Therapeutic drug monitoring kit and method and system thereof

Abstract

The invention provides a therapeutic drug monitoring kit and method and system, a magnetic bead extraction method is combined with LC-MS / MS detection to be applied to human blood drug concentration monitoring, a mixed magnetic bead solution containing phospholipid removal magnetic beads and to-be-detected drug adsorption magnetic beads is utilized to perform adsorption and extraction pretreatment on a to-be-detected drug in a sample, automatic treatment equipment is matched, and the concentration of the to-be-detected drug in the to-be-detected drug in the to-be-detected drug in the to-be-detected drug in the to-be-detected drug in the to-be-detected drug in the to-be-detected drug is detected. The pretreatment efficiency is high, the phospholipid content in the supernate is low, and the matrix effect and the chromatographic column washing pressure are greatly reduced. According to the therapeutic drug monitoring method provided by the invention, a unified pretreatment process is used, simultaneous extraction of multiple drugs is realized, and through a magnetic bead automatic extraction method, the pretreatment step of a sample can be rapidly completed, a therapeutic drug monitoring result can be rapidly obtained, the overall detection time is shortened, and the detection efficiency is improved. Therefore, a reliable laboratory examination basis is provided for individualized treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of medical diagnosis, and in particular to a therapeutic drug monitoring kit and a method and system thereof. Background Art

[0002] Therapeutic drug monitoring (TDM) is a technology that analyzes drugs, biomarkers, etc. in different patients and tailors dosing plans to achieve optimal efficacy and minimize adverse reactions, thereby achieving personalized treatment.

[0003] With almost identical drug dosages, the steady-state drug concentration in different individuals can differ by more than 20 times, resulting in different reactions such as effectiveness, ineffectiveness, and poisoning. The main reason for this is that differences in drug absorption, distribution, metabolism, and excretion result from differences in comorbidities, age, concomitant medications, and genetic characteristics among patients. Therefore, quantitative determination of drug concentrations in plasma or serum for individual patient dose titration to achieve optimal efficacy, better tolerability, and reduce the risk of poisoning is a necessary means to achieve personalized treatment.

[0004] Drugs that require TDM can be basically divided into the following five categories: 1. Drugs with a low therapeutic index, a narrow safety range, and a therapeutic concentration range that is very close to the toxic concentration, such as cardiac glycosides and aminoglycoside antibiotics. 2. Drugs that require long-term drug treatment but lack obvious, observable therapeutic endpoints or indicators, and no timely, easily observable, and predictable clinical indicators of efficacy to adjust the dosage, such as anti-epileptic drugs and immunosuppressants. 3. Drugs with nonlinear kinetic characteristics. 4. Drugs with large individual differences in pharmacokinetics and strong pharmacological activity. Due to genetic factors, there are great differences in individualized drug metabolism, such as tricyclic antidepressants. 5. Failure of drug treatment can lead to serious consequences, such as immunosuppressants and some antibiotics.

[0005] Currently, TDM mainly uses immunoassays and chromatographic analysis. Immunoassays are characterized by short detection cycles, low sample requirements, simple operation, and a high degree of automation. However, there are also problems with the limited types of drugs in the detection kits, which may cross-react with the metabolites of the original drug. It is necessary to develop a corresponding kit for each drug, which is not suitable for new drug research. Chromatographic analysis solves the pain points of immunoassays. It can quickly design new methods, has good flexibility, accurate quantification, good selectivity, high sensitivity, and high precision, making it the most widely used therapeutic drug monitoring analysis method. For example, LC-MS / MS is a chromatographic analysis method currently recognized at home and abroad as having better sensitivity, repeatability, accuracy, and a higher dynamic linear range, which can provide more reliable test data for clinical diagnosis.

[0006] Currently, the most commonly used pretreatment method for therapeutic drug concentration monitoring (TDM) using LC-MS / MS is protein precipitation, which involves using acids, metal salts, organic solvents, etc. as precipitants, mixing them with the sample in a certain proportion, vortexing, and centrifuging to obtain the supernatant or diluting the supernatant before testing on the machine. This method has a relatively obvious matrix effect, and when the chromatographic separation is insufficient, it will affect the sensitivity and accuracy of the test results. In addition, the drug concentration is diluted, and some low-concentration drugs cannot meet the requirements of the test, requiring further nitrogen concentration and then redissolution, or the use of more complex pretreatment enrichment methods, such as solid-phase extraction. At the same time, automation is an important part of the development of detection technology. Through the automation platform, the efficiency and accuracy of sample processing can be effectively improved, personnel costs can be saved, and better project management can be achieved.

[0007] Looking at the development of TDM pretreatment technology research, there are many phospholipid removal plates available on the market for impurity removal. Waters' Ostro protein-removal phospholipid plate and Biotage's The PLD+ protein and phospholipid removal plate uses a simple solvent precipitation and filtration step to remove more than 99% of the proteins and phospholipids in plasma that cause ion suppression effects. The pretreatment operation is usually: first add a precipitant (such as 1% formic acid acetonitrile), then add the biological sample, and aspirate or shake to mix thoroughly; use a 96-well positive pressure instrument for positive pressure filtration, and collect the filtrate for subsequent direct sampling or concentration. The use of the phospholipid removal plate is simple to operate and has good results in removing proteins and phospholipids, but the phospholipid removal plate is relatively expensive and the pretreatment time is prolonged, making it unsuitable for daily drug monitoring in China.

[0008] In terms of automation, Shimadzu recently launched the CLAM-2030, a fully automated pretreatment system that has been applied in drug monitoring and forensic screening. This system automatically precipitates proteins from biological samples, eliminating human error in manual pretreatment, improving reproducibility and analytical efficiency. It also enhances the efficiency of protein precipitation methods, and features a specially designed sample tube and negative pressure filtration system. While expensive, matrix effects cannot be eliminated.

[0009] Therefore, there is an urgent need to find a simpler, fully automated sample pretreatment method that is compatible with various drug monitoring processes, so as to build a more efficient and convenient therapeutic drug monitoring system with more accurate and reliable monitoring results. Summary of the Invention

[0010] In response to the problems existing in the prior art, the present invention provides a therapeutic drug monitoring kit and its method and system, which combines magnetic bead extraction with LC-MS / MS detection for the concentration monitoring of therapeutic drugs, and utilizes a mixed magnetic bead solution containing phospholipid-removing magnetic beads and magnetic beads adsorbing the drug to be tested to perform adsorption and extraction pretreatment on the drug to be tested in the sample. With the use of automated processing equipment, the pretreatment efficiency is high, the phospholipid content in the supernatant is low, and the matrix effect and chromatographic column flushing pressure are greatly reduced. The therapeutic drug monitoring method provided by the present invention uses a unified pretreatment process to achieve simultaneous extraction of multiple drugs, and through the magnetic bead automated extraction method, the pretreatment steps of the sample can be quickly completed, and the therapeutic drug blood concentration monitoring results can be quickly given, shortening the overall detection time, thereby providing a reliable laboratory examination basis for achieving individualized treatment.

[0011] In one aspect, the present invention provides a therapeutic drug monitoring kit, comprising a phospholipid removal magnetic bead solution containing magnetic beads for removing phospholipids from a sample.

[0012] LC-MS / MS TDM analysis technology has been widely used in drug monitoring, but technical bottlenecks still exist in the sample pretreatment process. Currently, most sample pretreatment methods use protein precipitation, which has the following problems: 1. Matrix effects still exist, and large molecules such as phospholipids in the sample are not effectively removed, which will affect the sample detection data and chromatographic column life; 2. The sample is diluted during the pretreatment process. For drugs with low content, nitrogen blowing and concentration are required, which is time-consuming and cumbersome; 3. It involves multiple tools and multi-step operations such as oscillation mixing and centrifugation, making it difficult to automate.

[0013] The magnetic bead-based automated sample pretreatment and LC-MS / MS analysis method provided by the present invention utilizes the specific binding properties of magnetic particles to target molecules to efficiently and selectively separate target components from complex biological samples, improve accuracy and consistency, reduce matrix effects, and provide feasibility for automated operation. It can solve the bottleneck problems in the existing technology and provide reliable technical support for the rapid and efficient analysis of TDM samples.

[0014] In order to improve the pretreatment efficiency of TDM and increase the level of automation while ensuring the accuracy of monitoring results, the present invention adopts original phospholipid-removing magnetic beads for sample pretreatment. The phospholipid-removing magnetic beads can be used alone or mixed with magnetic beads that adsorb the drug to be tested, so that the sample extraction and impurity removal processes can be carried out simultaneously. Moreover, the pretreatment process can be fully automated through a magnetic bead pretreatment instrument, which is suitable for the simultaneous selective extraction of multiple drugs, greatly improving the sample pretreatment efficiency. Up to hundreds of drugs can be extracted at a single time, greatly improving the detection efficiency while reducing human operation errors, making the therapeutic drug monitoring process simpler and more efficient, and also improving the monitoring accuracy.

[0015] The surface of the phospholipid-removing magnetic beads is modified with functional groups or molecules that can specifically bind to phospholipids, and can selectively bind to phospholipids in the sample to form a magnetic bead-phospholipid complex. Under the action of an external magnetic field, the magnetic bead-phospholipid complex is rapidly separated, and unbound substances remain in the solution, achieving separation and realizing high-throughput fully automatic sample processing.

[0016] Furthermore, the kit also includes a magnetic bead solution for adsorbing the drug to be tested, which is combined with a phospholipid-removing magnetic bead solution to form a mixed magnetic bead solution; the magnetic bead solution for adsorbing the drug to be tested contains magnetic beads for adsorbing and extracting the drug to be tested from the sample.

[0017] It is understood that the phospholipid removal magnetic beads and the test drug adsorption magnetic beads can be used separately and sequentially, for example, first removing phospholipids and then adsorbing the test drug, or they can be used in combination. In the present invention, the phospholipid removal magnetic beads and the test drug adsorption magnetic beads are preferably combined into a mixed magnetic bead solution and used simultaneously. Using them simultaneously can not only simplify the operation process, but also improve the adsorption of the test drug and the removal of phospholipids.

[0018] Furthermore, the phospholipid removal magnetic beads include any one or more of phospholipid removal magnetic beads, mixed mode magnetic beads, TiO2 silica gel magnetic beads, and ZrO2 silica gel magnetic beads.

[0019] Furthermore, the magnetic beads for adsorbing the drug to be tested include any one or more of C18 magnetic beads, C8 magnetic beads, phenyl magnetic beads, silica magnetic beads, PS magnetic beads, HLB magnetic beads, MAX magnetic beads, and MCX magnetic beads.

[0020] Furthermore, the phospholipid-removing magnetic beads in the mixed magnetic bead solution include ZrO2 silica gel magnetic beads, and the magnetic beads adsorbing the drug to be tested include HLB magnetic beads.

[0021] When phospholipid-removing magnetic beads and magnetic beads adsorbing the drug to be tested are used simultaneously, there may be mutual interference. The main reasons may be the following: 1. Competitive binding. The two magnetic beads may competitively bind to the same molecule, resulting in a decrease in the recovery rate of the target; 2. Steric hindrance. A large number of magnetic beads may cause steric hindrance, affecting the effective binding of the target to the magnetic beads; 3. Nonspecific adsorption. The phospholipid-removing magnetic beads may nonspecifically adsorb the drug to be tested, or the magnetic beads adsorbing the drug to be tested may nonspecifically adsorb phospholipids, affecting the experimental results; 4. Separation efficiency. The difference in the physical properties of the two magnetic beads may lead to reduced separation efficiency, affecting the recovery and purity of the target. Therefore, it is necessary to select appropriate magnetic bead characteristics (including appropriate surface modification and appropriate particle size, etc.) and screen for appropriate pretreatment conditions to eliminate mutual interference and reduce the impact.

[0022] Studies have shown that the use of surface-bonded ZrO2-linked phospholipid-removing magnetic beads and HLB magnetic beads mixed for TDM sample pretreatment can effectively prevent mutual interference due to the strong binding specificity of the two magnetic beads. There is no problem of competitive binding and nonspecific adsorption. The steric hindrance is small and the separation efficiency is high. It can significantly improve the effect of pretreatment and the accuracy of therapeutic drug detection results.

[0023] Furthermore, the drugs to be tested include any one or more of sedative-hypnotic drugs, antidepressant drugs, antipsychotic drugs, antiepileptic drugs, antibiotic drugs, antitumor drugs, cardiovascular drugs and poison screening drugs.

[0024] Studies have shown that the mixed magnetic bead pretreatment method provided by the present invention is suitable for the simultaneous selective extraction of most drugs, mainly including sedatives and hypnotic drugs, antidepressants, antipsychotics, antiepileptic drugs, antibiotics, antitumor drugs, cardiovascular drugs and toxicology screening drugs.

[0025] Furthermore, the sedative and hypnotic drugs include any one or more of alprazolam, clonazepam, midazolam, lorazepam, zopiclone, temazepam, bromazepam, nitrazepam, 6-hydroxybuspirone, buspirone, zaleplon, memantine, donepezil, tandospirone, diazepam, nordiazepam, oxazepam, zolpidem, and estazolam;

[0026] The antidepressant drugs include any one or more of sertraline, fluoxetine, norfluoxetine, escitalopram, fluvoxamine, paroxetine, venlafaxine, O-desmethylvenlafaxine, duloxetine, mirtazapine, trazodone, milnacipran, amitriptyline, nortriptyline, doxepin, vortioxetine, desmethylclomipramine, clomipramine, agomelatine, bupropion, mianserin, nordoxepin, and hydroxybupropion;

[0027] The antipsychotic drugs include any one or more of olanzapine, clozapine, paliperidone, risperidone, dehydroaripiprazole, aripiprazole, amisulpride, quetiapine, chlorpromazine, ziprasidone, N-desmethylclozapine, haloperidol, perphenazine, sulpiride, norquetiapine, fluphenazine, thioridazine, atomoxetine, lurasidone, blonanserin, maprotiline, methylphenidate, rivastigmine, perolanzapine, carbamazepine epoxide, norsertraline, norcitalopram, and normirtazapine;

[0028] The anti-epileptic drugs include any one or more of oxcarbazepine, lamotrigine, levetiracetam, 10-hydroxycarbamazepine, carbamazepine, phenytoin sodium, topiramate, primidone, gabapentin, pregabalin, rufinamide, stiripentol, perampanel, zonisamide, lacosamide, valproic acid, and phenobarbital;

[0029] The antibiotic drugs include any one or more of moxifloxacin, vancomycin, tigecycline, norvancomycin, polymyxin, linezolid, ciprofloxacin, sulfamethoxazole, and levofloxacin;

[0030] The anti-tumor drugs include any one or more of cyclophosphamide, ifosfamide, methotrexate, 5-fluorouracil, capecitabine, irinotecan, paclitaxel, docetaxel, afatinib, apatinib, icotinib, erlotinib, gefitinib, crizotinib, regorafenib, vemurafenib, imatinib, N-desmethylimatinib, alectinib, and osimertinib;

[0031] The cardiovascular drugs include any one or more of metoprolol, bisoprolol, nifedipine, amlodipine, atorvastatin calcium, 2-hydroxyatorvastatin, rosuvastatin, losartan, losartan metabolites, valsartan, irbesartan, telmisartan, clopidogrel metabolites, salicylic acid, ticagrelor, ticagrelor metabolite M8, etc.;

[0032] The poison screening drugs include rodenticides (brodifacoum, bromadiolone, difacoum, chlordiquinone, warfarin, fludioxonone, coumatetralyl, fluoroacetic acid, fumarate, fumarate, thiadiazole, chlordiquinone, chlordiquinone, difacoum), pesticides (199 kinds), psychotropic drugs (piroxicam, acetaminophen, o-ethoxybenzoate, paracetamol, sulindac, dihydroergotamine, ketorolac tromethamine, ketoprofen, isopropyl antipyrine, diphenhydramine, loxacin), any one or more of: fentanyl, flunitrazepam, ropivacaine, pethidine, procaine, hydroxydihydrocodeinone, tramadol, normorphine, ethylmorphine, dextropropoxyphene, lidocaine), biotoxins (aconitine, solanine, colchicine, amygdalin, strophanthin, tetrodotoxin, amanitine, aflatoxin).

[0033] The method provided by the present invention can simultaneously pre-process and detect more than 300 therapeutic drugs.

[0034] Furthermore, the magnetic bead solution contains acid, and the acid includes any one or more of formic acid, acetic acid, and citric acid.

[0035] The surface of HLB magnetic beads contains a specific ratio of hydrophilic and hydrophobic groups: the hydrophobic divinylbenzene structure retains non-polar compounds, while the hydrophilic N-vinylpyrrolidone structure retains polar compounds, resulting in good applicability. However, given the significant differences in polarity and acidity / alkalinity among different drugs, the acidity / alkalinity of the solution or the organic phase ratio must be strictly controlled during HLB magnetic bead extraction to achieve optimal extraction efficiency and universal applicability.

[0036] In some approaches, the addition of acid can be used to suppress the ionization of acidic compounds, thereby improving the extraction rate of acidic drugs such as valproic acid and methotrexate using HLB magnetic beads. Valproic acid, a common anti-epileptic drug, has a Pka of 4.6 (at 25°C). Valproic acid is fully ionized in the neutral environment of serum, making it unretainable on HLB magnetic beads and resulting in a very low extraction rate. By adding a certain concentration of acid to the solution to suppress the ionization of the carboxyl group and maintain a neutral molecular state, the extraction rate of HLB magnetic beads can be significantly improved.

[0037] In some cases, changes in acidity and alkalinity can also modulate drug-protein binding and facilitate drug extraction. For example, after absorption, the anti-tumor drug vemurafenib binds to human albumin and α-1 acid glycoprotein plasma proteins at a rate of >99% and is virtually insoluble in water. Acidity regulation modulates hydrogen bonds within protein molecules, reducing vemurafenib's binding to proteins and promoting drug migration into the HLB material.

[0038] Surprisingly, in the mixed magnetic beads, acid not only facilitates drug adsorption and extraction but also helps the phospholipid-removing magnetic beads more efficiently adsorb phospholipids, thereby effectively reducing matrix effects and ensuring the accuracy of test results. However, if phosphoric acid is used in this process, the phospholipid adsorption efficiency of the phospholipid-removing magnetic beads will be reduced. At the same time, phospholipids in the sample will be non-specifically adsorbed to the surface of the HLB magnetic beads. Therefore, the addition of a non-phosphoric acid acidic substance is required during the extraction process.

[0039] Furthermore, the mass concentration of the acid is 0.1-2%.

[0040] In some embodiments, it is preferred to add 1% formic acid (FA) or 1 M citric acid (CA) to the diluent.

[0041] Furthermore, an eluent is included, and the eluent includes formic acid and methanol.

[0042] Since the eluent is used to elute the mixed magnetic beads simultaneously, it must be able to smoothly elute the drug to be tested from the magnetic beads that adsorb the drug to be tested, but it must not elute the phospholipids from the phospholipid-removing magnetic beads. At the same time, the process of eluting the drug to be tested cannot be affected by the phospholipid-removing magnetic beads to prevent cross-influence and improve the specific elution effect. It can be seen that using a suitable eluent can also help improve the accuracy and sensitivity of the test results. During the elution process after adsorption and extraction of the mixed magnetic beads, acid helps to improve the elution efficiency of the analyte. However, when investigating the effects of different acids during the elution process, it was found that if phosphoric acid is added during the elution process, the phospholipid content in the eluent will increase, thereby affecting the life of the chromatographic column and causing contamination of the mass spectrometer detector. Therefore, phosphoric acid should be avoided in the eluent.

[0043] In some approaches, it is preferred to use 0.1% formic acid in methanol as the eluent.

[0044] Furthermore, it also includes a balancing liquid, a diluent and a liquid chromatography mobile phase, the balancing liquid and the diluent are both aqueous formic acid solutions; the liquid chromatography mobile phase includes mobile phase A and mobile phase B, the mobile phase A is an aqueous solution containing a mobile phase additive, and the mobile phase B is a methanol solution containing a mobile phase additive, and the mobile phase additive is one or a mixture of formic acid and ammonium acetate.

[0045] In another aspect, the present invention provides a method for detecting a drug, wherein the method is based on non-disease diagnosis purposes and comprises the following steps:

[0046] (1) treating the sample with a mixed magnetic bead solution, wherein the mixed magnetic bead solution includes phospholipid removal magnetic beads and test drug adsorption magnetic beads, and simultaneously adsorbs phospholipids and test drugs in the sample;

[0047] (2) eluting the drug to be tested with an eluent, and analyzing the content of the drug to be tested by liquid chromatography-tandem mass spectrometry;

[0048] The drugs to be tested include any one or more of sedative-hypnotic drugs, antidepressant drugs, antipsychotic drugs, antiepileptic drugs, antibiotic drugs, antitumor drugs, cardiovascular drugs and poison screening drugs.

[0049] Furthermore, the mixed magnetic bead solution in step (1) contains acid, and the acid includes any one or more of formic acid, acetic acid, and citric acid; and the eluent in step (2) includes formic acid methanol.

[0050] The TDM analysis technology based on magnetic bead method and LC-MS / MS provided by the present invention can realize the simultaneous monitoring of more than 300 therapeutic drugs with the simplest and most efficient operation process, effectively eliminating matrix effects and human influences, and ensuring the accuracy and sensitivity of the monitoring results.

[0051] On the other hand, the present invention provides a therapeutic drug monitoring system, which includes a drug detection module, a data input / output interface and a data analysis module; the drug detection module uses the above-mentioned method to obtain the detection value of the object to be detected, the data input / output interface is used to input the detection value of at least one drug, and the data analysis module is used to analyze the detection value of the drug, wherein the drug includes any one or more of sedative and hypnotic drugs, antidepressant drugs, antipsychotic drugs, antiepileptic drugs, antibiotic drugs, antitumor drugs, cardiovascular drugs and toxicology screening drugs; after analysis by the data analysis module, the data input / output interface is used to output the therapeutic drug monitoring results relative to the effective concentration range of the drug.

[0052] On the other hand, the present invention provides a use of a mixed magnetic bead solution for preparing a reagent for improving the accuracy of therapeutic drug monitoring, wherein the mixed magnetic bead solution includes a phospholipid-removing magnetic bead solution and a test drug adsorption magnetic bead solution, wherein the phospholipid-removing magnetic bead solution contains magnetic beads for removing phospholipids from a sample; and the test drug adsorption magnetic bead solution contains magnetic beads for adsorbing and extracting the test drug from the sample.

[0053] Furthermore, the phospholipid-removing magnetic beads include ZrO2 silica gel magnetic beads, and the magnetic beads for adsorbing the drug to be tested include HLB magnetic beads.

[0054] The therapeutic drug detection kit, method, and system constructed by the present invention have the following beneficial effects:

[0055] 1. The use of mixed magnetic beads for automated extraction can fully remove impurities (proteins, phospholipids, salts, etc.) and purify samples, thereby reducing matrix effects and shortening single-injection sample analysis time, thereby improving detection efficiency and extending chromatographic column life, thereby improving the accuracy of test results. This solves the problems of difficult impurity removal, high matrix effects, and easy damage to chromatographic columns in the protein precipitation method of existing TDM analysis technology.

[0056] 2. Hybrid magnetic bead extraction combined with LC-MS / MS is used for therapeutic drug monitoring. Pretreatment and analysis methods that are flexible and compatible with hundreds of drugs can be established quickly and efficiently. The high degree of automation eliminates human errors and inconsistencies between batches. The high specificity of the detection method can eliminate cross-reactions of metabolites with the original drug, which is beneficial to the new drug development process and is suitable for concentration detection of hundreds of drugs.

[0057] 3. In the existing technology, the sample concentration is indiscriminately diluted during the protein precipitation process, which will challenge the detection sensitivity of individual drugs. The supernatant needs to be further concentrated by nitrogen blowing and then re-dissolved before injection, which is time-consuming and labor-intensive. In the process of magnetic bead automated sample pretreatment, the sensitivity and concentration information of the drug being tested can be taken into account. By adjusting the volume of the eluent, the sample can be diluted or enriched, especially in cases where the drug being tested needs to be concentrated, such as in toxicology screening. This greatly improves the pretreatment and detection efficiency and has very strong adaptability.

[0058] 5. A combination of magnetic beads with surface-bonded HLB silica gel material and phospholipid-removing magnetic beads was screened to adsorb and extract the test drugs in the test serum samples. Combined with automated processing equipment, the pretreatment efficiency is high, phospholipid interference is reduced, and the extraction efficiency of some drugs is better than the traditional protein precipitation method. The pretreatment time for processing 96 test samples is only 8 minutes, and no equipment such as vortex analyzer and centrifuge is required.

[0059] 6. This technical solution has been declared as an in vitro diagnostic TDM pre-packaged kit. The declared type of kit includes a magnetic bead suspension, an activation solution, a sample diluent (with internal standard), a washing solution and an eluent. Before conducting TDM analysis, it is only necessary to tear off the sealing film of the pre-packaged kit, add appropriate amounts of the sample to be tested, calibration products, and quality control products to the sample diluents in different wells of the pre-packaged kit, start the automatic extraction process of the magnetic bead instrument, and the eluent can be obtained after 8 minutes. According to the size of the sample, you can choose the appropriate magnetic bead extractor and pre-packaged kit specifications, and you can extract up to 32 samples, 48 samples, and 96 samples at a time. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is the detection spectrum of imatinib, cyclophosphamide, and paclitaxel;

[0061] Figure 2This is the detection spectrum of osimertinib, duloxetine, and carbamazepine;

[0062] Figure 3 Detection chart of ziprasidone, lurasidone and quetiapine

[0063] Figure 4 This is the detection spectrum of valproic acid and stiripentol. DETAILED DESCRIPTION

[0064] To describe the present invention in more detail, the technical solution of the present invention is described in detail below with reference to the accompanying drawings and specific embodiments. These descriptions are merely intended to illustrate how the present invention is implemented and are not intended to limit the specific scope of the present invention. The scope of the present invention is defined in the claims.

[0065] Example 1: Method for monitoring therapeutic drugs using magnetic bead method combined with LC-MS / MS detection

[0066] 1. Solution preparation:

[0067] Magnetic bead suspension: Weigh 1 g of HLB magnetic beads (HLB Magnetic Extraction Beads; supplier: 3PBiosolutions, product number MB001-1; particle size 20-40 μm, specific surface area approximately 600-750 m 2 / g, pore size of about 80A) was added to 100 mL of 50% ethanol to prepare a 10 mg / ml HLB magnetic bead suspension, which was stored at 2-8°C.

[0068] Phospholipid Depletion Magnetic Beads: Weigh 1 g of phospholipid depletion magnetic beads (Phospholipid Depletion Magnetic Beads; Supplier: 3P Biosolutions, Catalog No. MB003-1; Particle size 20-40 μm, specific surface area approximately 600-750 m 2 / g, pore size of about 80A) was added to 100 mL of methanol to prepare a 10 mg / ml phospholipid-removing magnetic bead suspension, which was stored at 2-8°C.

[0069] Mixed magnetic bead solution: Take 1 mL of HLB magnetic bead suspension and 1 mL of phospholipid-removed magnetic bead suspension, mix them evenly to obtain a mixed magnetic bead solution.

[0070] Equilibrium solution: Pipette 1 ml of formic acid into 1000 ml of ultrapure water and mix well to obtain the equilibrium solution. The storage condition is 2-8°C.

[0071] Dilution: Pipette 1 ml of formic acid into 1000 ml of ultrapure water and mix well to obtain the dilution. The storage condition is 2-8°C.

[0072] Eluent 1 / Eluent 2: ultrapure water.

[0073] Eluent: 0.1% formic acid in methanol.

[0074] 2. Sample testing:

[0075] 1. Magnetic bead method pretreatment

[0076] 1) Sample addition: Add 20 μL of calibrator, quality control, or sample to be tested to column 3 / 9 of a 96-well plate, followed by 200 μL of diluent containing the internal standard.

[0077] 2) Add pretreatment reagents: According to Table 1, add 200 μL of magnetic bead suspension, 200 μL of equilibration solution, 200 μL of eluent 1, 200 μL of eluent 2, and 100 μL of eluent.

[0078] Table 1. Placement of pretreatment reagents

[0079] Column 1(7) Column 2(8) Column 3(9) Column 4(10) Column 5(11) Column 6(12) Magnetic bead suspension Balanced solution sample Eluent 1 Eluent 2 eluent Magnetic bead suspension Balanced solution sample Eluent 1 Eluent 2 eluent Magnetic bead suspension Balanced solution sample Eluent 1 Eluent 2 eluent Magnetic bead suspension Balanced solution sample Eluent 1 Eluent 2 eluent Magnetic bead suspension Balanced solution sample Eluent 1 Eluent 2 eluent Magnetic bead suspension Balanced solution sample Eluent 1 Eluent 2 eluent Magnetic bead suspension Balanced solution sample Eluent 1 Eluent 2 eluent Magnetic bead suspension Balanced solution sample Eluent 1 Eluent 2 eluent

[0080] 3) Place the 96-well plate into an automated magnetic bead extractor for sample extraction. The magnetic bead extractor's operating procedures are shown in Table 2. The pre-treatment time for each batch of samples is 8 minutes. The currently used magnetic bead extractor can handle two 96-well plates in parallel, with a pre-treatment throughput of 32 samples per batch.

[0081] Table 2. Operation steps of magnetic bead extractor

[0082] Serial number instruction 96-well plate columns Mixing time (S) Solvent volume (ul) Magnetic attraction time (S) 1 activation 1(7) 30 100 30 2 activation 2(8) 30 200 30 3 Loading 3(9) 60 200 30 4 rinse 4(10) 30 200 30 5 rinse 5(11) 30 200 30 6 Elution 6(12) 60 100 30 7 Waste discharge 1(7) 10 100 0

[0083] The main steps include:

[0084] Step 1: Place the magnetic bar in the activation tube and stir it up and down to activate the magnetic beads so that they are adsorbed on the surface of the magnetic bar and ready for subsequent operations.

[0085] Step 2: Transfer the separation magnetic bead material carried by the magnetic rod to the sample tube and continue to stir up and down to ensure that the magnetic bead material is fully mixed with the sample and adsorbs and extracts the target substance and impurities.

[0086] Step 3: Transfer the magnetic rod carrying the magnetic beads that have adsorbed the target and impurities to the elution tube, and stir up and down to achieve elution and remove impurities.

[0087] Step 4: Transfer the magnetic beads carried by the magnetic rod that have adsorbed the target to the elution tube and stir up and down to achieve elution, separation and washing of the target.

[0088] Step 5: The magnetic rod absorbs and takes away the residual separation magnetic beads and phospholipid removal magnetic beads, and transfers them to the waste well (original activation tube), completing the entire pre-treatment extraction process.

[0089] 4) After the magnetic bead extraction is complete, transfer 40 μL of the eluate from columns 6 and 12 of the 96-well plate to a 96-well sample plate, add 160 μL of ultrapure water, vortex to mix, and then analyze on the instrument.

[0090] 2. LC-MS / MS detection

[0091] Liquid chromatography-tandem mass spectrometry analysis was performed using gradient elution. Reversed-phase chromatography separation conditions were established as follows: a DISIGNS Column-003, 2.6 μm, 50 x 3 mm column, a flow rate of 0.7 mL / min, and a column temperature of 40°C. Mobile phase A consisted of an aqueous solution containing mobile phase additives, and mobile phase B consisted of a methanol solution containing mobile phase additives. The gradient program is shown in Table 3.

[0092] Table 3. Gradient elution program

[0093] Time (min) Flow rate (mL / min) Mobile phase A (%) Mobile phase B (%) 0 0.7 95 5 0.2 0.7 95 5 0.3 0.7 75 25 2 0.7 65 35 4.8 0.7 15 85 4.9 0.7 0 100 5.5 0.7 0 100 5.6 0.7 95 5 6 0.7 95 5

[0094] When performing mass spectrometry detection, a triple quadrupole mass spectrometer was used for quantitative detection. The instrument model was CalQuant-U, which was independently developed by CalSpec. The positive and negative ion modes and multiple reaction monitoring (MRM) mode of the electrospray ion source were used for mass spectrometry detection. The corresponding mass spectrometry parameters are shown in Table 4.

[0095] Table 4. Mass spectrometry parameters for quantitative detection of drugs

[0096]

[0097]

[0098]

[0099] The ion source conditions are shown in Table 5.

[0100] Table 5. Ion source conditions

[0101]

[0102] The internal standard method was used to establish a standard curve. The linear relationship verification record is shown in Table 6 below. The unit of measurement is ng / mL. Figures 1 to 4 shown.

[0103] Table 6. Standard curve information for quantitative detection of drugs

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110] A standard curve was prepared in a human serum-like matrix and processed simultaneously with the sample to be tested. A summary of the non-quantitative screening drug testing information is shown in Table 7.

[0111] Table 7. Summary of non-quantitative screening drug testing information

[0112]

[0113]

[0114]

[0115]

[0116]

[0117] Example 2: Comparison of different pretreatment methods

[0118] Blood drug concentration monitoring using protein precipitation methods most commonly uses methanol / acetonitrile as a precipitant, or a mixed precipitant with the addition of an acid or inorganic salt. This method is simple and efficient, but the corresponding injection solution contains high concentrations of phospholipids, which is currently considered a major factor affecting accuracy, precision, and instrument contamination in liquid chromatography-mass spectrometry. Magnetic bead pretreatment, benefiting from the use of phospholipid-depleting magnetic beads, allows phospholipids of various polarities to be firmly adsorbed by the beads. This significantly reduces the phospholipid content in the injection solution, thereby alleviating phospholipid interference with the assay and alleviating the burden of column flushing, ensuring rapid detection.

[0119] This example uses the mixed magnetic bead method and protein precipitation method provided in Example 1 for sample pretreatment. The protein precipitation method uses a precipitant of methanol / acetonitrile (1:1). Specifically, 250 μL of a methanol / acetonitrile mixed precipitant containing an isotope internal standard was added to 50 μL of sample. The phospholipid removal effects of the two methods were investigated for human serum samples 1 to 4. The mass spectrometry parameters and ion source parameters for various phospholipid detection are shown in Tables 8 and 9, and the detection results are shown in Table 10.

[0120] Table 8. Mass spectrometry parameters for phospholipid removal magnetic beads

[0121]

[0122]

[0123] Table 9. Ion source conditions

[0124]

[0125] Table 10. Comparison of phospholipid removal effects between mixed magnetic bead method and protein precipitation method

[0126]

[0127] According to Table 10, it can be seen that the effect of removing phospholipids by the mixed magnetic bead method provided in Example 1 is significantly better than that of the phospholipid removal method by the precipitation protein method. After removing phospholipids, the phospholipid detection result is lower, which shows that more phospholipids have been removed. In addition, the mixed magnetic bead method is simpler and more efficient in removing phospholipids, can realize fully automated batch processing, eliminate human differences, and has a significant improvement effect.

[0128] Example 3: Combination Effects of Different Phospholipid-Removing Magnetic Beads and Different Drug-Absorbing Magnetic Beads

[0129] Combining different phospholipid-removing magnetic beads with different magnetic beads that adsorb the drug to be tested for pretreatment of therapeutic drug monitoring can result in significant differences in treatment efficacy. In this example, different magnetic bead combinations, as shown in Table 10, were selected for sample pretreatment and testing according to the methods provided in Example 1 to investigate the effects of different magnetic bead combinations on phospholipid removal and drug detection. Phospholipid content was detected using the same method as in Example 2.

[0130] The supplier of HLB magnetic beads is 3P Biosolutions, supplier: 3P Biosolutions, product number MB001-1; particle size 20-40 μm, specific surface area approximately 600-750 m 2 / g, pore size approximately 80A; MCX magnetic beads, supplier: 3P Biosolutions, product number MB002-1; particle size 20-40μm, specific surface area approximately 600-750m 2 / g, pore size approximately 80A; MAX magnetic beads, supplier: 3PBiosolutions, product number MB003-1; particle size 20-40μm, specific surface area approximately 600-750m 2 / g, pore size is about 80A;

[0131] TiO2 magnetic beads and ZrO2 magnetic beads are customized phospholipid removal magnetic beads with a particle size of 20-40μm and a specific surface area of approximately 600-750m 2 / g, with a pore size of approximately 80 Å. TiO2 and ZrO2 were bonded to the surface as active components for phospholipid removal. The test sample was a serum sample. Table 11 lists the test results for four therapeutic drugs whose extraction effects were significantly affected by different magnetic bead combinations, with precipitated protein used as a control.

[0132] Table 11. Effects of different magnetic bead combinations on test results

[0133]

[0134] Table 11 shows that different magnetic bead combinations can produce varying results in phospholipid removal and drug adsorption. This may be due to competitive binding, steric hindrance, nonspecific adsorption, and separation efficiency. Taking all factors into consideration, the most preferred magnetic bead combination is HLB extraction beads plus ZrO2 phospholipid removal beads. This not only maintains excellent phospholipid removal (minimizing phospholipid content in the supernatant), reduces matrix effects, and enhances test accuracy, but also increases the peak area of the drug being tested, improving detection sensitivity.

[0135] This example also verified other therapeutic drugs, and found that for each therapeutic drug, the use of HLB extraction magnetic beads combined with ZrO2 phospholipid removal magnetic beads has a better phospholipid removal effect and improves detection accuracy and sensitivity.

[0136] Example 4: Effect of acid addition

[0137] In order to achieve optimal extraction efficiency and better universality, the acid-base properties of the solution or the organic phase ratio need to be strictly controlled when using ZrO2 dephospholipid magnetic beads and HLB magnetic beads for extraction.

[0138] In this example, pure water or water with formic acid, citric acid, and phosphoric acid added was used as the diluent, and magnetic bead extraction and detection were performed according to the method provided in Example 1 under the same other conditions. The sample to be tested was a human serum sample. The test results are shown in Table 12. The research results show that the addition of acid has a certain effect of increasing the peak area of almost all therapeutic drugs in the sample, especially for acidic drugs therein; in addition, in addition to phosphoric acid, the addition of acid also helps to improve the effect of phospholipid removal magnetic beads on phospholipid adsorption, but the addition of phosphoric acid will relatively reduce the effect of phospholipid removal magnetic beads on phospholipid adsorption, because phosphoric acid can competitively bind to the binding sites of phospholipid removal magnetic beads, removing phospholipids from the phospholipid removal magnetic beads, thereby causing the HLB magnetic beads to loosely bind to phospholipids, thereby affecting the phospholipid content in the final eluate. Table 12 lists the test results of four acidic therapeutic drugs with more obvious effects, and uses precipitated protein as a control to calculate the percentage of improvement or reduction in the test results.

[0139] Table 12. Effect of adding acid to diluent on test results

[0140]

[0141]

[0142] Table 12 shows that adding acid to the diluent not only increases the detection peak area of the therapeutic drug but also effectively enhances phospholipid removal, effectively reducing matrix content and improving the accuracy of the test results. The lower the residual phospholipid content in the supernatant, the better the phospholipid adsorption efficiency of the phospholipid removal beads. Comparing formic acid (FA) and citric acid (Citric Acid), it can be seen that the addition of formic acid further enhances the phospholipid removal efficiency of the phospholipid removal beads in the mixed magnetic beads.

[0143] The test results for valproic acid, quetiapine, ziprasidone, and stiripentol show that when pure water is used as the diluent, the peak areas of these substances measured by magnetic bead extraction are significantly lower than those after protein precipitation. This may be due to an unsuitable extraction environment, which severely impacts the extraction efficiency of these substances. For example, valproic acid contains a carboxyl group in its structure and has a pKa of approximately 4.6. When pure water is used as the diluent, the pH of the solution during magnetic bead extraction is approximately 7. Valproic acid exists in the solution in an ionic state, making it difficult to fully extract with the HLB magnetic beads.

[0144] By adding 1% formic acid (FA) or citric acid to the diluent, the extraction rate of the three drugs by HLB magnetic beads in the mixed magnetic beads can be improved. The reason may be that the acidic conditions can significantly inhibit the ionization of carboxyl groups, maintain a neutral molecular state, and significantly improve the extraction rate of HLB magnetic beads. At the same time, the change of acidity and base can also regulate the microenvironment of drug-protein binding, promote the dissociation of drugs from proteins, and thus accelerate the drug extraction process.

[0145] It can also be seen that for different drugs, the extraction effects are not exactly the same when different acids are added. When formic acid is added, the detection results of the four drugs are compared with the detection results after precipitation treatment, and the general response is improved. Among them, valproic acid and ziprasidone have relatively more obvious improvements; when phosphoric acid is added, the detection results of stiripentol are significantly reduced compared with formic acid, and slightly reduced compared with the protein precipitation method. It is speculated that the reason for the increased response of valproic acid is the result of the pH value combined with the interference of phospholipids; ziprasidone is mainly protein-bound (>99%), and sufficient protein dissociation is required in an acidic environment. Therefore, it is most preferred that formic acid be added to the diluent to improve the detection accuracy of the four drugs at the same time.

[0146] Example 5: Optimization of magnetic bead extraction method

[0147] When optimizing magnetic bead extraction conditions in this example, it was found that most drugs were extracted quickly and required minimal mixing. However, for drugs with strong protein binding or low polarity, such as lurasidone, ziprasidone, cyclosporine, and paclitaxel, sufficient mixing and shaking were required to ensure adequate contact between the magnetic beads and the sample, as well as sufficient dissociation between the sample and the protein. Using different extraction conditions, as shown in Table 13, directly impacted the extraction efficiency of low-polarity drugs like cyclosporine and paclitaxel, as well as the extraction of highly protein-bound drugs like lurasidone and ziprasidone. Due to the consistent trends observed for similar drugs, this example only lists the test results for paclitaxel and ziprasidone. Sample pretreatment and testing were performed using the methods provided in Example 1. The effects of different extraction conditions on the test results were examined, with all other conditions remaining the same. See Table 13 for details.

[0148] Table 13. Effects of different extraction conditions on the detection of paclitaxel and ziprasidone

[0149]

[0150] According to Table 13, the most preferred extraction condition is mixing for 120 seconds, and accurate detection can be achieved without standing. The effect of phospholipid removal by phospholipid removal magnetic beads is less affected, and the extraction effect of paclitaxel and ziprasidone is the best, with the highest detection peak area.

[0151] Example 6: Effect of Magnetic Bead Dosage and Usage Method on Detection Results

[0152] 1. Optimization of magnetic bead dosage

[0153] In this example, the dosage of magnetic beads is optimized. The dosage of magnetic beads is the dosage of mixed magnetic beads (the concentration of mixed magnetic beads is 10 mg / ml, so 0.5 mg is equivalent to 0.05 ml, 1 mg is equivalent to 0.1 ml, 2 mg is equivalent to 0.2 ml, and 3 mg is equivalent to 0.3 ml). The sample volume is 20 μL. The method provided in Example 1 is used for sample pretreatment and detection. Under the same conditions, the effects of different magnetic bead dosages on the therapeutic drug detection results are investigated. It is found that the magnetic bead dosage has the greatest impact on cyclophosphamide, imatinib, and osimertinib. Since the impact on other drugs is not significant, this example only lists the detection results of cyclophosphamide, imatinib, and osimertinib. See Table 14 for details.

[0154] Table 14. Effect of Magnetic Bead Amount

[0155]

[0156] According to Table 14, it can be seen that some drugs are not sensitive to different dosages of magnetic beads, and the extraction rate fluctuates within a certain range, such as cyclophosphamide. For most drugs, the extraction rate first increases and then decreases slightly with the increase of the dosage of magnetic beads, such as imatinib. The extraction rate of very small amounts of drugs will decrease significantly when the amount of magnetic beads is further increased, such as osimertinib. It is speculated that the reason for this difference may be related to the binding strength between the magnetic beads and the drugs combined with the extraction conditions of each drug. Finally, a dosage of 1 mg (0.1 ml, 100 μL) of magnetic beads was selected. At this time, the phospholipid removal effect is also the best, the matrix effect is the lowest, and the detection results are more accurate.

[0157] 2. Use of extraction magnetic beads alone or in combination with dephospholipid magnetic beads

[0158] This example also investigated: 1. Extraction with HLB magnetic beads alone; 2. The simultaneous use of HLB magnetic beads and dephospholipidated magnetic beads; and 3. The dephospholipidated magnetic beads were used to remove phospholipids first, followed by extraction with HLB magnetic beads. The extraction effects were compared, and it was found that the simultaneous use with dephospholipidated magnetic beads had significant beneficial effects, and the magnitude of the effects on the various test indicators varied. The greatest effects were found on quetiapine, carbamazepine, and duloxetine. Therefore, this example only lists the test results for quetiapine, carbamazepine, and duloxetine, as shown in Table 15.

[0159] Table 15. Effect of using phospholipid-free magnetic beads

[0160]

[0161]

[0162] According to Table 15, HLB magnetic beads can also extract the drug under test without the use of phospholipid removal magnetic beads. However, the peak area of the remaining phospholipids (PC (16:0 / C18:2) in the supernatant without the use of phospholipid removal magnetic beads is approximately 30 times that of the phospholipid response when the "phospholipid removal magnetic beads" are used. The response of duloxetine is strongly affected by phospholipids, and the response is reduced by about 10 times without the use of phospholipid removal magnetic beads.

[0163] In addition, compared with first using phospholipid removal magnetic beads to remove phospholipids and then using HLB magnetic beads for extraction, it can be found that the combination of HLB magnetic beads and phospholipid removal magnetic beads also improves the phospholipid removal effect and significantly improves the extraction of the analytes. This may be because the simultaneous use of HLB magnetic beads and phospholipid removal magnetic beads makes the charge microenvironment of the solution more suitable for extraction, and the extraction activity of both magnetic beads for phospholipids and analytes is enhanced, indicating that there is a certain synergistic effect when using them together. Moreover, simultaneous use is more convenient and simple to operate. Therefore, the combination of HLB magnetic beads and phospholipid removal magnetic beads is the most preferred method. It can not only effectively reduce the phospholipid content of the supernatant, but also helps to improve the mass spectrometry response of some test indicators.

[0164] This example also verified other therapeutic drugs, and found that for each therapeutic drug, the combination of HLB magnetic beads and phospholipid-removing magnetic beads had a better phospholipid removal effect and improved detection accuracy and sensitivity.

[0165] Example 7: Screening of eluent

[0166] The eluent for the mixed magnetic beads must meet the following requirements: 1. Efficiently elute the test drug from the HLB magnetic beads; 2. Avoid eluting phospholipids from the phospholipid-removing magnetic beads; and 3. Eliminate interference from the phospholipid-removing magnetic beads during the elution of the test drug from the HLB magnetic beads. This example employed the eluent formulas provided in Table 16, and performed pretreatment, elution, and drug detection according to the methods provided in Example 1. The goal was to identify an eluent with the most effective and simplest formula to enhance TDM analysis. Because the elution efficiency of the eluent varies with different drugs, this example uses lurasidone, a drug with a more pronounced effect, as an example. The test results are shown in Table 16.

[0167] Table 16. Comparison of the effects of different eluents

[0168]

[0169]

[0170] Studies have found that pure methanol can completely elute most drugs extracted from HLB, leaving minimal residual amounts. However, some drugs, such as lurasidone, exhibit strong binding and require the use of an acidified elution solvent. Considering volatility and compatibility with subsequent detection systems, 0.1% formic acid in methanol is the most preferred eluent. Citric acid and phosphoric acid are preferred because their boiling points are higher than formic acid, which can affect the ionization efficiency of mass spectrometry detection. Furthermore, phosphoric acid can effectively elute phospholipids bound to the phospholipid beads, thus affecting the phospholipid response in the supernatant.

[0171] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the scope defined by the claims.

Claims

1. A therapeutic drug monitoring kit, characterized in that: The invention comprises a phospholipid removal magnetic bead solution, wherein the phospholipid removal magnetic bead solution contains magnetic beads for removing phospholipids in a sample.

2. The kit according to claim 1, wherein It also includes a magnetic bead solution for adsorbing drugs to be tested, wherein the magnetic bead solution for adsorbing drugs to be tested and the phospholipid-removing magnetic bead solution are combined into a mixed magnetic bead solution; the magnetic bead solution for adsorbing drugs to be tested contains magnetic beads for adsorbing and extracting drugs to be tested from samples.

3. The kit according to claim 2, wherein The phospholipid removal magnetic beads include any one or more of phospholipid removal magnetic beads, mixed mode magnetic beads, TiO2 silica gel magnetic beads, and ZrO2 silica gel magnetic beads.

4. The kit according to claim 2, wherein The magnetic beads for adsorbing the drug to be tested include any one or more of C18 magnetic beads, C8 magnetic beads, phenyl magnetic beads, silica magnetic beads, PS magnetic beads, HLB magnetic beads, MAX magnetic beads, and MCX magnetic beads.

5. The kit according to claim 2, wherein The phospholipid-removing magnetic beads in the mixed magnetic bead solution include ZrO2 silica gel magnetic beads, and the magnetic beads adsorbing the drug to be tested include HLB magnetic beads.

6. The kit according to claim 2, wherein The drugs to be tested include any one or more of sedative-hypnotic drugs, antidepressant drugs, antipsychotic drugs, antiepileptic drugs, antibiotic drugs, antitumor drugs, cardiovascular drugs and poison screening drugs.

7. The kit according to claim 6, wherein The sedative and hypnotic drugs include any one or more of alprazolam, clonazepam, midazolam, lorazepam, zopiclone, temazepam, bromazepam, nitrazepam, 6-hydroxybuspirone, buspirone, zaleplon, memantine, donepezil, tandospirone, diazepam, nordiazepam, oxazepam, zolpidem, and estazolam; The antidepressant drugs include any one or more of sertraline, fluoxetine, norfluoxetine, escitalopram, fluvoxamine, paroxetine, venlafaxine, O-desmethylvenlafaxine, duloxetine, mirtazapine, trazodone, milnacipran, amitriptyline, nortriptyline, doxepin, vortioxetine, desmethylclomipramine, clomipramine, agomelatine, bupropion, mianserin, nordoxepin, and hydroxybupropion; The antipsychotic drugs include any one or more of olanzapine, clozapine, paliperidone, risperidone, dehydroaripiprazole, aripiprazole, amisulpride, quetiapine, chlorpromazine, ziprasidone, N-desmethylclozapine, haloperidol, perphenazine, sulpiride, norquetiapine, fluphenazine, thioridazine, atomoxetine, lurasidone, blonanserin, maprotiline, methylphenidate, rivastigmine, perolanzapine, carbamazepine epoxide, norsertraline, norcitalopram, and normirtazapine; The anti-epileptic drugs include any one or more of oxcarbazepine, lamotrigine, levetiracetam, 10-hydroxycarbamazepine, carbamazepine, phenytoin sodium, topiramate, primidone, gabapentin, pregabalin, rufinamide, stiripentol, perampanel, zonisamide, lacosamide, valproic acid, and phenobarbital; The antibiotic drugs include any one or more of moxifloxacin, vancomycin, tigecycline, norvancomycin, polymyxin, linezolid, ciprofloxacin, sulfamethoxazole, and levofloxacin; The anti-tumor drugs include any one or more of cyclophosphamide, ifosfamide, methotrexate, 5-fluorouracil, capecitabine, irinotecan, paclitaxel, docetaxel, afatinib, apatinib, icotinib, erlotinib, gefitinib, crizotinib, regorafenib, vemurafenib, imatinib, N-desmethylimatinib, alectinib, and osimertinib; The cardiovascular drugs include any one or more of metoprolol, bisoprolol, nifedipine, amlodipine, atorvastatin calcium, 2-hydroxyatorvastatin, rosuvastatin, losartan, losartan metabolites, valsartan, irbesartan, telmisartan, clopidogrel metabolites, salicylic acid, ticagrelor, ticagrelor metabolite M8, etc.; The poison screening drugs include rodenticides (brodifacoum, bromadiolone, difacoum, chlordiquinone, warfarin, fludioxonone, coumatetralyl, fluoroacetic acid, fumarate, fumarate, thiadiazole, chlordiquinone, chlordiquinone, difacoum), pesticides (219 common types, see Table 7), psychotropic drugs (piroxicam, acetaminophen, o-ethoxybenzoate, paracetamol, sulindac, dihydroergotamine, ketorolac tromethamine, ketoprofen, isopropyl antipyrine, diphenhydramine), , loxapine, penfluridol, benzhexol, naproxen, nikethamide, benzoylecgonine, buprenorphine, fentanyl, flunitrazepam, ropivacaine, pethidine, procaine, hydroxydihydrocodeinone, tramadol, normorphine, ethylmorphine, dextropropoxyphene, lidocaine), any one or more of biological toxins (aconitine, solanine, colchicine, amygdalin, strophanthin, tetrodotoxin, amanita muscaria, aflatoxin).

8. The kit according to claim 2, wherein The mixed magnetic bead solution contains acid, and the acid includes any one or more of formic acid, acetic acid, and citric acid.

9. The kit according to claim 2, wherein Also included is an eluent comprising formic acid and methanol.

10. The kit according to claim 2, wherein It also includes a balancing liquid, a diluent and a liquid chromatography mobile phase additive, wherein the balancing liquid and the diluent are both aqueous formic acid solutions; the liquid chromatography mobile phase additive includes additives for mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution containing mobile phase additives, and mobile phase B is a methanol solution containing mobile phase additives, and the mobile phase additives are one or a combination of formic acid and ammonium acetate.

11. A method for detecting a drug, characterized in that: The method is based on non-disease diagnosis purposes and comprises the following steps: (1) treating the sample with a mixed magnetic bead solution, wherein the mixed magnetic bead solution includes phospholipid removal magnetic beads and test drug adsorption magnetic beads, and simultaneously adsorbs phospholipids and test drugs in the sample; (2) eluting the drug to be tested with an eluent, and analyzing the content of the drug to be tested by liquid chromatography-tandem mass spectrometry; The drugs to be tested include any one or more of sedative-hypnotic drugs, antidepressant drugs, antipsychotic drugs, antiepileptic drugs, antibiotic drugs, antitumor drugs, cardiovascular drugs and poison screening drugs.

12. A therapeutic drug monitoring system, characterized in that: The system includes a drug detection module, a data input / output interface and a data analysis module; the drug detection module uses the method as described in claim 11 to obtain the drug detection value, the data input / output interface is used to input the detection value of at least one drug, and the data analysis module is used to analyze the drug detection value, and the drugs include any one or more of sedatives and hypnotic drugs, antidepressants, antipsychotics, antiepileptic drugs, antibiotics, antitumor drugs, cardiovascular drugs and toxicology screening drugs; after analysis by the data analysis module, the data input / output interface is used to treat drug monitoring results.

13. Use of a mixed magnetic bead solution for preparing a reagent for improving the accuracy of therapeutic drug monitoring, characterized in that: The mixed magnetic bead solution includes a phospholipid removal magnetic bead solution and a drug adsorption magnetic bead solution. The phospholipid removal magnetic bead solution contains magnetic beads for removing phospholipids from the sample; the drug adsorption magnetic bead solution contains magnetic beads for adsorbing and extracting the drug from the sample.

14. The use according to claim 13, characterized in that The phospholipid-removing magnetic beads include ZrO2 silica gel magnetic beads, and the magnetic beads for adsorbing the drug to be tested include HLB magnetic beads.

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