5-HTR2A cell membrane chromatography solid-phase microextraction material, preparation method thereof and application of 5-HTR2A cell membrane chromatography solid-phase microextraction material in atypical antipsychotic drug TDM

Abstract

The invention discloses a 5-HTR2A cell membrane chromatographic solid-phase microextraction material, a preparation method thereof and application of the 5-HTR2A cell membrane chromatographic solid-phase microextraction material in an atypical antipsychotic drug TDM, and belongs to the technical field of chromatographic science. Benzylguanine is bonded to silica gel, then thioether covalent bonds are used for specific recognition of high-expression 5-HTR2A cell membrane fragments with SNAP-Tag, and stable covalent binding is carried out on the fragments and the silica gel, so that cell membranes are fixed on the surface of the silica gel, and the SNAP-Tag bridged high-expression 5-HTR2A cell membrane chromatography solid-phase microextraction material is obtained. According to the cell membrane chromatography solid-phase extraction material, a carrier and a target membrane receptor are combined through a firm chemical bond, and compared with a traditional physical adsorption cell membrane chromatography stationary phase material, the stability is remarkably improved. Therefore, the cell membrane chromatography stationary phase material can be used as a solid-phase extraction adsorbent, so that the specificity of a solid-phase extraction technology is improved, and a target component can be quickly screened and pre-concentrated from a complex sample.

Description

Technical Field

[0001] The invention belongs to the field of affinity chromatography science and technology, and specifically relates to a 5-HTR2A cell membrane chromatography solid phase microextraction material and a preparation method thereof and application thereof in atypical antipsychotic drug TDM. Background Art

[0002] Therapeutic Drug Monitoring (TDM) refers to the process of collecting blood (sometimes urine, saliva and other fluids) from patients at regular intervals to measure the drug concentration and explore the absorption and metabolism of drugs in the body during clinical drug treatment. This is to develop an individualized dosing regimen based on the specific conditions of the patient. Atypical antipsychotics (also known as second-generation antipsychotics, atypical antipsychotics, AAs) include clozapine, olanzapine, risperidone, quetiapine, aripiprazole and ziprasidone. Compared with traditional antipsychotics, these drugs have the advantages of a broad spectrum of action, good efficacy and good safety, and can slow down extrapyramidal reactions and improve the quality of life of patients. The main pharmacological action of atypical antipsychotics is the blockade of serotonin 2A (5-HT2A) and dopamine D2 receptors. It is generally believed in clinical practice that their effects not only have an anti-hallucination and delusion effect, but also have an activation effect, thereby effectively improving the negative symptoms of schizophrenia patients. Therefore, being able to effectively conduct TDM of atypical antipsychotics is of great significance for the treatment of patients taking atypical antipsychotics.

[0003] At present, the TDM method for atypical antipsychotic drugs is mainly high performance liquid chromatography equipped with mass spectrometry detector (LC-MS), especially tandem mass spectrometry detector (LC-MS / MS). However, the disadvantage is that most traditional solid phase extraction materials can only separate components of different properties according to the strength of polarity, and the interaction mode with the target component is mainly non-specific adsorption, resulting in low lifespan and specificity of traditional solid phase extraction methods. Therefore, a highly stable and highly specific bioaffinity solid phase extraction material is needed.

[0004] Cell Membrane Chromatography (CMC) is a method of fixing the cell membrane to a carrier to form a Cell Membrane Stationary Phase (CMSP). Chromatographic technology is used to study the interaction between drugs and receptors in the mobile phase. It is also a technology that integrates chromatographic separation and activity identification. It also has unique advantages in the screening of active ingredients in complex systems. The cell membrane chromatography stationary phase has the ability to specifically screen target substances. The principle is that the target substance can interact with the relevant receptors through specific receptor-ligand interactions. It has been widely used in the screening of active ingredients in traditional Chinese medicine and the study of ligand-receptor interactions, but there has been no report on its use in TDM of atypical antipsychotic drugs. Summary of the invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a 5-HTR2A cell membrane chromatography solid phase microextraction material and a preparation method thereof and an application in TDM of atypical antipsychotic drugs, so as to solve the problem of low accuracy and sensitivity of the existing TDM method of atypical antipsychotic drugs.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention discloses a 5-HTR2A cell membrane chromatography solid phase microextraction material, which is prepared by fixing a high-expressing cell membrane of a 5-HTR2A protein C-terminus or N-terminus fused with a SNAP-Tag on a benzylguanine-modified silica gel.

[0007] The second aspect of the present invention discloses a method for preparing the above-mentioned 5-HTR2A cell membrane chromatography solid phase microextraction material, wherein benzylguanine-modified silica gel is mixed with a high-expression cell membrane suspension of 5-HTR2A protein fused with SNAP-Tag at the C-terminus or N-terminus to obtain the 5-HTR2A cell membrane chromatography solid phase microextraction material.

[0008] Preferably, the mass ratio of the benzylguanine-modified silica gel to the high-expressing cell membrane of the 5-HTR2A protein C-terminus or N-terminus fused with SNAP-Tag is 125:4.

[0009] Preferably, the preparation method of benzylguanine-modified silica gel is: mixing cyanuric chloride silica gel with benzylguanine to obtain benzylguanine-modified silica gel.

[0010] More preferably, when preparing the benzylguanine-modified silica gel, DMF is used as the solvent.

[0011] Preferably, cyanuric chloride silica gel is mixed with benzylguanine, filtered, washed with DMF, repeatedly washed with anhydrous ethanol, and the filter cake is dried to obtain benzylguanine-modified silica gel.

[0012] Preferably, the preparation method of the high-expression cell membrane suspension of 5-HTR2A protein fused with SNAP-Tag at the C-terminus or N-terminus is: crushing and centrifuging the overexpressing stable cell precipitate of 5-HTR2A protein fused with SNAP-Tag at the C-terminus or N-terminus, taking the supernatant, and centrifuging again to obtain a cell membrane precipitate; mixing the cell membrane precipitate with ultrapure water to obtain a high-expression cell membrane suspension of 5-HTR2A protein fused with SNAP-Tag at the C-terminus or N-terminus.

[0013] Preferably, the method for preparing stably transfected cells overexpressing SNAP-Tag fused to the C-terminus or N-terminus of the 5-HTR2A protein is as follows: a vector plasmid containing SNAP-Tag fused to the C-terminus or N-terminus of the 5-HTR2A protein is packaged by lentivirus and then transfected into HEK293T cells, and the transfected cells are screened using the resistance gene in the plasmid to obtain stably transfected cells overexpressing SNAP-Tag fused to the C-terminus or N-terminus of the 5-HTR2A protein.

[0014] Preferably, the cell precipitate is broken and centrifuged as follows: the cell precipitate is added with Tris-HCl and suspended on ice, and then broken with a cell disruptor at 4°C, 1000 × g Centrifuge for 10 min, transfer the supernatant and incubate at 4°C at 12000 × g Centrifuge for 20 min.

[0015] Preferably, the benzylguanine-modified silica gel is mixed with a high-expression cell membrane suspension of 5-HTR2A protein fused with SNAP-Tag at the C-terminus or N-terminus, shaken at 37° C. and 1500 rpm for 30 min, and repeatedly washed three times with PBS to obtain a 5-HTR2A cell membrane chromatography solid phase microextraction material.

[0016] The third aspect of the present invention discloses the use of the above 5-HTR2A cell membrane chromatography solid phase microextraction material in the screening and identification of atypical antipsychotic drugs.

[0017] The fourth aspect of the present invention discloses the use of the above-mentioned 5-HTR2A cell membrane chromatography solid phase microextraction material in the pre-concentration of target components of atypical antipsychotic drugs.

[0018] Preferably, the target components of the atypical antipsychotic drugs are pre-concentrated and then combined with HPLC for use in TDM of atypical antipsychotic drugs.

[0019] The fifth aspect of the present invention discloses a method for monitoring the blood concentration of an atypical antipsychotic drug, comprising injecting an atypical antipsychotic drug solution into the above-mentioned 5-HTR2A cell membrane chromatography solid phase microextraction material, eluting, centrifuging to obtain the supernatant, filtering, and measuring the eluate by HPLC to calculate the blood concentration of the atypical antipsychotic drug in the blood.

[0020] Preferably, the mass ratio of the atypical antipsychotic drug to the 5-HTR2A cell membrane chromatography solid phase microextraction material is 4:25.

[0021] Preferably, during elution, acetonitrile and 0.1% triethylamine aqueous solution in a volume ratio of 1:1 are selected as the eluent.

[0022] Preferably, the liquid phase detection conditions for atypical antipsychotic drugs are: detection using a Diamonsil C18, 150 × 4.6mm id, 5 μm chromatographic column; the mobile phase conditions are set as: 70% A phase-30% B isocratic elution, A phase is 0.1% triethylamine aqueous solution, B phase is acetonitrile; the flow rate is 0.8 mL / min; the detection wavelength is 215 nm.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a 5-HTR2A cell membrane chromatography solid phase microextraction material, which selects 5-HTR2A protein as the target receptor of atypical antipsychotic drugs, bonds benzylguanine (BG) to silica gel, and reacts specifically with the high-expression cell membrane of SNAP-Tag fused to the C-terminus or N-terminus of the 5-HTR2A protein. Under the covalent binding action of the thioether bond, the SNAP-Tag can specifically covalently bond with the benzylguanine, thereby directionally covalently fixing the 5-HTR2A protein on the surface of the modified silica gel. The 5-HTR2A cell membrane chromatography solid phase microextraction material has the following characteristics: 1) under covalent bonding, it can realize the covalent bonding of protein tag-target receptor fusion protein cell membrane fragments and small molecule modified silica gel carriers, effectively preventing membrane shedding and improving the stability of the stationary phase; 2) using covalent bonding reaction to retain target activity; compared with the traditional physical adsorption immobilized cell membrane chromatography stationary phase, the nonspecific adsorption is low; at the same time, the specific reaction of SNAP-Tag and benzylguanine also further screens the immobilized cell membrane, so that only cell membrane fragments fused with and expressing SNAP-Tag can be fixed, and the specificity of the stationary phase is further improved. Therefore, the 5-HTR2A cell membrane chromatography solid phase microextraction material can be used as a solid phase extraction adsorbent, which greatly improves the shortcomings of traditional solid phase extraction materials, improves the selectivity and specificity of the extraction process, and solves the problems of low lifespan and specificity of traditional physical adsorption immobilized cell membranes. It can be used in the study of ligand-receptor interactions and the screening and identification of potential active components (especially the screening and identification of potential active components acting on specific receptors in complex systems), providing a theoretical basis for the subsequent realization of clinical blood drug concentration monitoring and expanding the application scope of cell membrane chromatography technology.

[0024] The present invention provides a method for preparing a 5-HTR2A cell membrane chromatography solid phase microextraction material. The 5-HTR2A cell membrane chromatography solid phase microextraction material is prepared by a directional immobilization method using a SNAP-Tag protein labeling technology, and a carrier is combined with a target membrane receptor through a strong chemical bond. The preparation conditions are mild and the operation is simple. The prepared 5-HTR2A cell membrane chromatography solid phase microextraction material has high stability and high specificity.

[0025] The present invention provides a method for monitoring the blood concentration of an atypical antipsychotic drug. The atypical antipsychotic drug solution is directly injected into a 5-HTR2A cell membrane chromatography solid phase microextraction material for offline extraction and elution, and then the blood concentration is detected in combination with HPLC, thereby avoiding problems such as background interference caused by direct detection of biological samples on an HPLC system. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A flow chart for preparing the 5-HTR2A cell membrane chromatography solid phase microextraction material of the present invention; Figure 2 This is the expression plasmid map in the construction of 5-HTR2A-SNAP-Tag HEK293T cells of the present invention; Figure 3 This is a map of the lentiviral backbone vector used in the 5-HTR2A-SNAP-Tag HEK293T cell construction of the present invention; Figure 4 Transmission electron micrographs of the benzylguanine-modified silica gel (SiO2-BG) and SNAP-Tag directional covalently fixed 5-HTR2A cell membrane chromatography solid phase microextraction material (5-HTR2A-SNAP-Tag / CMSP) of the present invention; wherein, (a) and (b) have a scale of 5 μm, representing SiO2-BG and 5-HTR2A-SNAP-Tag / CMSP, respectively; (c) and (d) have a scale of 50 nm, representing SiO2-BG and 5-HTR2A-SNAP-Tag / CMSP, respectively; Figure 5 The 5-HTR2A cell membrane chromatography solid phase microextraction material of the present invention is a graph for investigating the adsorption performance of clozapine; wherein (a) is a static adsorption result; (b) is a dynamic adsorption result; Figure 6 This is a graph showing the elution solvents for the 5-HTR2A cell membrane chromatography solid phase microextraction material of the present invention when it adsorbs clozapine; wherein (1) acetonitrile: water = 2:8 ( v / v ); (2) acetonitrile: water = 5:5 ( v / v ); (3) acetonitrile: water = 8:2 ( v / v ); (4) acetonitrile; (5) methanol: water = 2:8 ( v / v ); (6) methanol: water = 5:5 ( v / v ); (7) methanol: water = 8:2 (v / v); (8) methanol; (9) acetonitrile: 0.1% triethylamine aqueous solution = 2:8 ( v / v ); (10) acetonitrile: 0.1% triethylamine aqueous solution = 8:2 ( v / v ); (11) acetonitrile: 0.1% triethylamine aqueous solution = 5:5 ( v / v ); (12) methanol: 0.1% triethylamine aqueous solution = 2:8 ( v / v ); (13) methanol: 0.1% triethylamine aqueous solution = 5:5 ( v / v ); (14) methanol: 0.1% triethylamine aqueous solution = 8:2 ( v / v ); (15) water; (16) 0.1% triethylamine aqueous solution; Figure 7The results of the investigation of the application ability of the 5-HTR2A cell membrane chromatography solid phase microextraction material of the present invention to simulated clinical samples are shown in FIG. 1 ; wherein (a) is a 350 ng / mL clozapine aqueous solution sample; (b) is a 600 ng / mL clozapine aqueous solution sample; (c) is a 350 ng / mL clozapine serum sample; and (d) is a 600 ng / mL clozapine serum sample; Figure 8 The stability investigation diagram of the 5-HTR2A cell membrane chromatography solid phase microextraction material of the present invention; wherein, (a) is the change in the recovery rate of clozapine content detected by HPLC after solid phase extraction of clozapine samples for 5 consecutive days; (b) is the change in the recovery rate of clozapine content detected by HPLC after solid phase extraction of clozapine samples for 5 consecutive times on the same day. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to understand the characteristics and effects of the present invention, the following is a general description and definition of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used in the text are the common meanings understood by those skilled in the art for the present invention. In the event of a conflict, the definition in this specification shall prevail.

[0028] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0029] In this article, all features such as values, quantities, contents and concentrations defined in the form of numerical ranges or percentage ranges are for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0030] In this document, unless otherwise specified, “includes,” “including,” “contains,” “has,” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0031] In this document, in order to make the description concise, not all possible combinations of various technical features in various embodiments or examples are described. Therefore, as long as there is no contradiction in the combination of these technical features, the various technical features in various embodiments or examples can be combined arbitrarily, and all possible combinations should be considered to be within the scope of this specification.

[0032] The present invention provides a 5-HTR2A cell membrane chromatography solid phase microextraction material (5-HTR2A-SNAP-Tag / CMSP), which is prepared by covalent binding reaction between silica gel modified with benzylguanine and a high-expression cell membrane suspension of 5-HTR2A protein C-terminus or N-terminus fused with SNAP-Tag. The 5-HTR2A cell membrane chromatography solid phase microextraction material can be used for TDM of atypical antipsychotic drugs.

[0033] The present invention also provides a method for preparing the above-mentioned 5-HTR2A cell membrane chromatography solid phase microextraction material, such as Figure 1 As shown, the following steps are included: 1. Modify amino silica gel to obtain cyanuric chloride silica gel; mix cyanuric chloride silica gel with benzylguanine to obtain benzylguanine-modified silica gel. At the same time, culture overexpression cell lines with SNAP-Tag fused to the C-terminus or N-terminus of 5-HTR2A protein to prepare high-expression cell membrane suspension with SNAP-Tag fused to the C-terminus or N-terminus of 5-HTR2A protein.

[0034] 2. The high-expression cell membrane suspension with SNAP-Tag fused to the C-terminus or N-terminus of 5-HTR2A protein was mixed with silica gel modified with benzylguanine. SNAP-Tag can form a thioether covalent bond with benzylguanine through a specific reaction, so that the target receptor (5-HTR2A protein) is directionally covalently fixed on the silica gel surface to prepare 5-HTR2A cell membrane chromatography solid phase microextraction material.

[0035] The present invention also provides a method for monitoring the blood concentration of an atypical antipsychotic drug, comprising adding an atypical antipsychotic drug solution to the above-mentioned 5-HTR2A cell membrane chromatography solid phase microextraction material, vortexing thoroughly, centrifuging to discard the supernatant, eluting with 0.2 mL of acetonitrile: 0.1% triethylamine aqueous solution (volume ratio of 1:1), centrifuging to obtain the supernatant, filtering, and detecting the eluate using a chromatographic column with a theoretical plate number of >7000 / m, and calculating the concentration of the atypical antipsychotic drug in the blood based on the detection result.

[0036] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0037] The following examples use conventional instruments and equipment in the art. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, are conventional commercial products, and their specifications are conventional specifications in the art.

[0038] 1. Preparation of 5-HTR2A-SNAP-Tag / CMSP 1. Preparation of benzylguanine-modified silica gel (SiO2-BG) 1) Preparation of cyanuric chloride silica gel (SiO2-TCT): Weigh 1.4 g of amino silica gel (SiO2-NH2; model: Innova, 5μm, 100 Å, 3.5% carbon loading) and place it in a clean, anhydrous reaction round-bottom flask, add 14 mL of tetrahydrofuran and a stirring magnet in sequence, then place the reaction round-bottom flask in an ice-water bath environment and place it on a magnetic stirrer, keep the entire reaction in the ice-water bath and continue stirring, then slowly add 1.4 mL of N, N-diisopropylethylamine and 0.36 g of cyanuric chloride, and continue stirring for 2 h. After the reaction is completed, wash the stationary phase product with anhydrous ethanol several times to remove excess cyanuric chloride, and dry it in an oven overnight to obtain SiO2-TCT.

[0039] 2) Preparation of SiO2-BG: Weigh 50 mg of 6-[[4-(aminomethyl)phenyl]methoxy]-7H-purine-2-amine (BG) and place it in a clean, anhydrous reaction round-bottom flask, then add 50 mL of N, N-dimethylformamide (DMF), sonicate to disperse the reaction system evenly, dissolve as much BG as possible in the system, then add all the SiO2-TCT products obtained in the previous step, sonicate until evenly dispersed, add a stirring magnet, stir and reflux at 95°C for 8 h, cool to room temperature, filter, wash the product with DMF, then repeatedly wash the product with anhydrous ethanol several times, and dry to obtain SiO2-BG.

[0040] 2. Preparation of 5-HTR2A-SNAP-Tag / CMSP 1) Preparation of high-expression cell membrane suspension of 5-HTR2A C-terminal fused SNAP-Tag (referred to as 5-HTR2A-SNAP-Tag cell membrane suspension): Genetic engineering technology was used to construct Figure 2 The plasmid containing SNAP-Tag fused to the C-terminus of 5-HTR2A protein was expressed as Figure 3 The lentiviral vector shown was packaged and transfected into HEK293T cells. The transfected cells were screened using the Blasticdin (3 μg / mL) resistance gene in the plasmid to obtain stably transfected HEK293T cells overexpressing SNAP-Tag fused to the C-terminus of the 5-HTR2A protein (constructed by Shanghai Nobai Biotechnology Co., Ltd.). Cells in the logarithmic growth phase (no less than 5 × 10 7 ), 4°C, 1000× gAfter centrifugation for 10 min, the culture medium was discarded, the cells were washed twice with saline, the precipitate was suspended with hypotonic solution, and the membrane was broken by ultrasound for 30 min before homogenization. The suspension was then heated to 1000 × g Centrifuge for 10 min, take the supernatant, and centrifuge at 12000 × g Centrifuge for 20 min, discard the supernatant, add 5 mL of hypotonic solution to resuspend the precipitate, and centrifuge again at 12000 × g Centrifuge. Take the cell membrane pellet and resuspend it in 400 μL pre-cooled ultrapure water to obtain 5-HTR2A-SNAP-Tag cell membrane suspension for later use. This process was performed on ice throughout.

[0041] 2) Preparation of 5-HTR2A-SNAP-Tag / CMSP: Mix the 5-HTR2A-SNAP-Tag cell membrane suspension (containing 1.6 mg 5-HTR2A-SNAP-Tag cell membrane) with 50 mg SiO2-BG prepared in step 1, shake at 37°C, 1500 rpm for 30 min, and wash three times with PBS to obtain 5-HTR2A-SNAP-Tag / CMSP.

[0042] 2. Structural characterization of 5-HTR2A-SNAP-Tag / CMSP In order to verify the synthesized solid phase microextraction materials, we used transmission electron microscopy (TEM) to characterize SiO2-BG and 5-HTR2A-SNAP-Tag / CMSP, respectively.

[0043] The results are as follows Figure 4 As shown in the figure, compared with SiO2-BG, the presence of cell membrane can be more obviously seen on the surface of 5-HTR2A-SNAP-Tag / CMSP, which indicates the successful preparation of 5-HTR2A-SNAP-Tag / CMSP.

[0044] 3. Evaluation of adsorption performance of 5-HTR2A-SNAP-Tag / CMSP 1. Draw a standard curve About 10 mg of clozapine was accurately weighed and placed in a 100 mL volumetric flask. 20 mL of methanol was added. After ultrasonic dissolution, it was diluted to the mark with methanol. After shaking, a clozapine stock solution with a concentration of 100 μg / mL was obtained. The clozapine stock solution was diluted to different concentrations (0.625, 1.250, 2.500, 5.000, 10.000 μg / mL) with methanol. The clozapine standard curve was prepared with the clozapine concentration as the abscissa and the corresponding clozapine liquid chromatography peak area as the ordinate. The liquid phase detection conditions of clozapine were as follows: Diamonsil C18 (150 × 4.6 mm id, 5 μm) column was used for detection; the mobile phase conditions were set as: 30% B isocratic elution (Phase A was 0.1% triethylamine aqueous solution, Phase B was acetonitrile); the flow rate was 0.8 mL / min; the detection wavelength was 215 nm. In this experiment, the external standard method was used for the quantification of clozapine.

[0045] The obtained standard curve of clozapine is: y = 63094 x - 34540, r The linear correlation is 0.9991, which is high and can be used for quantitative analysis.

[0046] 2. Determination of adsorption concentration 5 mg of the prepared 5-HTR2A-SNAP-Tag / CMSP was added to a 2 mL centrifuge tube, and then 1 mL of clozapine aqueous solution with different concentrations (300, 400, 500, 600, 800, 1000 ng / mL) was added in sequence. After vortexing for 5 min, 1000 × g After centrifugation for 3 min, the supernatant was collected and filtered through a water-based microporous filter membrane with a pore size of 0.45 μm. The concentration of clozapine in the supernatant was determined by HPLC. 0.2 mL of acetonitrile: 0.1% triethylamine aqueous solution (volume ratio of 1:1) was used as the eluent. The HPLC detection conditions were as follows: a Diamonsil C18 (150 × 4.6 mm id, 5 μm) column was used, and the mobile phase conditions were set as: 70% A-30%B isocratic elution (Phase A was 0.1% triethylamine aqueous solution, Phase B was acetonitrile); the flow rate was 0.8 mL / min; and the detection wavelength was 215 nm.

[0047] The results are as follows Figure 5As shown in (a), as the concentration of clozapine increases, the adsorption of clozapine by 5-HTR2A-SNAP-Tag / CMSP increases. However, after reaching adsorption equilibrium, the adsorption rate decreases as the concentration of clozapine continues to increase. When the concentration of clozapine solution reaches 1.0 μg / mL, the adsorption rate decreases significantly, indicating that within the actual clinical application range of clozapine blood drug concentration (350 ng / mL~600 ng / mL), the adsorption performance of 5-HTR2A-SNAP-Tag / CMSP can meet the requirements.

[0048] 3. Adsorption time determination 5 mg of the prepared 5-HTR2A-SNAP-Tag / CMSP was added to a 2 mL centrifuge tube, and 1 mL of clozapine at a concentration of 600 ng / mL was added. After vortexing for different time periods (5, 30, and 60 s), the centrifuge tubes at the corresponding time periods were removed and 1000 × g After centrifugation for 3 min, the supernatant was collected and filtered through a water-based microporous filter membrane with a pore size of 0.45 μm. The concentration of clozapine in the supernatant was determined by HPLC (the HPLC determination conditions were the same as those in step 2). The adsorption time was then investigated.

[0049] The results are as follows Figure 5 As shown in (b), the adsorption equilibrium was reached when the sample was vortexed with 5-HTR2A-SNAP-Tag / CMSP for about 30 s, indicating that the target substance in the sample combined with 5-HTR2A-SNAP-Tag / CMSP rapidly, which greatly saved the sample loading time and achieved the purpose of rapid extraction of the target substance.

[0050] IV. 5-HTR2A-SNAP-Tag / CMSP Methodological Evaluation The present invention provides a method for monitoring the blood drug concentration of an atypical antipsychotic drug. The general steps are as follows: 1 mL of clozapine drug solution of different concentrations is added to a 5-HTR2A cell membrane chromatography solid phase microextraction material, fully vortexed, centrifuged to discard the supernatant, added 1 mL of water for elution, then eluted with 0.2 mL of eluent, centrifuged to obtain the supernatant, filtered, and the content of clozapine in the eluate is determined by HPLC. The HPLC conditions are: using a Diamonsil C18 (150 × 4.6 mm id, 5 μm) chromatographic column; 70% A-30%B isocratic elution (Phase A is 0.1% triethylamine aqueous solution, Phase B is acetonitrile); the flow rate is 0.8 mL / min; the detection wavelength is 215 nm, the recovery rate is calculated according to the detection result, and the solid phase microextraction method is evaluated with the recovery rate of clozapine as an indicator.

[0051] Methodological evaluation includes the following aspects: 1) elution conditions; 2) application ability to simulate clinical samples; 3) precision; 4) repeatability; 5) stability. The specific steps are as follows: 1. Selection of elution conditions 5 mg of the prepared 5-HTR2A-SNAP-Tag / CMSP was added to a 2 mL centrifuge tube, and 1 mL of clozapine at a concentration of 600 ng / mL was added. After vortexing for 60 s, 1000 × g After centrifugation for 3 min, transfer the supernatant to a new centrifuge tube. The recovery rate of substances in solid phase extraction is mainly affected by the type of eluent. The experiment investigated the types of eluents and compared 16 eluents (acetonitrile: water = 2:8 ( v / v ); acetonitrile: water = 5:5 ( v / v ); acetonitrile: water = 8:2 ( v / v ); acetonitrile; methanol: water = 2:8 ( v / v ); methanol: water = 5:5 ( v / v ); methanol: water = 8:2 ( v / v ); methanol; acetonitrile: 0.1% triethylamine aqueous solution = 2:8 ( v / v ); acetonitrile: 0.1% triethylamine aqueous solution = 8:2 ( v / v ); acetonitrile: 0.1% triethylamine aqueous solution = 5:5 ( v / v ); methanol: 0.1% triethylamine aqueous solution = 2: 8 ( v / v ); methanol: 0.1% triethylamine aqueous solution = 5:5 ( v / v ); methanol: 0.1% triethylamine aqueous solution = 8:2 ( v / v ); water; 0.1% triethylamine aqueous solution). Add 0.2 mL of the corresponding eluent solution to the material adsorbed with clozapine and vortex thoroughly. g After centrifugation for 3 min, the supernatant was taken and filtered through a microporous membrane with a pore size of 0.45 μm. The concentration of clozapine in the eluent and elution was determined by HPLC. The HPLC determination conditions were the same as those in the adsorption performance evaluation experiment, so as to determine the elution rate of different eluents. Results The optimal elution solvent was selected mainly based on the recovery rate of clozapine.

[0052] The results of the test are as follows Figure 6 As shown, it can be seen that acetonitrile: 0.1% triethylamine aqueous solution = 5:5 ( v / v ) has the best elution effect, so acetonitrile: 0.1% triethylamine aqueous solution = 5:5 ( v / v ) as the elution solution to elute the target substance from the material.

[0053] 2. Evaluation of the application ability of 5-HTR2A-SNAP-Tag / CMSP on simulated clinical samples 5 mg of the prepared 5-HTR2A-SNAP-Tag / CMSP was added to a 2 mL centrifuge tube, and 1 mL of clozapine samples of different concentrations (10 μL of clozapine alcohol solution of different concentrations + 990 μL of water or fetal bovine serum) was added. After thorough vortexing, the supernatant was discarded after centrifugation. After 1 mL of water was added for elution, 200 μL of acetonitrile: 0.1% triethylamine aqueous solution = 5:5 ( v / v ) After elution, 1000 × g After centrifugation for 3 min, the supernatant was collected and filtered through a microporous filter membrane with a pore size of 0.45 μm. The concentration of clozapine was determined by HPLC. The HPLC determination conditions were the same as those in the adsorption performance evaluation experiment, so as to investigate the recovery rate of clozapine using aqueous solution and serum as the matrix.

[0054] The results are as follows Figure 7 As shown in the figure, after the 350 ng / mL and 600 ng / mL clozapine aqueous solution samples were treated by the above solid phase extraction method, the recovery rates reached more than 80%; after the 350 ng / mL and 600 ng / mL clozapine fetal bovine serum samples were treated by the above solid phase extraction method, the recovery rates reached more than 60%, among which the recovery rate of the 350 ng / mL fetal bovine serum sample reached 80%, which may be because the protein in the fetal bovine serum combined with the drug, resulting in a decrease in the recovery rate of the drug in the serum sample, but the method still has a relatively high recovery rate overall and meets the requirements of subsequent experiments.

[0055] 3. 5-HTR2A-SNAP-Tag / CMSP precision evaluation Under the optimal experimental conditions, 5-HTR2A-SNAP-Tag / CMSP was investigated as an adsorbent. There was a good linear relationship between the concentration of clozapine in the range of 0.625-10 μg / mL, and the standard curve formula was: y = 63094 x - 34540. r The value of the PCR product was 0.9971, the detection limit was 0.25 μg / mL, and the quantification limit was 0.5 μg / mL. In order to evaluate the repeatability of this method, samples were tested 6 times a day for intra-day precision evaluation, and samples were tested for 3 consecutive days for inter-day precision evaluation.

[0056] Table 1 Results of precision and repeatability investigation of solid phase extraction method

[0057] The results are shown in Table 1. The intra-day and inter-day relative standard deviations of the 350 ng / mL clozapine sample extracted were 1.15% and 3.65%, respectively; the intra-day and inter-day relative standard deviations of the 600 ng / mL clozapine sample extracted were 1.35% and 1.83%, respectively, indicating that the method has good precision.

[0058] 4. 5-HTR2A-SNAP-Tag / CMSP repeatability evaluation The reproducibility of 5-HTR2A-SNAP-Tag / CMSP from 6 different batches and the same batch was investigated.

[0059] The results are shown in Table 1. The relative standard deviations of clozapine samples extracted by 5-HTR2A-SNAP-Tag / CMSP at 350 ng / mL and 600 ng / mL from 6 different batches were 6.79% and 9.18%, respectively; the relative standard deviations of clozapine samples extracted by 5-HTR2A-SNAP-Tag / CMSP at 350 ng / mL and 600 ng / mL from 6 groups of the same batch were 3.14% and 2.76%, respectively. The above results all indicate that the method has good repeatability.

[0060] 5. 5-HTR2A-SNAP-Tag / CMSP stability evaluation The changes in the recovery rate of clozapine content detected by HPLC were compared after solid phase extraction of clozapine samples by 5-HTR2A-SNAP-Tag / CMSP of the same batch (n = 3) for 5 consecutive days and 5 consecutive times on the same day, in order to evaluate the stability of the material.

[0061] The results are as follows Figure 8 As shown in the figure, the recovery rate did not decrease significantly after extracting two concentrations of clozapine samples for 5 consecutive days; the recovery rate did not decrease significantly after extracting two concentrations of clozapine samples for 5 consecutive times on the same day. This shows that the solid phase microextraction material has good stability and can be used in subsequent solid phase extraction determination.

[0062] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A 5-HTR2A cell membrane chromatography solid phase microextraction material, characterized in that: The method is prepared by fixing the high-expressing cell membrane of 5-HTR2A protein with SNAP-Tag fused to the C-terminus or N-terminus on silica gel modified with benzylguanine.

2. The method for preparing a 5-HTR2A cell membrane chromatography solid phase microextraction material according to claim 1, characterized in that: The benzylguanine-modified silica gel is mixed with a high-expression cell membrane suspension of 5-HTR2A protein fused with SNAP-Tag at the C-terminus or N-terminus to obtain a 5-HTR2A cell membrane chromatography solid phase microextraction material.

3. The method for preparing a 5-HTR2A cell membrane chromatography solid phase microextraction material according to claim 2, characterized in that: The mass ratio of benzylguanine-modified silica gel to the high-expression cell membrane of 5-HTR2A protein fused with SNAP-Tag at the C-terminus or N-terminus was 125:

4.

4. The method for preparing a 5-HTR2A cell membrane chromatography solid phase microextraction material according to claim 2 or 3, characterized in that: The preparation method of benzylguanine-modified silica gel is as follows: cyanuric chloride silica gel is mixed with benzylguanine to obtain benzylguanine-modified silica gel.

5. The method for preparing a 5-HTR2A cell membrane chromatography solid phase microextraction material according to claim 2, characterized in that: The preparation method of the high-expression cell membrane suspension of 5-HTR2A protein C-terminus or N-terminus fused with SNAP-Tag is as follows: crushing and centrifuging the overexpression stable cell precipitate of 5-HTR2A protein C-terminus or N-terminus fused with SNAP-Tag, taking the supernatant, and centrifuging again to obtain the cell membrane precipitate; mixing the cell membrane precipitate with ultrapure water to obtain the high-expression cell membrane suspension of 5-HTR2A protein C-terminus or N-terminus fused with SNAP-Tag.

6. Use of the 5-HTR2A cell membrane chromatography solid phase microextraction material according to claim 1 in screening and identifying atypical antipsychotic drugs.

7. Use of the 5-HTR2A cell membrane chromatography solid phase microextraction material according to claim 1 in pre-concentration of target components of atypical antipsychotic drugs.

8. The use according to claim 7, characterized in that: The target components of the atypical antipsychotic drugs are pre-concentrated and then used in combination with HPLC in the TDM of atypical antipsychotic drugs.

9. A method for monitoring the blood concentration of an atypical antipsychotic drug, characterized in that: The atypical antipsychotic drug solution is injected into the 5-HTR2A cell membrane chromatography solid phase microextraction material of claim 1, eluted, centrifuged to obtain the supernatant, filtered, and the eluate is measured by HPLC to calculate the blood concentration of the atypical antipsychotic drug in the blood.

10. A method for monitoring the blood concentration of atypical antipsychotic drugs according to claim 9, characterized in that: The mass ratio of atypical antipsychotic drugs to 5-HTR2A cell membrane chromatography solid phase microextraction materials is 4:25.

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