of Py-BPy-COF; (i) O 1s plot of Py-BPy-COF.

[0017] Figure 4 For the optimization of SPE conditions in this embodiment of the invention, (a) sample solution pH, (b) sample loading rate, (c) type of eluent, (d) acetonitrile ratio of eluent, (e) elution rate, and (f) eluent volume.

[0018] Figure 5 The figure shows the changes in the recovery rates of Ang I, Ang II, Ang III and Aldo during the solid-phase extraction cycle in this embodiment of the invention.

[0019] Figure 6 Py-BPy in this embodiment of the invention 2+ -Adsorption isotherms of COF for four substances.

[0020] Figure 7 The Langmuir and Freundlich adsorption models for Aldo, Ang I, Ang II, and Ang III are shown in the embodiments of the present invention.

[0021] Figure 8 Py-BPy in this embodiment of the invention 2+ Adsorption kinetics curves of -COF for Aldo, Ang I, Ang II, and Ang III.

[0022] Figure 9These are the fitting curves of the pseudo-first-order and pseudo-second-order adsorption kinetic models of Aldo, AngⅠ, AngⅡ, and AngⅢ in the embodiments of the present invention.

[0023] Figure 10 The optimal configuration in this embodiment of the invention is (a) Py-BPy. 2+ -COF; (b) Ang I and Ang II fragment molecules; (c) Ang III fragment molecules; (d) Aldo.

[0024] Figure 11 The adsorption conformation simulation in this embodiment of the invention is (a) Py-BPy 2+ -COF with Ang I and Ang II fragment molecules; (b)Py-BPy 2+ -COF and AngIII fragment molecules; (c)Py-BPy 2+ -COF and Aldo; (d) Mapping function symbols in the IGMH diagram ( λ 2) ρ Common explanations of coloring methods. Detailed Implementation

[0025] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for the purpose of describing specific embodiments and not for limiting the scope of protection of the present invention.

[0027] In a typical embodiment of the present invention, a cationic COF material is provided, which is Py-BPy. 2+ -COF was prepared as follows: Py-TA and 2,2'-BPy-DCA were added to a first mixed solution containing mesitylene, 1,4-dioxane, and acetic acid. After degassing, the solution was purified by a single heating reaction to obtain Py-BPy-COF material. The Py-BPy-COF material was then dispersed in a second mixed solution of nitrobenzene and dibromoethane, and purified by a second heating reaction to obtain Py-BPy. 2+ -COF material.

[0028] A target material with quaternary ammonium salt cationic functional groups was successfully synthesized via a two-step method. The preparation process was controllable, and the post-modification strategies were highly compatible, effectively preserving the basic framework of the material. The cationic COF material exhibits a stacked rod-like microstructure, high specific surface area, and good thermal stability. The quaternary ammonium salt cationic functional groups are strongly positively charged and can specifically bind negatively charged angiotensin via electrostatic interactions, providing a structural basis for targeted adsorption. The stacked rod-like morphology increases the contact area between the material and the sample, improving adsorption efficiency; while the high specific surface area provides abundant adsorption sites, enhancing the enrichment capacity of the target analyte; the good thermal stability allows it to withstand solvent elution and centrifugation during solid-phase extraction, and it is also suitable for the high-temperature detection conditions of subsequent LC-MS / MS, preventing material degradation and sample contamination.

[0029] The mass-to-volume ratio of Py-TA and 2,2'-BPy-DCA to the first mixed solution is 50-60:40-50:1-10, mg / mg / mL; preferably 56.7:42.5:5.5. This ratio range effectively avoids the problems of excessively high monomer concentration leading to agglomeration and excessively low concentration leading to low yield, ensuring the uniformity of the framework bonding; thus, it enables high crystallinity of the COF framework and improves the pore order of the material.

[0030] The volume ratio of mesitylene, 1,4-dioxane, and acetic acid is 1-10:1-10:1, preferably 5:5:1. Mesitylene and 1,4-dioxane are good mixed solvents, and acetic acid is used as a Schiff base reaction catalyst. Optimizing the ratio of the three can accelerate the reaction process, shorten the skeleton synthesis time, and effectively improve the crystallinity and pore regularity of the product.

[0031] The conditions for a single heating reaction are: reacting at 100-150℃ for 1-5 days, preferably at 120℃ for 3 days.

[0032] The secondary heating reaction conditions are: reacting at 200-250℃ for 12-36 h, preferably at 210℃ for 24 h.

[0033] In this invention, the initial heating temperature and time range conform to the kinetic characteristics of the Schiff base reaction, avoiding incomplete reactions due to excessively low temperatures and side reactions due to excessively high temperatures. The optimized conditions enable complete bonding of the COF basic framework, ensuring the stability of the framework structure. The secondary heating is a quaternization modification reaction, which effectively activates the alkylation reaction between dibromoethane and the pyridine nitrogen sites of Py-BPy-COF, achieving efficient grafting of cationic functional groups. The optimized reaction time enables full coverage of the quaternization modification, ensuring the density of positive potential points on the material surface and improving subsequent adsorption performance.

[0034] The purification process includes acetone washing and drying. For example, the acetone can be washed 2-3 times, and the drying can be vacuum drying, such as vacuum drying at 30-50°C (preferably 40°C) for 1-5 days (preferably 2 days). No specific limitations are specified here.

[0035] In another specific embodiment of the present invention, a solid-phase extraction adsorbent is provided, wherein the solid-phase extraction adsorbent comprises at least the above-mentioned cationic COF material. In practical applications, the solid-phase extraction adsorbent can be loaded in a solid-phase extraction plate or a solid-phase extraction column, and no specific limitation is made herein.

[0036] In another specific embodiment of the present invention, the application of the above-mentioned cationic COF material or solid-phase extraction adsorbent is provided in the enrichment and / or detection of angiotensin and aldosterone. The angiotensin includes Ang I, Ang II, and Ang III.

[0037] The aforementioned SPE material effectively overcomes the limitations of single or few components, and can simultaneously enrich the four core components of the RAAS system: Ang I, Ang II, Ang III, and aldosterone. At the same time, the material's targeted adsorption characteristics can effectively remove matrix interferences such as proteins and lipids in plasma, improve the purity of the target analytes, and reduce matrix effects for subsequent LC-MS / MS detection.

[0038] In another specific embodiment of the present invention, a method for joint detection of angiotensin and aldosterone is provided, the method comprising: performing solid-phase extraction on a pretreated plasma sample using the above-mentioned cationic COF material or solid-phase extraction adsorbent, and detecting it based on liquid chromatography-mass spectrometry.

[0039] The method for pretreating plasma includes adding plasma to a working buffer containing soybean trypsin inhibitor and benzyl sulfonyl chloride for incubation and then terminating the incubation. The incubation temperature is 30-40℃, and the incubation time is 1-5 hours. Soybean trypsin inhibitor (SBIT) inhibits the activity of trypsin in plasma, and benzyl sulfonyl chloride (PMSF) inhibits the activity of serine proteases. The two work synergistically to effectively prevent angiotensin in plasma from being degraded by proteases, ensuring the recovery rate of the target analyte.

[0040] During solid-phase extraction adsorption, the pH of the loading solution is 7.5-9.5 (preferably 8.5), and a 50-90% acetonitrile aqueous solution (preferably 80% acetonitrile aqueous solution) is selected as the eluent. Furthermore, the eluent also contains 0.05-0.5% (preferably 0.1%) formic acid, which helps to adjust the pH of the eluent solution and prevents adsorption and degradation. Therefore, the eluent is an 80% acetonitrile aqueous solution containing 0.1% formic acid.

[0041] Liquid chromatography conditions: C18 column; mobile phase: phase A is 0.05-0.5% (preferably 0.1%) formic acid aqueous solution, phase B is acetonitrile solution; gradient elution program: 0-0.25 min, phase A 95%, phase B 5%; 0.25-2 min, phase B increases from 5% to 95%, then holds for 2 min; 4-4.1 min, phase B decreases from 95% to 5%, then holds for 1.4 min; The flow rate is 0.1-0.5 mL / min. -1 (Preferably 0.3 mL min) -1 The column temperature is 35-45℃ (preferably 40℃), and the injection volume is 10-30 μL (preferably 15 μL).

[0042] Mass spectrometry conditions: Ion source: electrospray ionization ESI (-); curtain gas (CUR): 10 psi; ionization voltage (IS): -3800 V; temperature (TEM): 350 °C; spray gas (GS1): 40 psi; scan mode: multiple reaction monitoring (MRM).

[0043] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0044] Example 1 Experimental Methods 1.1 Synthesis and Characterization of Materials 1.1.1 Material synthesis Synthesis of Py-BPy-COF: Py-TA (56.7 mg) and 2,2'-BPy-DCA (42.5 mg) were added to a 35 mL Pierrex tube containing a mixture of trimethylbenzene / 1,4-dioxane / 6 M acetic acid (volume ratio 5 / 5 / 1; 5.5 mL). The solution was then degassed by three freeze-evacuation-thawing cycles. The reaction was carried out at 120 °C for 3 days. After the reaction, the sample was washed three times with acetone (30 mL each time) and dried under vacuum at 40 °C for 48 hours to obtain the yellow Py-BPy-COF material.

[0045] Py-BPy 2+ Synthesis of Py-BPy-COF: 50 mg of Py-BPy-COF was dispersed in a mixed solution of nitrobenzene and dibromoethane (1.4 mol equivalents). The mixture was placed at 210 °C and reacted for 24 h. After the reaction was complete, the mixture was washed three times with acetone (30 mL each time) and dried under vacuum at 40 °C for 48 h to obtain dark brown Py-BPy. 2+ -COF material.

[0046] 1.1.2 Material Characterization To observe Py-BPy-COF and Py-BPy 2+ The morphological characteristics of COF were characterized using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images obtained using a SUPPA™ 55 (Zeiss, Germany) and an HT-7700 (Hitachi New Technology, Japan). Images from 500 to 4000 cm⁻¹ were recorded using a Nicolet 710 Fourier transform infrared spectrometer (Thermo Scientific, USA). -1 FTIR spectra within the specified wavelength range were obtained. X-ray diffraction (XRD) data were recorded using a D / max-Rb diffractometer (Rigaku, Japan) to observe the crystal structure of both materials. X-ray energy dispersive spectroscopy (EDS) data were recorded using an EACALAB Xi+ photoelectron spectrometer (Thermo Scientific, USA) to observe the content of each constituent element. The specific surface area (BET) and pore size distribution of TPB-DMTP-COF were measured using an ASAP2460 fully automated surface area and porosity analyzer (Micromeritics, USA). Thermogravimetric analysis (TG) was performed using a thermogravimetric analyzer to investigate the thermal stability of the material. The stepwise synthesis of Py-BPy was measured using a Zeta potentiometer (Malvern Panalytical, UK). 2+ The potential change of -COF ensures the expected structure Py-BPy. 2+ -COF was successfully prepared.

[0047] 1.2 Establishment of a 96-well solid-phase extraction plate and optimization of SPE conditions The dried Py-BPy 2+ -COF and polypropylene gaskets self-assemble into a 96-well plate. First, 1 mg of Py-BPy is loaded into each well. 2+-COF material was gently tapped in a 96-well plate to ensure even distribution. A polypropylene gasket was then placed on top of the material. After preparing the 96-well plate, following the basic solid-phase extraction procedure, the column was first rinsed with 400 μL of methanol for pre-activation, followed by equilibration with 400 μL of a weakly alkaline solution. The prepared test solution was then loaded through a 96-well positive pressure device. After loading, the column was rinsed with 200 μL of 80% acetonitrile and 0.1% formic acid solution for elution. The eluted solution was filtered through a 0.22 µm microporous membrane. Finally, 100 μL was added to 200 μL of pure water, diluted three times, and stored in a sample vial for later analysis. The experiment optimized several influencing factors during loading and elution (elution solvent type, volume, loading rate, elution rate, pH of the loading solution, and acetonitrile ratio in the eluent). Four substances, Ang I, Ang II, Ang III, and Aldo, were selected and spiking concentrations of 1.5, 0.15, 0.15, and 0.3 ng / mL were prepared. -1 The above spiking solution was added to a solution containing 1 mg Py-BPy. 2+ In a 96-well plate filled with COF packing material, after an SPE elution step, three replicates were performed for each concentration to investigate the recovery rate of the standard material spiked under different conditions.

[0048] 1.3 Establishment of LC-MS / MS method (1) Preparation of standard solutions and internal standard solutions: Weigh 1 mg each of Ang I, Ang II, and Ang III standards using a precision electronic balance of 0.0001 g / mL, and add 1 mL of 20% acetonitrile solution (containing 0.1% formic acid). Weigh 1 mg of Ald standard and add 1 mL of acetonitrile solution to prepare solutions with a concentration of 1 mg / mL for each standard. -1 Single standard stock solutions were prepared. Different volumes of the single standard stock solutions were transferred and diluted with 20% acetonitrile solution (containing 0.1% formic acid) to obtain Ang I, Ang II, Ang III, and Aldo concentrations of 2 μg / mL. -1 400, 400, 800 ng / mL -1 A mixed standard stock solution. Prepare Ang I at a concentration of 250 ng / mL. -1 The concentrations of Ang II and Ang III were 50 ng / mL. -1 Aldo concentration was 100 ng / mL -1 The mixed standard solution was serially diluted with 20% acetonitrile solution (containing 0.1% formic acid) to prepare Ang I concentrations of 0.05, 0.25, 0.5, 2.5, 5.0, 25.0, 50.0, 125, and 250.0 ng / mL. -1The concentrations of Ang II and Ang III were 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 25, and 50.0 ng / mL, respectively. -1 The standard solutions were prepared with Aldo concentrations of 0.02, 0.1, 0.2, 1.0, 2.0, 10.0, 20.0, 50.0, and 100.0 ng / mL. -1 A standard curve was plotted using gradient solutions of Ang I-[ 13 C6, 15 N4]、Ang II-[ 13 C6, 15 N4] and AngⅢ-[ 13 C6, 15 [N4] 0.1 mg of each standard was dissolved in 2 mL of 20% acetonitrile solution to prepare a solution with a concentration of 50 μg / mL. -1 For the single-compound internal standard stock solution, 0.1 mg of Aldo-d7 was weighed and dissolved in 1 mL of acetonitrile to prepare a solution with a concentration of 100 μg / mL. -1 Aldo internal standard stock solution. A suitable volume of the above single internal standard stock solution was transferred and diluted with 20% acetonitrile solution to obtain a solution containing Ang I-[ 13 C6, 15 [N4] (20 ng mL) -1 ), Ang II-[ 13 C6, 15 [N4] (4 ng mL) -1 ), AngⅢ-[ 13 C6, 15 [N4] (12 ng mL) -1 ) and Aldo-d7 (4 ng mL -1 The mixed internal standard stock solution was prepared by further diluting the mixed internal standard stock solution with 20% acetonitrile solution to finally prepare the internal standard working solution, wherein Ang I-[ 13 C6, 15 N4]、Ang II-[ 13 C6, 15 N4]、AngⅢ-[ 13 C6, 15 The concentrations of N4 and Aldo-d7 were 5 ng / mL. -1 1 ng mL -1 3 ng mL -1 and 1 ng mL -1 .

[0049] (2) Instrument conditions: This experiment uses a triple quadrupole mass spectrometer (TSQ Altis plus) for liquid chromatography, optimizes the instrument parameters, and establishes a liquid chromatography-mass spectrometry coupling method.

[0050] Column: Thermo Scientific™ Hypersil GOLD TM C18; Column temperature: 40℃; Flow rate: 0.3 mL / min -1 Mobile phase: 0.1% formic acid aqueous solution (A) - acetonitrile (B); Injection volume: 15 µL, gradient elution program is shown in Table 1.

[0051] Table 1 Gradient elution program

[0052] Mass spectrometry conditions: Ion source: electrospray ionization (ESI-); Curtain gas (CUR): 10 psi; Ionization voltage (IS): -3800 V; Temperature (TEM): 350 °C; Spray gas (GS1): 40 psi; Scan mode: Multiple reaction monitoring (MRM). The mass spectrometry parameters of the 8 substances are shown in Table 2.

[0053] Table 2 Mass Spectrometry Parameters of Eight Substances

[0054] 1.4 Sample Preprocessing Preparation of sample incubation buffer and stop solution: (1) Buffer solution: Accurately weigh 13.46 g Tris and 8.22 g EDTA, add to 88.8 mL of water, sonicate to dissolve, adjust the pH to 5.45-5.50 with acetic acid, and dilute to 100 mL. Store at 4℃. (2) Soybean trypsin inhibitor (SBIT) solution: Accurately weigh 1.0 mg SBIT and dissolve in 10 mL of water to prepare a solution with a concentration of 0.1 mg / mL. -1 The stock solution was aliquoted and stored at -20℃. (3) Benzyl sulfonyl chloride (PMSF) solution: Accurately weigh 0.175 g PMSF, dissolve it in 5 mL isopropanol, and prepare a solution with a concentration of 0.2 mol / L. -1 The stock solution should be stored at 4℃. (4) Working buffer solution: Take 10 μL, 0.1 mg / mL -1 200 μL of SBIT solution and 0.2 mol L -1 (5) Incubation termination solution: Take 0.5 mL of 20% acetonitrile solution containing 0.1% formic acid and add 49.5 mL of 2.5% (V / V) ammonia solution to obtain the incubation termination solution.

[0055] Take 1 mL of plasma and add 1 mL of working buffer. Vortex mix and incubate at 37°C for 3 hours. Then add 1 mL of incubation stop solution and vortex mix to stop the reaction, obtaining a pretreated plasma sample.

[0056] 1.5 Validation of the method for determining angiotensin and aldosterone in plasma using LC-MS / MS To obtain method calibration curves, plasma was used as the sample matrix for experimentation, and the method was validated using the internal standard method. Ang I was prepared at concentrations of 0.25, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, 15, and 25 ng / mL. -1 Ang II and Ang III at doses of 0.025, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75, 1, 1.5, and 2.5 ng / mL. -1 Aldo 0.05, 0.1, 0.15, 0.2, 0.5, 1, 1.5, 2, 3 and 5 ng mL -1 The sample solution. Ang I-[ 13 C6, 15 N4]、Ang II-[ 13 C6, 15 N4]、AngⅢ-[ 13 C6, 15 The concentrations of N4 and Aldo-d7 were 5 ng / mL. -1 1 ng mL -1 3 ng mL -1 and 1 ng mL -1 The internal standard solution was prepared. After sample loading and analysis, the plasma was finally analyzed by liquid chromatography-mass spectrometry. A calibration curve was plotted with concentration ratio on the x-axis and peak area ratio on the y-axis to obtain the linear range and correlation coefficient. The limit of detection (LOD) and limit of quantitation (LOQ) were defined using 3-fold and 10-fold signal-to-noise ratios, and the intra-day and inter-day relative standard deviations (RSDs) were determined. The results were obtained by measuring Ang I, Ang II, Ang III, and Aldo (1, 0.1, 0.1, 0.2 ng / mL). -1 Precision assessment was performed on samples at different concentration levels. Samples at each concentration level were measured 5 times per day for 5 consecutive days. The relative standard deviations of the 5 samples (within batch) and 5 batches (between batches) within the same batch were calculated.

[0057] Different concentrations of Ang I, Ang II, Ang III, and Aldo standards were added to normal low-value plasma to prepare test samples at low, medium, and high concentration levels. The concentrations of Ang I were 2.0, 10.0, and 20.0 ng / mL, respectively. -1The concentrations of Ang II were 0.2, 1.0, and 2.0 ng / mL, respectively. -1 The concentrations of Ang III were 0.2, 1.0, and 2.0 ng / mL, respectively. -1 The concentrations of Aldo were 0.4, 2.0, and 4.0 ng / mL, respectively. -1 Three concentration samples were tested on the same day, with each concentration sample tested three times, and blank plasma concentration was measured simultaneously. The recovery rate for each sample was calculated as (measured concentration / theoretical concentration) × 100%.

[0058] 1.6 Determination of actual samples To verify the practical applicability of the method, 10 clinically normal healthy volunteers (aged 20-40 years, 5 males and 5 females) without underlying RAAS-related diseases were randomly selected using the established method. Fasting venous blood was collected in the morning in a seated position into EDTA-K2 anticoagulant tubes and immediately centrifuged (25 ℃, 10 min, centrifugal force 3000×g). The concentrations of Ang I, Ang II, Ang III and Aldo in each plasma sample were detected and analyzed using the constructed SPE-LC-MS / MS separation and analysis method.

[0059] 1.7 Determination of Adsorbent Performance Py-BPy 2+ -COF was used as a solid-phase extraction adsorbent, and its performance was studied in this experiment. The sample solution used in the experiment was plasma, and the spiking concentrations of the mixed standard solution of Ang I, Ang II, Ang III, and Aldo were 1.5, 0.15, 0.15, and 0.3 ng / mL. -1 Each experiment was conducted in triplicate, with repeated SPE recycling experiments to study the effect of adsorbent reuse on the recovery rate of four substances.

[0060] 1.8 Study on Adsorption Mechanism 1.8.1 Isothermal Adsorption Experiment With Py-BPy 2+ SPE columns were prepared using COF as the adsorbent, with each column containing 5 mg of packing material. The columns were subjected to sample loading and elution according to the basic SPE procedure. The loading concentration gradient of Ang I was 3, 6, 12, 18, 24, 30, 36, and 42 ng / mL. -1 The loading concentration gradients of Ang II and Ang III were 0.6, 1.2, 2.4, 3.6, 4.8, 6, 7.2, and 8.4 ng / mL. -1 The loading concentration gradients for Aldo were 1.2, 2.4, 4.8, 7.2, 9.6, 12, 14.4, and 16.8 ng / mL. -1After filtration through a 0.22 μm filter membrane and dilution by 3 times, the concentrations of angiotensin and aldosterone in the filtrate were determined by LC-MS / MS. The adsorption capacity Qe (μg g) was calculated based on the concentrations of angiotensin and aldosterone in the solution before and after adsorption. -1 The calculation formula is as follows: Qe =(C0-Ce)V / m① Wherein, C0 and Ce are the concentrations (ng / mL) of the four substances in the solution before and after adsorption, respectively. -1 V is the volume (mL) of the mixed solution of Ang I, Ang II, Ang III, and Aldo; m is the mass (mg) of the adsorbent; Qe is the equilibrium adsorption capacity (μg / g) of the adsorbent for Ang I, Ang II, Ang III, and Aldo. -1 ).

[0061] 1.8.2 Adsorption Kinetics Experiment By setting different adsorption times of 0, 2, 5, 8, 10, 15, 20, and 40 min, the adsorption capacity Q at each time point was calculated. Based on the adsorption experimental data obtained at different adsorption times, an adsorption capacity-time curve was plotted with adsorption time t as the abscissa and adsorption capacity Q as the ordinate to investigate the Py-BPy adsorption capacity. 2+ The time-dependent adsorption process of AngⅠ, AngⅡ, AngⅢ and Aldo by -COF.

[0062] 1.8.3 Adsorption Model Fitting Based on experimental data of adsorption capacity and adsorption kinetics, Langmuir and Freundlich adsorption isotherm models, as well as pseudo-first-order and pseudo-second-order kinetic models, were fitted. The pseudo-first-order kinetic model is ln(Qe–Qt)=lnQe–k1t, where Qe is the equilibrium adsorption capacity, Qt is the real-time adsorption capacity, and k1 is the first-order kinetic adsorption rate constant. A fitting equation was obtained by plotting t against ln(Qe–Qt). The pseudo-second-order kinetic model is t / Qt=t / k2(Qe)2+t / Qe, where Qe is the equilibrium adsorption capacity, Qt is the real-time adsorption capacity, and k2 is the second-order kinetic adsorption rate constant. A fitting equation was obtained by plotting t against t / Qt. By combining experimental data and model fitting data, Py-BPy was analyzed. 2+ -Adsorption mechanism of COF with Ang I, Ang II, Ang III and Aldo.

[0063] 1.8.4 Hershfield Allocation (IGMH) Analysis To further study Py-BPy 2+The interactions between -COF and Ang I, Ang II, Ang III, and Aldo molecules were analyzed using the IGMH method, identifying intermolecular interaction regions through electron density analysis. First, the initial structures of the adsorbate molecules and the adsorbent surface were constructed, and the geometry was optimized. An implicit solvent model was used for solvation, and an aqueous solution model was used for the solution. Frequency calculations were then performed after optimization. The electron density of the system was obtained through quantum chemical calculations, and the electron density was divided into molecular fragments to quantify the contribution of each fragment to the interactions. Using the IGMH scheme, the interaction forces were decomposed, and key sites dominating adsorption were identified. Stable adsorption conformations were determined through energy distribution and geometric parameter statistics. Geometry optimization and binding energy calculations were performed using Gaussian software.

[0064] 2 Results and Analysis 2.1 Material Synthesis and Characterization Results The prepared Py-BPy² was analyzed using SEM and TEM. + The microstructure of -COF and its precursor Py-BPy-COF was systematically characterized. For example... Figure 1 As shown in (c, d), the SEM images reveal that the material exhibits a stacked rod-like structure. In contrast, the post-modified Py-BPy²... + -COF exhibits an increased diameter and a larger lateral dimension. Further TEM characterization results ( Figure 1 Consistent with this, a) and b) provide more detailed structural morphology information. As can be observed from the figures, after Br ion modification, the material still maintains a clear stacked rod-like morphology, but the width of the rods is indeed increased compared to the precursor. This morphological change may originate from the Br ions introduced during the post-modification process. - The ions occupy the crystalline channels of the material, causing local expansion and size growth without disrupting the overall framework. This result indicates that the proposed post-modification strategy has good structural compatibility, effectively preserving the original morphological characteristics of the material while introducing the target functional groups. Scanning electron microscopy was used to study Py-BPy²... + Elements in -COF and Py-BPy-COF were subjected to EDS surface scan ( Figure 1 (em). As can be seen from the figure, Py-BPy² + The C, N, and O elements in the covalent organic frameworks of -COF and Py-BPy-COF are evenly distributed, and Py-BPy² + -COF is characterized by the uniform dispersion of Br elements in its structure, reflecting the successful modification of Br ions.

[0065] The Py-BPy spectrum was analyzed and verified using Fourier transform infrared spectroscopy. 2+-COF and Py-BPy-COF feature functional groups. Figure 2 (a) is Py-BPy 2+ FTIR comparison spectra of -COF and Py-BPy-COF. At 1618 cm⁻¹ -1 At the specified location, characteristic absorption peaks of imine (C=N) groups were observed in both materials, indicating that a covalent organic framework structure was formed between the monomers through a Schiff base reaction. In Py-BPy... 2+ In the -COF spectrum, at 2921 cm⁻¹ -1 and 2849 cm -1 The characteristic stretching vibration of the methylene (-CH2) group is observed at the position, indicating that 1,2-dibromoethane was successfully attached to the COF backbone after the quaternization reaction.

[0066] X-ray diffraction analysis of Py-BPy 2+ Phase and crystal structure of samples of -COF and Py-BPy-COF. From Figure 2 As can be seen in (b), the absorption peak of Py-BPy-COF at a diffraction angle of 3.16° corresponds to the reflection from the (001) crystal plane. After the introduction of bromide ions, Py-BPy 2+ The crystal structure and electron density of -COF have changed, possibly due to the insertion of bromide ions leading to changes in interlayer spacing or reduced symmetry, which disrupts the diffraction conditions of this crystal plane, resulting in peak disappearance. The absorption peaks at diffraction angles of 6.42°, 8.72°, and 12.98° are located in Py-BPy². + The enhancement in -COF may be related to the strong scattering factor (more electrons) of bromide ions. When they are embedded in specific positions in the lattice, they increase the electron density of these crystal planes. Moreover, bromide ions may enhance the order of the material through electrostatic interaction, thereby enhancing the diffraction intensity of the corresponding crystal planes.

[0067] The Py-BPy adsorption-desorption experiment was used to analyze the Py-BPy composition. 2+ Specific surface area and porosity of -COF and Py-BPy-COF. From Figure 2 As can be seen from (cf), the BET values ​​for the two materials are 520.8 m. 2 g -1 and 2744.7m 2 g -1 This change may stem from the degree of functionalization of the material. As an unfunctionalized original COF framework, its high specific surface area originates from its inherently highly ordered porous crystal structure, providing abundant channels and a large internal surface area. Py-BPy... 2+The bromide ion moiety modified in -COF occupies the internal space or surface of the pores, introducing steric hindrance and slightly affecting the crystallinity of the framework, resulting in a decrease in specific surface area compared to the original COF. Surface charge properties were analyzed using zeta potentials. Figure 2 As shown in (i), the Zeta potential of Py-BPy-COF is approximately -17 mV, indicating that its surface carries a negative charge. This negative charge mainly originates from the high electron density of the imine bonds and pyridine nitrogen atoms in its framework, which may be due to slight deprotonation or preferential adsorption of anions in solution. After quaternization, Py-BPy 2+ The zeta potential of -COF shifted further negatively to approximately -25 mV. This phenomenon indicates that although a positively charged cyclic diquaternary ammonium salt structure was successfully introduced into the framework, the overall apparent charge of the material remained negative. This may be due to the fact that the numerous introduced positive charge centers acted as anchor points, more strongly adsorbing and enriching negatively charged counterions (such as Br⁻) in the solution. - The presence of ions and other anions leads to a dominance of negative charge contribution at the sliding surface measured at the Zeta potential. The significant change in the Zeta potential confirms that the quaternization reaction successfully altered the surface chemistry of the materials. The thermal stability of both materials was tested by thermogravimetric analysis. Figure 2 As shown in (g, h), Py-BPy 2+ The TG curves of Py-BPy-COF and Py-BPy-COF show a slight mass loss of about 1%-3% at approximately 100°C, mainly attributed to the evaporation of adsorbed moisture and the removal of residual solvent. With increasing temperature, Py-BPy... 2+ - The quality of COF materials decreases above 300°C. This stage mainly corresponds to the breaking of imine bonds, partial decomposition of the organic framework, and thermal decomposition of functional groups, but the quality can still be maintained above 80%.

[0068] Surface elements and chemical states were analyzed using XPS characterization. Figure 3 The middle ae graph is Py-BPy 2+ XPS characterization results of Py-BPy-COF: C 1s bands show two peaks at 284.8 eV and 285.8 eV, corresponding to C=N and CN, respectively. N 1s bands show three peaks at 399.5 eV, 400.8 eV, and 401.8 eV, corresponding to C=N, CN, and NH, respectively. O 1s bands show two peaks at 531.6 eV and 533.2 eV, corresponding to C=O and CO, respectively. Br bands show two peaks at 67.6 eV and 70.5 eV, attributed to bromide anions and brominated alkanes, respectively, confirming successful Br ion modification on the Py-BPy-COF surface. Figure 3The figure in Figure 1 shows the XPS characterization results of Py-BPy-COF. The C 1s peaks are divided into two peaks at 284.8 eV and 285.5 eV, corresponding to C=N and CN, respectively. The N 1s peaks are divided into two peaks at 399.3 eV and 400.3 eV, corresponding to C=N and CN, respectively. The O 1s peaks are divided into two peaks at 532.5 eV and 533.2 eV, corresponding to C=O and CO, respectively. From these characterization results, it can be seen that Py-BPy... 2+ -COF materials possess excellent structure and thermal stability, as well as high specific surface area, providing a solid materials science basis for solid-phase extraction applications.

[0069] 2.2 Establishment of the SPE-LC-MS / MS method 2.2.1 Optimization of SPE conditions This method optimizes several influencing factors during sample loading and analysis. The spiking concentrations of the four substances Ang I, Ang II, Ang III, and Aldo are 1.5 ng / mL. -1 0.15 ng mL -1 0.15 ng mL -1 0.3 ng mL -1 Three replicates were performed for each concentration to examine the recovery rate of the standard substance spiked and recovered samples under different conditions.

[0070] Ang I, Ang II, and Ang III have similar molecular configurations and are polypeptide compounds formed by the dehydration condensation of 7-10 amino acids, exhibiting amphoteric dissociation properties. When the solution pH is below the isoelectric point, the carboxyl group binds a proton, giving the compound a positive charge; conversely, when the solution pH is above the isoelectric point, the amino group donates a proton, resulting in a negative charge. Therefore, the pH of the loading solution is an important factor to consider. The experiment selected a pH range of 7.5-9.5, and the results are as follows... Figure 4 As shown in (a). For the four substances, a higher recovery rate was achieved at pH 8.5, which is due to Py-BPy. 2+ -COF carries a positive charge at this pH value, and it attracts the negative charge from the dissociation of angiotensin, thus generating adsorption affinity. Therefore, a pH value of 8.5 was chosen.

[0071] The type and amount of eluent affect the elution efficiency of the target analyte. This method selected three organic solvents (methanol, ethanol, and acetonitrile) as eluents. Analysis of adsorption forces necessitates considering both the polarity of the organic solvents and the elution strength. Figure 4(c) It can be seen that acetonitrile has a higher elution capacity for the four substances than methanol, while ethanol has the lowest elution capacity. This may be because acetonitrile is a moderately polar but strongly hydrophobic solvent, which can effectively break the interaction force between the material and the target substance, resulting in the strongest elution capacity. Therefore, acetonitrile was chosen as the eluent. To maintain the biological activity of angiotensin and obtain good elution results, the proportion of acetonitrile in the eluent was optimized in this method, using aqueous solutions of acetonitrile with volume percentages of 50%, 60%, 70%, 80%, and 90% as the eluent. Figure 4 (d) It can be seen that for the four substances, a higher recovery rate can be obtained by eluting with 80% acetonitrile. Therefore, the elution solution selected in the experiment is 80% acetonitrile, with 0.1% formic acid added. Adding formic acid can adjust the pH value of the elution solution. In addition, it can prevent adsorption and degradation during preparation, storage and transfer. Therefore, the final elution solvent is an 80% acetonitrile solution containing 0.1% formic acid.

[0072] Furthermore, the elution buffer volume was optimized in the experiment, with four volume gradients selected: 0.1, 0.2, 0.3, and 0.4 mL. Figure 4 (f) It can be seen that complete elution is not achieved when the eluent volume is 0.1 mL, while 0.2 mL, 0.3 mL, and 0.4 mL all achieve high recovery rates. Considering that the principle of optimizing the eluent volume is to reduce the amount of solvent while ensuring recovery, the eluent volume was determined to be 0.2 mL. This method investigated the effects of loading flow rate and elution flow rate on the solid-phase extraction effect, with the loading flow rate set in the range of 0.1–0.9 mL / min. -1 The elution flow rate should be set within the range of 0.05-0.4 mL / min. -1 The result is as follows Figure 4 (b) and Figure 4 As shown in (e), both figures demonstrate that the recovery rate of all substances is affected by the flow rate. As the flow rate increases, the recovery rate decreases. This is because increased flow rate leads to insufficient adsorption and desorption, while excessively slow flow rate affects the efficiency of sample pretreatment. Considering all factors, a loading rate and elution rate of 0.5 mL / min were selected. -1 and 0.2 mL min -1 .

[0073] 2.2.2 Methodological Validation of SPE-LC-MS / MS This method established the linear equation, linear range, LOD, LOQ, and precision for the determination of Ang I, Ang II, Ang III, and Aldo using SPE-LC-MS / MS. The data in the table show that Ang I, Ang II, Ang III, and Aldo are within the range of 0.25–25 ng / mL.-1 0.025-2.5 ng / mL -1 0.025-2.5 ng / mL -1 0.05-5 ng / mL -1 The method exhibited good linearity across the entire concentration range, with correlation coefficients ranging from 0.9905 to 0.9923. The LOD values ​​ranged from 0.005 to 0.01 ng / mL. 1 The LOQ value of the method ranged from 0.02 to 0.17 ng / mL. -1 The relative standard deviations within and between days were 3.6%-4.8% and 4.1%-5.1%, respectively, while the relative standard deviations within and between batches were 5.5%-8.1% and 8.0%-9.5%, respectively. This indicates that the method has a wide linear range, low detection limit, and high sensitivity.

[0074] Table 3 Analytical data using the LC-MS / MS method

[0075] 2.2.3 Spike Recovery Experiment This method was used to determine the blank content of plasma matrix, and recovery tests were conducted at low, medium, and high concentrations. As shown in Table 4, after deducting the background value of the blank sample, the recoveries of Ang I, Ang II, Ang III, and Aldo ranged from 83.8% to 94.9%, 80.1% to 88.0%, 81.0% to 90.4%, and 82.1% to 92.9%, respectively. This method demonstrated good accuracy and precision.

[0076] Table 4. Spike recovery experiments of Ang I, Ang II, Ang III, and Aldo.

[0077] 2.3 Determination of actual samples Ten healthy volunteers with normal clinical appearance and no underlying RAAS-related diseases were included in the experiment. Blood samples were collected from all subjects while they were seated, and the method was used for detection. Table 5 shows that the concentration ranges of Ang I, Ang II, Ang III, and Aldo in adult plasma were 5.31-12.8 ng / mL. -1 0.012-0.066 ng / mL -1 0.010-0.028 ng / mL -1 and 0.046-0.11 ng mL -1 Actual sample analysis results show that this method has satisfactory analytical results and can achieve combined quantitative analysis of Ang I, Ang II, Ang III and Aldo in plasma.

[0078] Table 5. Determination of Ang I, Ang II, Ang III, and Aldo levels in plasma

[0079] -: Not detected 2.4 Adsorbent Performance Experiment With Py-BPy 2+ Using COF as the adsorbent, this experiment investigated the reusability of the developed 96-well solid-phase extraction plate. Figure 5 As shown, in the initial 10 experiments, the recoveries of the four substances remained at a high level, ranging from 81.4% to 115.5%. However, with increasing usage, the recoveries of the four substances dropped below 80%. This decrease in recovery may be related to other non-target compounds present in the plasma matrix, which non-specifically adsorb onto the material, occupying binding sites and being difficult to remove by conventional elution steps. Furthermore, each washing, centrifugation, and filtration step causes a small amount of material loss. Although the loss is small in a single instance, after 10 cycles, this cumulative loss becomes quite significant, leading to a decrease in the total adsorption capacity. Therefore, the Py-BPy synthesized in this experiment... 2+ -COF material is used in the SPE sample pretreatment of plasma matrix and can be recycled up to 10 times while ensuring good recovery rate.

[0080] 2.5 Adsorption Mechanism Analysis 2.5.1 Isothermal Adsorption Experiment Py-BPy 2+ -The adsorption isotherms of COF for four substances are as follows: Figure 6 As shown. The concentration range of the four substances in the solution is 0-27.67 ng / mL. -1 Calculate the equilibrium adsorption capacity Qe (μg g) according to Formula 1. -1 Plotting Ce on the horizontal axis and Qe on the vertical axis, and using Langmuir (Equation 2) and Freundlich (Equation 3) models to perform nonlinear fitting on the experimental data.

[0081] from Figure 6 As can be seen from this, with the increase of the initial concentration, Py-BPy 2+ The adsorption capacity of the COF for the four substances gradually increased, ultimately reaching the maximum adsorption capacity at initial concentrations of 15, 4.8, 4.8, and 9.6 ng / mL. -1 Adsorption equilibrium was reached when the initial concentration was greater than 15, 4.8, 4.8, and 9.6 ng / mL. -1At this point, the adsorption capacity no longer changes, and the equilibrium adsorption capacities of Aldo, Ang I, Ang II, and Ang III are 9.16, 26.12, 5.18, and 5.02 μg g, respectively. -1 .

[0082] The experimental data were fitted using the Langmuir (Equation 2) and Freundlich (Equation 3) adsorption models, as shown in the following formulas: Ce / Qe = Ce / Qm + 1 / K L Qm② LnQe=lnK F + lnCe③ R L =1 / (1+K L ×C0)④ In the formula, Qe is the amount of adsorption at equilibrium, in μg / g. -1 ;K L These are Langmuir model constants, in mL and ng. -1 Ce represents the concentration of each substance in the equilibrium solution, in ng / mL. -1 Qm represents the maximum adsorption capacity calculated by the Langmuir model, in μg / g. -1 ;R L The feasibility of the adsorbent can be determined; K F is the Freundlich constant; n is the Freundlich constant, reflecting the adsorption strength.

[0083] The adsorption process was linearly fitted using Langmuir and Fredunlich methods with Ce as the x-axis and Ce / Qe as the y-axis, and ln Ce as the x-axis and ln Qe as the y-axis, respectively. R0 2 The ranges are 0.9799-0.9930 and 0.5320-0.7935, respectively, indicating that Py-BPy 2+ -COF adsorption of the four substances is more consistent with the Langmuir model ( Figure 7 This indicates that the four substances are in Py-BPy 2+ Adsorption on -COF is a monolayer with limited adsorption sites. Furthermore, Py-BPy... 2+ The maximum adsorption capacities calculated for the four substances in the COF adsorption experiments were 9.16, 26.12, 5.18, and 5.02 μg g, respectively. -1 The theoretical maximum adsorption capacities obtained by fitting the Langmuir model were 9.66, 28.2, 5.35, and 5.45 μg g. -1 (Table 6)

[0084] Table 6 Aldo, Ang I, Ang II, Ang III in Py-BPy 2+ Adsorption isotherm parameters on COF

[0085] R L Used to determine the feasibility of the adsorbent. When R L <0 indicates that the adsorption is irreversible; when 0 <R L <1 indicates that the adsorption is favorable, R L = 1 indicates that the adsorption is linear, when R = 1. L A value greater than 1 indicates that the adsorption is unfavorable. The R value calculated using Formula 4 is... L The values ​​range from 0.03 to 0.06 (Table 6), indicating that Py-BPy 2+ -COF exhibits good adsorption properties for Aldo, Ang I, Ang II, and Ang III. Furthermore, the strength factor (1 / n) in the Freundlich model reflects the ease of the adsorption process. Table 6 shows that the 1 / n values ​​(0.35-0.45) related to the strength of the adsorption driving force are less than 1, further confirming the effectiveness of Py-BPy. 2+ -COF readily adsorbs Aldo, Ang I, Ang II, and Ang III.

[0086] 2.5.2 Adsorption Kinetics Experiment At initial concentrations of 1.5, 0.15, 0.15, and 0.3 mg L... -1 In Aldo, Ang I, Ang II, and Ang III solutions, the concentrations of remaining Aldo, Ang I, Ang II, and Ang III after adsorption at different times (0-40 min) were measured, and the adsorption amount Qt (μg g) at a specific time was calculated. -1 Adsorption kinetics curves were plotted with time t (min) on the x-axis and Qt on the y-axis. Figure 8 shows the results of Py-BPy. 2+ -COF can achieve rapid adsorption of Aldo, Ang I, Ang II, and Ang III, with an equilibrium time of 5 min.

[0087] Simultaneously, the adsorption kinetic data were linearly fitted using pseudo-first-order kinetic (Equation 5) and pseudo-second-order kinetic (Equation 6) models, as shown in the following formulas: ln(Qe Qt)=lnQe k1t⑤ t / Qt=t / Qe+1 / k2 Qe 2 ⑥ Where Qe and Qt represent the equilibrium adsorption capacity and the adsorption capacity at t minutes, respectively (unit: μg g). -1 k1 and k2 are the adsorption rate constants for the pseudo-first-order and pseudo-second-order models, respectively.

[0088] Table 7 Aldo, Ang I, Ang II, and Ang III in Py-BPy 2+ Adsorption kinetic parameters on COF

[0089] Using ln(Qe) respectively Plotting Qt and t / Qt on the vertical axis and time t on the horizontal axis, linear fitting of pseudo-first-order and pseudo-second-order dynamic models is performed. For example... Figure 9 As shown in Table 7, the linear fit of the pseudo-second-order kinetic model (R² = 0.9701–0.9988) is better than that of the pseudo-first-order kinetic model (R² = 0.2781–0.6661). Furthermore, the Qe calibration values ​​of the pseudo-second-order kinetic model (11.54, 25.94, 5.40, 5.46 μg g) are also better. -1 The adsorption capacities are closer to the actual adsorption capacities (9.16, 26.12, 5.18, and 5.02 μg g). -1 Typically, pseudo-first-order kinetic models are used to describe adsorption mechanisms related to physisorption and diffusion, while pseudo-second-order kinetic models are used to describe chemisorption processes. Therefore, Py-BPy... 2+ The kinetic adsorption behavior of -COF on Aldo, Ang I, Ang II, and Ang III is essentially a relatively complex adsorption process, which may involve van der Waals forces, hydrogen bonds, and other forces, rather than simple physical adsorption.

[0090] 2.5.3 IGMH Analysis Because Ang I, Ang II, and Ang III have large molecular structures, a fragmentation method was used to simplify the conformational space and achieve theoretical calculations and simulations. Py-BPy was selected. 2+ The geometry of one Schiff base reaction unit of -COF, the common fragment molecules of Ang I and Ang II, the fragment molecules of Ang III, and the Aldo molecule was optimized. Figure 10 The optimal conformation of a molecule (i.e., the lowest energy structure) is determined and stabilized by its intricate network of weak interactions. Figure 10 Py-BPy involved 2+The optimal configurations of the -COF, Ang I, Ang II, Ang III, and Aldo systems all exhibit their own unique and synergistic weak interaction modes. Among them, hydrogen bonds and van der Waals interactions are the main contributing factors to maintaining the stability of these conformations. These interactions, through precise control of directionality, strength, and spatial matching in three-dimensional space, collectively stabilize the molecule in the lowest-energy conformational state.

[0091] from Figure 11 As can be seen, Ang I, Ang II, Ang III, and Aldo molecules enter the COF pores through intermolecular interactions. IGMH analysis revealed that the adsorption energies between Ang I and Ang II and COF are -130.76 kJ / mol. -1 The adsorption energy between AngⅢ and COF is -186.63 kJ mol. -1 The adsorption energy between Aldo and COF is -86.44 kJ / mol. -1 From the results, it can be seen that the absolute values ​​of the adsorption energies of the four substances with COF are ranked as follows: AngⅢ > AngⅠ, AngⅡ > Aldo. The larger the absolute value, the stronger the adsorption force. Based on the above, the possible adsorption mechanisms between COF adsorbent material and AngⅠ, AngⅡ, AngⅢ and Aldo are as follows: (1) Angiotensin has amphoteric dissociation characteristics. When the pH value of the solution is higher than the pKa value of angiotensin, the amino group of angiotensin dissociates with a negative charge, Py-BPy 2+ -COF has positively charged functional groups, and the positive and negative charges attract each other, resulting in ion interactions. (2) Py-BPy 2+ -COF is formed by the covalent connection of carbon-nitrogen bonds and aromatic rings, and has a unique π-π conjugated system and pore structure, which facilitates the entry of aldosterone molecules and generates strong hydrophobic interactions with them.

[0092] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A cationic COF material, which is Py-BPy 2+ -COF, characterized in that, The following method was used to prepare Py-BPy-COF material: Py-TA and 2,2'-BPy-DCA were added to a first mixed solution containing mesitylene, 1,4-dioxane, and acetic acid. After degassing, the mixture was purified by a single heating reaction to obtain Py-BPy-COF material. The Py-BPy-COF material was then dispersed in a second mixed solution of nitrobenzene and dibromoethane, and purified by a second heating reaction to obtain Py-BPy. 2+ -COF material.

2. The cationic COF material as described in claim 1, characterized in that, The mass-to-volume ratio of Py-TA and 2,2'-BPy-DCA to the first mixed solution is 50-60:40-50:1-10, mg / mg / mL.

3. The cationic COF material as described in claim 1, characterized in that, The volume ratio of mesitylene, 1,4-dioxane and acetic acid is 1-10:1-10:

1.

4. The cationic COF material as described in claim 1, characterized in that, The single heating reaction conditions are: reacting at 100-150℃ for 1-5 days; The secondary heating reaction conditions are: reacting at 200-250℃ for 12-36 h, preferably at 210℃ for 24 h.

5. The cationic COF material as described in claim 1, characterized in that, The purification process includes acetone washing and drying.

6. A solid-phase extraction adsorbent, characterized in that, The solid-phase extraction adsorbent comprises at least the cationic COF material as described in any one of claims 1-5.

7. The use of the cationic COF material according to any one of claims 1-5 or the solid-phase extraction adsorbent according to claim 6 in the enrichment and / or detection of angiotensin and aldosterone; wherein, The angiotensin I includes Ang I, Ang II, and Ang III.

8. A method for the combined detection of angiotensin and aldosterone, characterized in that, The method includes: performing solid-phase extraction on a pretreated plasma sample using the cationic COF material of any one of claims 1-5 or the solid-phase extraction adsorbent of claim 6, and detecting it based on a liquid chromatography-mass spectrometry method.

9. The method as described in claim 8, characterized in that, Methods for pretreating plasma include incubating the plasma in a working buffer containing soybean trypsin inhibitor and benzyl sulfonyl chloride and then terminating the incubation process. When performing solid-phase extraction adsorption, the pH of the sample loading solution should be 7.5-9.5; Elution is performed using a 50-90% acetonitrile aqueous solution as the eluent; further, the eluent also contains 0.05-0.5% formic acid.

10. The method as described in claim 8, characterized in that, Liquid chromatography conditions: C18 column; mobile phase: phase A is 0.05-0.5% formic acid aqueous solution, phase B is acetonitrile solution; gradient elution program: 0-0.25 min, phase A 95%, phase B 5%; 0.25-2 min, phase B increases from 5% to 95%, then holds for 2 min; 4-4.1 min, phase B decreases from 95% to 5%, then holds for 1.4 min; The flow rate is 0.1-0.5 mL / min. -1 The column temperature is 35-45℃, and the injection volume is 10-30 μL. Mass spectrometry conditions: Ion source: electrospray ionization (ESI-); curtain gas: 10 psi; ionization voltage: -3800 V; Temperature: 350℃; Spray gas: 40 psi; Scan mode: Multiple reaction monitoring mode.

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