Sample pretreatment methods, kits, and detection methods for antiepileptic drug testing

Abstract

This application relates to the field of analytical detection technology, specifically to sample pretreatment methods, kits, and detection methods for the detection of antiepileptic drugs. The sample pretreatment method for the detection of antiepileptic drugs in this application includes one or more of the following antiepileptic drugs: valproic acid, carbamazepine, phenytoin, levetiracetam, lamotrigine, and phenobarbital. The sample pretreatment method includes the following steps: mixing magnetic beads with an activation solution to prepare a magnetic bead suspension; mixing the sample to be tested, the magnetic bead suspension, and an internal standard solution, and separating the supernatant and the magnetic bead complex after magnetic adsorption treatment; adding an eluent to the magnetic bead complex, and collecting the magnetic bead-target complex after elution treatment; adding an elution buffer to the magnetic bead-target complex, and collecting the supernatant after elution treatment for detection. The above sample pretreatment method is simple to operate, time-saving, and low-cost, providing key technical support for the efficient quantitative detection of antiepileptic drugs.

Description

Technical Field

[0001] This application relates to the field of analytical detection technology, specifically to sample pretreatment methods, reagent kits, and detection methods for the detection of antiepileptic drugs. Background Technology

[0002] Epilepsy is a chronic neurological disorder caused by abnormal electrical discharges in the brain, characterized by recurrent epileptic seizures. Antiepileptic drugs (AEDs) are a class of medications used to prevent and control epileptic seizures. Their main mechanism of action is to regulate the excitability and inhibition of neurons, reducing the occurrence of abnormal discharges, thereby controlling or preventing epileptic seizures.

[0003] Because epilepsy requires long-term maintenance medication, and antiepileptic drugs have a narrow effective blood concentration range and significant individual metabolic variations, individualized dosing through monitoring blood drug concentrations is crucial for reducing adverse reactions and avoiding vicious cycles. Pretreatment of antiepileptic drugs is a key step in ensuring accurate analysis. Commonly used pretreatment methods include protein precipitation, liquid-liquid extraction, solid-phase extraction, and derivatization. While protein precipitation is simple to operate, it is difficult to automate; liquid-liquid extraction and solid-phase extraction have high extraction efficiency, but are time-consuming or costly; derivatization is complex and cannot meet the demands of high-throughput detection. Summary of the Invention

[0004] Based on this, this application provides a sample pretreatment method, kit, and detection method for the detection of antiepileptic drugs. The sample pretreatment method for the detection of antiepileptic drugs provided in this application is characterized by simple operation, short time consumption, low cost, and easy automation, and provides key technical support for the efficient quantitative detection of antiepileptic drugs.

[0005] A first aspect of this application provides a sample pretreatment method for detecting antiepileptic drugs, wherein the antiepileptic drugs include one or more of valproic acid, carbamazepine, phenytoin, levetiracetam, lamotrigine, and phenobarbital; the sample pretreatment method includes the following steps:

[0006] A magnetic bead suspension was prepared by mixing magnetic beads with an activation solution.

[0007] The sample to be tested, the magnetic bead suspension and the internal standard solution are mixed, and after magnetic adsorption treatment, the supernatant and the magnetic bead complex are separated.

[0008] Eluent was added to the magnetic bead complex, and after rinsing, the magnetic bead-target complex was collected.

[0009] An eluent was added to the magnetic bead-target complex, and after elution, the supernatant was collected for detection.

[0010] In one embodiment, the surface of the magnetic bead has a modifying group, which includes one or more of aromatic, pyrrolidone, and octadecyl groups.

[0011] In one embodiment, the internal standard solution includes an internal standard and an internal standard solvent.

[0012] In one embodiment, the correspondence between the internal standard and the types of antiepileptic drugs included in the test sample has one or more of the following characteristics:

[0013] (1) The antiepileptic drug is valproic acid, and the internal standard is a homologue of valproic acid;

[0014] (2) The antiepileptic drug is carbamazepine, and the internal standard is an impurity of oxcarbazepine. ;

[0015] (3) The antiepileptic drug is phenytoin, and the internal standard is a homologue of phenytoin;

[0016] (4) The antiepileptic drug is levetiracetam, and the internal standard is a homologue of levetiracetam;

[0017] (5) The antiepileptic drug is lamotrigine, and the internal standard is benzotriamine. ;

[0018] (6) The antiepileptic drug is phenobarbital, and the internal standard is a homologue of phenobarbital or a barbituric acid derivative.

[0019] In one embodiment, the activation solution is an aqueous isopropanol solution with a volume concentration of 40% to 60%; and / or, the rinsing solution includes one or more of water, an aqueous methanol solution with a volume concentration of 5% to 50%, an aqueous acetonitrile solution with a volume concentration of 5% to 50%, and an aqueous isopropanol solution with a volume concentration of 5% to 50%.

[0020] In one embodiment, the eluent includes one or more of methanol, acetonitrile, a methanol-formic acid mixture, a methanol-ammonia mixture, an acetonitrile-formic acid mixture, and an acetonitrile-ammonia mixture; wherein the methanol-formic acid mixture comprises methanol with a volume concentration of 99% to 99.95% and formic acid with a volume concentration of 0.05% to 1%; the methanol-ammonia mixture comprises methanol with a volume concentration of 99% to 99.95% and ammonia with a volume concentration of 0.05% to 1%; the acetonitrile-formic acid mixture comprises acetonitrile with a volume concentration of 99% to 99.95% and formic acid with a volume concentration of 0.05% to 1%; and the acetonitrile-ammonia mixture comprises acetonitrile with a volume concentration of 99% to 99.95% and ammonia with a volume concentration of 0.05% to 1%.

[0021] In one embodiment, the mass-volume concentration of the magnetic beads in the magnetic bead suspension is 40 mg / mL to 60 mg / mL.

[0022] In one embodiment, the sample to be tested is a serum sample.

[0023] In one embodiment, the volume-to-mass ratio of the sample to be tested and the magnetic beads included in the magnetic bead suspension is (10~500) μL : (0.25~0.5) mg.

[0024] In one embodiment, the volume ratio of the test sample to the internal standard solution is (10~500) μL: (100~500) μL.

[0025] In one embodiment, the process parameters for magnetic attraction include oscillation at 2000 rpm to 5000 rpm.

[0026] In one embodiment, the process parameters for the rinsing treatment include oscillation at 2000 rpm to 5000 rpm.

[0027] In one embodiment, the process parameters for the elution process include oscillation at 2000 rpm to 5000 rpm.

[0028] A second aspect of this application provides a pretreatment kit for detecting antiepileptic drugs, comprising: magnetic beads, activation solution, eluent, and elution solution; the pretreatment kit having one or more of the following characteristics:

[0029] (1) The surface of the magnetic beads has a modifying group, which includes one or more of aromatic group, pyrrolidone group and octadecyl group;

[0030] (2) The activation solution is an isopropanol aqueous solution with a volume concentration of 40%~60%;

[0031] (3) The rinsing solution includes one or more of the following: water, methanol aqueous solution with a volume concentration of 5% to 50%, acetonitrile aqueous solution with a volume concentration of 5% to 50%, and isopropanol aqueous solution with a volume concentration of 5% to 50%.

[0032] (4) The eluent includes one or more of methanol, acetonitrile, methanol-formic acid mixture, methanol-ammonia mixture, acetonitrile-formic acid mixture, and acetonitrile-ammonia mixture; wherein the methanol-formic acid mixture includes methanol with a volume concentration of 99%~99.95% and formic acid with a volume concentration of 0.05%~1%; the methanol-ammonia mixture includes methanol with a volume concentration of 99%~99.95% and ammonia with a volume concentration of 0.05%~1%; the acetonitrile-formic acid mixture includes acetonitrile with a volume concentration of 99%~99.95% and formic acid with a volume concentration of 0.05%~1%; and the acetonitrile-ammonia mixture includes acetonitrile with a volume concentration of 99%~99.95% and ammonia with a volume concentration of 0.05%~1%.

[0033] A third aspect of this application provides a method for detecting antiepileptic drugs, comprising the following steps:

[0034] After pretreating the sample to be tested using the sample pretreatment method for detecting antiepileptic drugs according to any one of the first aspects of this application, the collected supernatant is used as the test solution.

[0035] The peak intensity of the test solution was detected by ion mobility spectrometry, and the detection result was substituted into a standard curve reflecting the relationship between peak intensity and concentration to obtain the concentration of the antiepileptic drug in the test solution.

[0036] In one embodiment, the steps for constructing a standard curve reflecting the relationship between peak intensity and concentration include:

[0037] Standard working solutions of antiepileptic drugs at different concentrations were prepared using standard stock solutions. Ion mobility spectrometry was used to detect the standard working solutions containing internal standards, and a standard curve of antiepileptic drugs was constructed based on peak area and concentration.

[0038] In one embodiment, in the steps of detecting the peak intensity of the test solution using ion mobility spectrometry and detecting the standard working solution using ion mobility spectrometry, the conditions for ion mobility spectrometry each independently have one or more of the following characteristics:

[0039] (1) The ion source voltage is 1800V~2000V;

[0040] (2) The migration tube voltage is 8400V~9000V;

[0041] (3) The air inlet temperature is 170℃~190℃;

[0042] (4) The temperature of the migration tube is 230℃~240℃;

[0043] (5) The ion gate voltage is 45V~55V;

[0044] (6) The gate voltage pulse width is 90μs~110μs;

[0045] (7) The migration airflow velocity is 0.9 L / min to 1.2 L / min;

[0046] (8) The exhaust gas flow rate is 0.9 L / min to 1.2 L / min;

[0047] (9) The injection flow rate is 1.5 μL / min to 3 μL / min;

[0048] (10) The sampling frequency is 180000s -1 ~220000 s -1 .

[0049] In one embodiment, the mass-volume concentration of the antiepileptic drug varies in a gradient in the standard working solutions of the antiepileptic drug at different concentrations.

[0050] A fourth aspect of this application provides a kit for detecting antiepileptic drugs, comprising a pretreatment kit for detecting antiepileptic drugs, a standard working solution, and an internal standard solution;

[0051] Alternatively, it may include pretreatment kits, standard stock solutions, and internal standard solutions for the detection of antiepileptic drugs.

[0052] The internal standard solution includes an internal standard substance and an internal standard solvent.

[0053] The sample pretreatment method for detecting antiepileptic drugs provided in this application has at least the following beneficial effects:

[0054] The sample pretreatment method for antiepileptic drug detection provided in this application mixes activated magnetic beads with the sample to be tested. Based on the high specific adsorption capacity of the magnetic beads, the target drug can be rapidly enriched to ensure detection sensitivity. Furthermore, magnetic separation replaces the traditional centrifugation step, significantly simplifying the operation and effectively shortening the processing time. The rinsing and elution steps work synergistically to effectively remove matrix interference, ensuring the accuracy and stability of the detection results. Simultaneously, this pretreatment method is compatible with ion mobility spectrometry detection, enabling high-throughput sample processing. It combines low cost and high efficiency, laying a solid foundation for the accurate detection of antiepileptic drugs in clinical and research settings.

[0055] Therefore, the sample pretreatment method for antiepileptic drug detection provided in this application is characterized by simple operation, short time consumption, low cost, and easy automation, and can provide key technical support for the efficient quantitative detection of antiepileptic drugs. Attached Figure Description

[0056] Figure 1 This is a schematic diagram illustrating the working principle of ion mobility spectrometry.

[0057] Figure 2 The ion mobility spectrum was measured using the standard working solution with a mass / volume of 90 μg / mL in Example 1.

[0058] Figure 3 The ion mobility spectrum was measured for the standard working solution with a mass / volume of 40 μg / mL in Example 2.

[0059] Figure 4 The ion mobility spectrum was measured for the standard working solution with a mass / volume of 32 μg / mL in Example 3.

[0060] Figure 5 The ion mobility spectrum is obtained from the standard working solution with a mass / volume of 32 μg / mL in Example 4. Detailed Implementation

[0061] The sample pretreatment method, kit, and detection method for antiepileptic drug detection of this application are further described in detail below with reference to specific embodiments. This application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of this application.

[0062] It has been reported that there are significant differences in the absorption, metabolism, and elimination of antiepileptic drugs among different individuals. The relationship between drug dosage and pharmacological activity is further complicated by disease state, drug management, and patient complications. Therefore, monitoring the blood concentration of antiepileptic drugs is of great importance to help clinicians develop individualized dosing regimens and adjust dosages in a timely manner.

[0063] Traditional sample pretreatment methods for antiepileptic drugs mainly include protein precipitation and extraction. Protein precipitation primarily uses a methanol and acetonitrile mixture as a protein precipitant to pretreat plasma samples for antiepileptic drug detection. However, due to the poor stability of this pretreatment process, the detection results are easily affected by interference, resulting in large errors and low sensitivity. Extraction methods mainly use liquid-liquid extraction and solid-phase extraction to extract antiepileptic drugs from plasma samples for detection of the purified solution. Extraction methods have high extraction efficiency, but they are time-consuming.

[0064] Current sample pretreatment methods for antiepileptic drug testing suffer from drawbacks such as long processing times and reduced accuracy and stability of test results.

[0065] A first aspect of this application provides a sample pretreatment method for detecting antiepileptic drugs, wherein the antiepileptic drugs include one or more of valproic acid, carbamazepine, phenytoin, levetiracetam, lamotrigine, and phenobarbital.

[0066] Valproic acid (VPA) is the first-line monotherapy for many types of epilepsy, with good therapeutic effects. Carbamazepine is a derivative of imino-1,2-stilbene; due to differences in absorption and metabolism, individual responses to carbamazepine vary greatly. Phenytoin is a classic antiepileptic drug, belonging to the hydantoin class, which mainly controls seizures by inhibiting abnormal neuronal discharges. Levetiracetam belongs to the pyrrolidone class of derivatives; it mainly inhibits epileptiform discharges by selectively binding to synaptic vesicle protein SV2A and regulating presynaptic neurotransmitter release. Lamotrigine is a phenyltriazine antiepileptic drug that works by inhibiting voltage-dependent sodium channels and regulating glutamate release; it is an important drug in the clinical treatment of various types of epilepsy. Phenobarbital is a barbiturate antiepileptic drug with good sedative and antiepileptic effects, widely used for neonatal and pediatric seizures, as well as drug-resistant seizures and non-convulsive status epilepticus. Many of the above-mentioned antiepileptic drugs have nonlinear pharmacokinetic characteristics, resulting in significant differences in metabolism after ingestion. Therefore, optimizing pretreatment steps to monitor blood drug concentration in real time is crucial for guiding rational drug use in clinical practice.

[0067] Furthermore, the sample pretreatment method includes the following steps:

[0068] S10: Mix the magnetic beads with the activation solution to prepare a magnetic bead suspension.

[0069] S20: Mix the sample to be tested, the magnetic bead suspension and the internal standard solution, and after magnetic adsorption treatment, separate the supernatant and the magnetic bead complex.

[0070] S30: Add rinsing solution to the magnetic bead complex, and collect the magnetic bead-target complex after rinsing.

[0071] S40: Add elution solution to the magnetic bead-target complex, and after elution, collect the supernatant for detection.

[0072] The sample pretreatment method for antiepileptic drug detection provided in this application mixes activated magnetic beads with the sample to be tested. Based on the high specific adsorption capacity of the magnetic beads, the target drug can be rapidly enriched to ensure detection sensitivity. Furthermore, magnetic separation replaces the traditional centrifugation step, significantly simplifying the operation and effectively shortening the processing time. The rinsing and elution steps work synergistically to effectively remove matrix interference and achieve rapid separation of the target analyte and the magnetic beads. This ensures the accuracy and stability of the detection results while reducing pretreatment time. Simultaneously, this pretreatment method is compatible with ion mobility spectrometry detection, enabling high-throughput sample processing. It combines low cost and high efficiency, laying a solid foundation for the accurate detection of antiepileptic drugs in clinical and research scenarios. Therefore, the sample pretreatment method for antiepileptic drug detection provided in this application is characterized by simple operation, short processing time, low cost, and ease of automation, providing key technical support for the efficient quantitative detection of antiepileptic drugs.

[0073] In some examples, in step S10, the surface of the magnetic beads has modifying groups, which include one or more of aromatic, pyrrolidone, and octadecyl groups. For example, the magnetic beads are PLS magnetic beads or C18 magnetic beads. PLS magnetic beads comprise a matrix and modifying groups modified on the matrix surface, wherein the modifying groups of the PLS magnetic beads include phenyl and pyrrolidone groups. The matrix of the PLS magnetic beads comprises Fe3O4@SiO2. C18 magnetic beads comprise a matrix and modifying groups modified on the matrix surface, wherein the modifying groups of the C18 magnetic beads include octadecyl groups. The matrix of the C18 magnetic beads comprises Fe3O4@SiO2.

[0074] The aforementioned magnetic beads can be linked to antiepileptic drugs through surface modification groups to achieve the separation and detection of specific analytes.

[0075] In some examples, in step S10, the activation solution can be one or more of methanol-water solution, ethanol-water solution, and isopropanol-water solution. This is to ensure the activation efficiency of the functional groups on the surface of PLS ​​magnetic beads or C18 magnetic beads. Preferably, the activation solution is an isopropanol-water solution with a volume concentration of 40% to 60%. For example, the volume concentration of isopropanol in the activation solution includes, but is not limited to, 40%, 43%, 45%, 48%, 49%, 50%, 51%, 52%, 55%, 58%, or 60%, or any two of the above values ​​as endpoints.

[0076] The magnetic beads are activated by the above-mentioned activation solution. The isopropanol enhances the permeability of the magnetic beads to the pores, allowing the surface modification groups of the magnetic beads to fully expand and expose the adsorption sites. The polarity of water can also be used to prevent the magnetic bead matrix from being over-swelled or damaged, ensuring that the activated magnetic beads maintain stable and efficient performance in subsequent separation and other steps.

[0077] In some examples, in step S10, the mass-volume concentration of the magnetic beads in the magnetic bead suspension is 40 mg / mL to 60 mg / mL. Limiting the mass-volume concentration of the magnetic beads in the activation step ensures that the surface-modified groups are fully exposed and react uniformly with the activation solution through an appropriate magnetic bead density, while avoiding excessively high concentrations that could lead to bead aggregation and hinder the activation reaction, or excessively low concentrations that could reduce activation efficiency, thus achieving an optimal balance between activation effect and operational stability. For example, the mass-volume concentration of the magnetic beads in the magnetic bead suspension includes, but is not limited to, 40 mg / mL, 45 mg / mL, 48 mg / mL, 49 mg / mL, 50 mg / mL, 51 mg / mL, 52 mg / mL, 55 mg / mL, 58 mg / mL, or 60 mg / mL, or any two of the above values ​​as endpoints within a range.

[0078] In some examples, the sample pretreatment method of this application does not include the step of equilibrating the magnetic beads with water. The sample pretreatment method provided by this application, through the synergy of magnetic beads, activator, and internal standard solution, avoids the step of equilibrating the magnetic beads with purified water, thus having the advantages of short processing time and high processing efficiency.

[0079] In some examples, in step S20, the sample to be tested is a serum sample. Using the serum sample directly as the test sample can directly reflect the real-time concentration of the analyte, and the sampling operation is relatively convenient.

[0080] In some examples, in step S20, the volume-to-mass ratio of the test sample to the magnetic beads included in the magnetic bead suspension is (10~500) μL : (0.25~0.5) mg. For example, the volume-to-mass ratio of the test sample to the magnetic beads included in the magnetic bead suspension includes, but is not limited to, 10 μL : 0.25 mg, 20 μL : 0.25 mg, 50 μL : 0.25 mg, 70 μL : 0.25 mg, 90 μL : 0.25 mg, 100 μL : 0.25 mg, 100 μL : 0.3 mg, 120 μL : 0.3 mg, 150 μL : 0.3 mg, and 170 μL. : 0.3mg, 190μL: 0.3mg, 200μL: 0.3mg, 300μL: 0.4mg, 320μL: 0.4mg, 350μL: 0.4mg, 370μL: 0.4mg, 390μL: 0.4mg, 400μL: 0.4mg, 490μL: 0.5mg or 500μL: 0.5mg, or any two of the above point values ​​as endpoints within the range.

[0081] The volume-to-mass ratio of the sample to be tested and the magnetic beads in the magnetic bead suspension is limited to the above range. This ensures that the adsorption sites on the surface of the magnetic beads are fully combined with the antiepileptic drugs in the sample to achieve efficient enrichment, while avoiding excessive use of magnetic beads that could lead to increased non-specific adsorption or insufficient use that could affect the separation efficiency.

[0082] In some of these examples, in step S20, the internal standard solution includes an internal standard and an internal standard solvent.

[0083] The correspondence between the internal standard and the types of antiepileptic drugs included in the test sample has one or more of the following characteristics:

[0084] (1) The antiepileptic drug is valproic acid, and the internal standard is a homologue of valproic acid;

[0085] (2) The antiepileptic drug is carbamazepine, and the internal standard is an impurity of oxcarbazepine. ;

[0086] (3) The antiepileptic drug is phenytoin, and the internal standard is a homologue of phenytoin;

[0087] (4) The antiepileptic drug is levetiracetam, and the internal standard is a homologue of levetiracetam;

[0088] (5) The antiepileptic drug is lamotrigine, and the internal standard is benzotriamine. ;

[0089] (6) The antiepileptic drug is phenobarbital, and the internal standard is a homologue of phenobarbital or a barbituric acid derivative.

[0090] This application selects homologues or parent nucleus fragments of antiepileptic drugs as internal standards. These internal standards are obtained by modifying the target analyte molecule with methyl, ethyl, or other groups with similar physicochemical properties. By utilizing the similar chemical properties and chromatographic behavior of the internal standard to the target analyte, matrix effects, injection errors, and instrument fluctuations can be simultaneously corrected during sample pretreatment and detection, thereby significantly improving the accuracy and repeatability of quantitative analysis. Furthermore, the selection of these internal standards avoids the high cost of isotope labeling and reduces environmental pollution.

[0091] Further, the antiepileptic drug is valproic acid, and the internal standard is 2-propylhexanoic acid. The structural formula of 2-propylhexanoic acid is... It is a homologue of valproic acid.

[0092] Furthermore, the antiepileptic drug is carbamazepine, and the internal standard is an impurity of oxcarbazepine. This internal standard has a similar parent nucleus to carbamazepine.

[0093] Further, the antiepileptic drug is phenytoin, and the internal standard is 5-(4-methylphenyl)5-phenylhydantoin. The structural formula of 5-(4-methylphenyl)5-phenylhydantoin is... It is a homologue of phenytoin.

[0094] Further, the antiepileptic drug is levetiracetam, and the internal standard is a homologue of levetiracetam. For example, the internal standard for levetiracetam could be pirenzeracetam.

[0095] (5) The antiepileptic drug is lamotrigine, and the internal standard is benzotricyanate. The structural formula of benzotricyanate is: .

[0096] (6) The antiepileptic drug is phenobarbital, and the internal standard is a homologue of phenobarbital or a barbituric acid derivative. For example, the internal standard for phenobarbital is pentobarbital.

[0097] In some examples, the internal standard solvent includes an aqueous solution of formic acid with a volume concentration of 0.05% to 0.5%, an aqueous solution of acetic acid with a volume concentration of 0.05% to 0.5%, an aqueous solution of phosphoric acid with a volume concentration of 0.05% to 0.5%, an aqueous solution of ammonium acetate with a molar concentration of 10 mmol / L to 50 mmol / L, an aqueous solution of ammonium formate with a molar concentration of 10 mmol / L to 50 mmol / L, or an aqueous solution of ammonia with a volume concentration of 0.08% to 0.15%. The volume concentration of the aqueous formic acid solution in the above internal standard solvents includes, but is not limited to, 0.05%, 0.08%, 0.1%, 0.11%, 0.13%, 0.15%, 0.2%, 0.4%, or 0.5%. The volume concentration of the aqueous acetic acid solution in the above internal standard solvents includes, but is not limited to, 0.05%, 0.08%, 0.1%, 0.11%, 0.13%, 0.15%, 0.2%, 0.4%, or 0.5%. Among the internal standard solvents mentioned above, the volume concentration of the phosphoric acid aqueous solution includes, but is not limited to, 0.05%, 0.08%, 0.1%, 0.11%, 0.13%, 0.15%, 0.2%, 0.4%, or 0.5%. The molar concentration of the ammonium acetate aqueous solution is 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, or 50 mmol / L. The molar concentration of the ammonium formate aqueous solution is 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, or 50 mmol / L. The volume concentration of the ammonia aqueous solution includes, but is not limited to, 0.08%, 0.1%, 0.12%, or 0.15%.

[0098] Further, in the internal standard solution, the mass-volume concentration of the internal standard is 0.4 μg / mL to 12 μg / mL. For example, the mass-volume concentration of the internal standard in the internal standard solution includes, but is not limited to, 0.4 μg / mL, 0.8 μg / mL, 2 μg / mL, 4 μg / mL, 6 μg / mL, 6.2 μg / mL, 6.3 μg / mL, 6.5 μg / mL, 7 μg / mL, 7.5 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 11 μg / mL, or 12 μg / mL.

[0099] In some examples, in step S20, the volume ratio of the test sample to the internal standard solution is (10~500) μL:(100~500) μL. The volume ratio of the test sample to the internal standard solution includes, but is not limited to, 50 μL:400 μL, 50 μL:450 μL, 50 μL:500 μL, 90 μL:400 μL, 100 μL:400 μL, 110 μL:400 μL, 180 μL:400 μL, 190 μL:400 μL, 200 μL:400 μL, or 210 μL:400 μL.

[0100] As a further example, the antiepileptic drug included in the test sample is valproic acid, and the volume ratio of the test sample to the internal standard solution is (40~60) μL: (440~460) μL.

[0101] As a further example, the antiepileptic drug included in the test sample is lamotrigine, and the volume ratio of the test sample to the internal standard solution is (180~220) μL: (380~420) μL.

[0102] As a further example, the antiepileptic drug included in the test sample is phenytoin, and the volume ratio of the test sample to the internal standard solution is (80~120) μL: (380~420) μL.

[0103] As a further example, the antiepileptic drug included in the test sample is carbamazepine, and the volume ratio of the test sample to the internal standard solution is (80~120) μL: (380~420) μL.

[0104] In some examples, in step S20, the process parameters for the magnetic attraction process include oscillation at 2000 rpm to 5000 rpm.

[0105] In some examples, the magnetic adsorption treatment time in step S20 is 0.8 min to 2 min. For example, the magnetic adsorption treatment time includes, but is not limited to, 0.8 min, 0.9 min, 1 min, 1.2 min, 1.5 min, or 2 min. During the magnetic adsorption treatment step, the modifying groups on the surface of the magnetic beads can specifically bind to the target analyte to rapidly separate the magnetic bead complex and the supernatant.

[0106] Since the magnetic bead complex in step S20 contains impurities in addition to the target analyte, it is necessary to use an elution solution to remove the impurities in order to further eliminate interference from the impurities.

[0107] In some examples, in step S30, the eluent comprises one or more of the following: water, a methanol aqueous solution with a volume concentration of 5% to 50%, an acetonitrile aqueous solution with a volume concentration of 5% to 50%, and an isopropanol aqueous solution with a volume concentration of 5% to 50%. The volume concentration of the methanol aqueous solution in the eluent includes, but is not limited to, 5%, 6%, 8%, 10%, 14%, 15%, 16%, 20%, 30%, 40%, or 50%. The volume concentration of the acetonitrile aqueous solution includes, but is not limited to, 5%, 8%, 10%, 15%, 20%, 40%, or 50%. The volume concentration of the isopropanol aqueous solution includes, but is not limited to, 5%, 8%, 10%, 20%, 40%, or 50%.

[0108] In some examples, in step S30, the process parameters for the rinsing treatment include oscillation at 2000 rpm to 5000 rpm.

[0109] In some examples, the rinsing time is 0.8 min to 2 min. For example, the rinsing time includes, but is not limited to, 0.8 min, 0.9 min, 1 min, 1.2 min, 1.5 min, or 2 min. During the rinsing step, the strong solubility and affinity between the rinsing solution and the impurity components are utilized to detach the impurities from the magnetic bead composite and carry them away by the rinsing solution, thereby achieving the purpose of impurity removal.

[0110] In some examples, in step S40, the eluent includes one or more of methanol, acetonitrile, a methanol-formic acid mixture, a methanol-ammonia mixture, an acetonitrile-formic acid mixture, and an acetonitrile-ammonia mixture; wherein the methanol-formic acid mixture comprises methanol with a volume concentration of 99% to 99.95% and formic acid with a volume concentration of 0.05% to 1%. For example, in the methanol-formic acid mixture, the volume concentration of methanol includes, but is not limited to, 99.5%, 99.8%, 99.9%, 99.92%, or 99.95%; and the volume concentration of formic acid includes, but is not limited to, 0.5%, 0.2%, 0.1%, 0.08%, or 0.05%. The methanol-ammonia mixture comprises methanol with a volume concentration of 99% to 99.95% and ammonia with a volume concentration of 0.05% to 1%. For example, in the methanol-ammonia mixture, the volume concentration of methanol includes, but is not limited to, 99.5%, 99.8%, 99.9%, 99.92%, or 99.95%; the volume concentration of ammonia includes, but is not limited to, 0.5%, 0.2%, 0.1%, 0.08%, or 0.05%. For example, the acetonitrile-formic acid mixture includes acetonitrile with a volume concentration of 99% to 99.95% and formic acid with a volume concentration of 0.05% to 1%. In the acetonitrile-formic acid mixture, the volume concentration of acetonitrile includes, but is not limited to, 99.5%, 99.8%, 99.9%, 99.92%, or 99.95%; the volume concentration of formic acid includes, but is not limited to, 0.5%, 0.2%, 0.1%, 0.08%, or 0.05%. In the acetonitrile-ammonia mixture, the volume concentration of acetonitrile includes, but is not limited to, 99.5%, 99.8%, 99.9%, 99.92%, or 99.95%; and the volume concentration of ammonia includes, but is not limited to, 0.5%, 0.2%, 0.1%, 0.08%, or 0.05%.

[0111] In some of these examples, the elution process parameters include oscillation at 2000 rpm to 5000 rpm.

[0112] In some examples, the elution time is 0.8 min to 2 min. Examples include, but are not limited to, 0.8 min, 0.9 min, 1 min, 1.2 min, 1.5 min, or 2 min. Through the eluent and elution process, rapid separation of the magnetic beads and the analyte can be achieved, thereby enabling the detection of the analyte.

[0113] A second aspect of this application provides a pretreatment kit for detecting antiepileptic drugs, comprising: magnetic beads, activation solution, rinsing solution, and elution solution.

[0114] In some of these examples, the surface of the magnetic beads has modifying groups, including one or more of aromatic groups, pyrrolidone groups, and octadecyl groups.

[0115] In some of these examples, the activating solution is an aqueous solution of isopropanol with a volume concentration of 40% to 60%.

[0116] In some examples, the rinsing solution includes one or more of water, a methanol aqueous solution with a volume concentration of 5% to 50%, an acetonitrile aqueous solution with a volume concentration of 5% to 50%, and an isopropanol aqueous solution with a volume concentration of 5% to 50%.

[0117] In some examples, the eluent comprises one or more of methanol, acetonitrile, a methanol-formic acid mixture, a methanol-ammonia mixture, an acetonitrile-formic acid mixture, and an acetonitrile-ammonia mixture; wherein the methanol-formic acid mixture comprises methanol with a volume concentration of 99% to 99.95% and formic acid with a volume concentration of 0.05% to 1%; the methanol-ammonia mixture comprises methanol with a volume concentration of 99% to 99.95% and ammonia with a volume concentration of 0.05% to 1%; the acetonitrile-formic acid mixture comprises acetonitrile with a volume concentration of 99% to 99.95% and formic acid with a volume concentration of 0.05% to 1%; and the acetonitrile-ammonia mixture comprises acetonitrile with a volume concentration of 99% to 99.95% and ammonia with a volume concentration of 0.05% to 1%.

[0118] Understandably, the above-mentioned pretreatment kit can be used not only to process the test samples. In ion mobility spectrometry, after preparing standard working solutions or quality control solutions using bovine serum albumin (BSA) as a solvent, the same pretreatment method or kit can also be used for pretreatment.

[0119] It is understood that the properties of the magnetic beads, activation solution, rinsing solution and elution solution included in the pretreatment kit for detecting antiepileptic drugs described in the second aspect of this application are the same as those in the first aspect of this application, and therefore will not be repeated here.

[0120] In some examples, the pretreatment kit for antiepileptic drug detection further includes an internal standard solution. The internal standard solution comprises an internal standard and an internal standard solvent. The internal standard solvent includes an aqueous formic acid solution with a volume concentration of 0.05%–0.5%, an aqueous acetic acid solution with a volume concentration of 0.05%–0.5%, an aqueous phosphoric acid solution with a volume concentration of 0.05%–0.5%, an ammonium acetate solution with a molar concentration of 10 mmol / L–50 mmol / L, an ammonium formate solution with a molar concentration of 10 mmol / L–50 mmol / L, or an ammonia solution with a volume concentration of 0.08%–0.15%. The internal standard in the internal standard solution has a mass-volume concentration of 8 μg / mL–12 μg / mL.

[0121] The pretreatment kit of this application utilizes reagents that work synergistically to overcome the limitations of traditional pretreatment methods, such as complexity, time-consuming processes, and low extraction efficiency. Specifically, the activation solution, through optimized formulation, ensures full exposure of active sites on the magnetic bead surface, enabling the magnetic beads to specifically adsorb the target analyte; the internal standard solution, leveraging the homologue properties of the target analyte, accurately corrects experimental errors; the eluent efficiently removes impurities, and the elution buffer completely releases the target analyte. The entire pretreatment process is simple to operate, with compact steps, enabling rapid sample processing.

[0122] Furthermore, this pretreatment kit is suitable for the deployment and implementation of automated detection platforms and is applicable to the pretreatment requirements of ion mobility spectra. This provides precise data support for the formulation of individualized clinical medication regimens, reduces the incidence of adverse reactions such as neurotoxicity caused by drug overdose, and enables precise control of blood drug concentration within the therapeutic window, thereby playing an important role in improving the efficacy / risk ratio of epilepsy treatment.

[0123] Existing detection techniques for antiepileptic drugs include high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS / MS), immunoassays (such as homogeneous enzyme immunoassay and fluorescence polarization immunoassay), and gas chromatography (GC). These methods often require complex sample pretreatment (such as extraction, centrifugation, and derivatization), resulting in long pretreatment times (30 minutes to several hours) and the potential for introducing errors. They also have long detection cycles (several minutes to over ten minutes), making real-time monitoring difficult.

[0124] Based on this, a third aspect of this application provides a method for detecting antiepileptic drugs, comprising the following steps:

[0125] P10: After pretreating the sample to be tested using the sample pretreatment method for detecting antiepileptic drugs according to any one of the first aspects of this application, the collected supernatant is used as the test solution.

[0126] P20: The peak intensity of the test solution is detected by ion mobility spectrometry, and the detection result is substituted into the standard curve reflecting the relationship between peak intensity and concentration to obtain the concentration of the antiepileptic drug in the test solution.

[0127] This application employs ion mobility spectrometry for the quantitative detection of antiepileptic drugs, which offers a faster detection rate with a detection cycle of approximately 20-30 seconds, effectively overcoming the drawback of long detection cycles in traditional techniques. For details, please refer to... Figure 1Ion mobility spectrometry (IMS) refers to the principles, methods, and instruments used to characterize substances based on the velocities of a group of substances (defined as gaseous ion clusters) in an electric field and drift gas environment. The sample to be tested passes through a "desolvation region" to allow ions to escape the solvent, and after ionization, enters a migration tube filled with drift gas, where they move under the influence of an electric field. Due to differences in mass, charge, and shape, different ions exhibit varying migration rates in the drift gas, thus achieving separation and reaching the ion detector at different drift times. Therefore, the characteristic peaks of the analyte can be determined based on the drift time, and the relative relationship between the peak area of ​​the characteristic peak and a standard curve enables rapid quantitative analysis of antiepileptic drugs.

[0128] In some of these examples, step P20, which involves constructing a standard curve reflecting the relationship between peak intensity and concentration, includes:

[0129] Standard working solutions of antiepileptic drugs at different concentrations were prepared, and an internal standard solution was added to the standard working solutions to prepare a standard working solution containing the internal standard. The standard working solution containing the internal standard was detected by ion mobility spectrometry, and a standard curve of the antiepileptic drugs was constructed based on the peak area and concentration.

[0130] In some of these examples, the mass-volume concentration of the antiepileptic drug varies in a gradient within the standard working solutions of the different concentrations of the antiepileptic drug.

[0131] According to the *Chinese Pharmacopoeia*, the *Consensus Guidelines for Monitoring Neuropsychopharmacological Therapeutic Drugs: 2017 Edition*, and the *Expert Consensus on Monitoring Therapeutic Drugs in Children*, the effective blood concentrations of valproic acid are (50~100) μg / mL. The effective blood concentrations of phenytoin are (10~20) μg / mL. The effective blood concentrations of carbamazepine are (4~12) μg / mL. The effective blood concentrations of lamotrigine are (3~15) μg / mL. The effective blood concentrations of levetiracetam are (12~46) μg / mL. As can be seen, the effective blood concentration ranges of different antiepileptic drugs vary. Therefore, to ensure that the standard working solution accurately covers the effective concentration range of each drug and meets the linear range requirements for quantitative detection, its concentration needs to be differentiated according to the type of drug.

[0132] For example, the antiepileptic drug is valproic acid, and the mass-volume concentration of valproic acid in the standard working solution is independently 20 μg / mL to 200 μg / mL, and the mass-volume concentration of valproic acid in the standard working solution varies in a gradient. Further, the antiepileptic drug is valproic acid, and the mass-volume concentration of valproic acid in the standard working solutions of different concentrations of the antiepileptic drug is (20~22) μg / mL, (28~32) μg / mL, (48~52) μg / mL, (88~92) μg / mL, (148~152) μg / mL, and (198~200) μg / mL, respectively. Even further, the antiepileptic drug is valproic acid, and the mass-volume concentration of valproic acid in the standard working solutions of different concentrations of the antiepileptic drug is 20 μg / mL, 30 μg / mL, 50 μg / mL, 90 μg / mL, 150 μg / mL, or 200 μg / mL, respectively.

[0133] For example, the antiepileptic drug is phenytoin, and the mass-volume concentration of phenytoin in the standard working solution is independently 4 μg / mL to 50 μg / mL, and the mass-volume concentration of valproic acid in the standard working solution varies in a gradient. Further, the antiepileptic drug is phenytoin, and the mass-volume concentration of phenytoin in the standard working solutions of different concentrations of the antiepileptic drug is (4~6) μg / mL, (7~9) μg / mL, (14~18) μg / mL, (30~34) μg / mL, (38~42) μg / mL, and (48~52) μg / mL, respectively. Even further, the antiepileptic drug is phenytoin, and the mass-volume concentration of phenytoin in the standard working solutions of different concentrations of the antiepileptic drug is 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 40 μg / mL, and 50 μg / mL, respectively.

[0134] For example, the antiepileptic drug is carbamazepine, and the carbamazepine concentration in the standard working solution is independently 2 μg / mL to 32 μg / mL, with the valproic acid concentration in the standard working solution exhibiting a gradient. Further, for example, the antiepileptic drug is carbamazepine, and the carbamazepine concentrations in the standard working solutions of different concentrations of the antiepileptic drug are (2~3) μg / mL, (4~6) μg / mL, (7~9) μg / mL, (13~17) μg / mL, (22~26) μg / mL, and (30~34) μg / mL, respectively. Even further, for example, the antiepileptic drug is carbamazepine, and the carbamazepine concentrations in the standard working solutions of different concentrations of the antiepileptic drug are 2 μg / mL, 5 μg / mL, 8 μg / mL, 15 μg / mL, 24 μg / mL, and 32 μg / mL, respectively.

[0135] For example, the antiepileptic drug is lamotrigine, and the mass-volume concentration of lamotrigine in the standard working solution is independently 2 μg / mL to 40 μg / mL, and the mass-volume concentration of valproic acid in the standard working solution varies in a gradient. Further, the antiepileptic drug is lamotrigine, and the mass-volume concentration of lamotrigine in the standard working solutions of different concentrations of the antiepileptic drug is (2~2.3) μg / mL, (2.4~3) μg / mL, (8~12) μg / mL, (16~20) μg / mL, (30~34) μg / mL, and (38~42) μg / mL, respectively. Even further, the antiepileptic drug is lamotrigine, and the mass-volume concentration of lamotrigine in the standard working solutions of different concentrations of the antiepileptic drug is 2 μg / mL, 2.5 μg / mL, 10 μg / mL, 18 μg / mL, 32 μg / mL, and 40 μg / mL, respectively.

[0136] In some of these examples, the preparation of the standard working solution includes: (1) diluting the antiepileptic drug with methanol to prepare standard stock solutions with different concentration gradients; and (2) taking bovine serum albumin and adding it to standard stock solutions with different concentration gradients to prepare standard working solutions.

[0137] In some examples, the pretreatment step of the standard working solution includes: processing the standard working solution using the pretreatment method described in the first aspect of this application, the main difference being that the test sample is replaced with the standard working solution. Understandably, the pretreatment steps for the standard working solution and the test sample are the same, and the internal standard solution added is also the same. Therefore, it will not be described in detail here. In some examples, the step of constructing a standard curve for antiepileptic drugs based on peak area and concentration includes: using the concentrations of standard working solutions of different concentrations of antiepileptic drugs as the abscissa, and the ratio of the peak area of ​​the characteristic peak of the antiepileptic drug measured in the standard working solution containing the internal standard to the peak area of ​​the characteristic peak of the internal standard as the ordinate, to construct a standard curve for antiepileptic drugs based on peak area and concentration.

[0138] In the above detection method, an internal standard solution is added to both the test sample and the standard working solution, and a standard curve is constructed with the peak area ratio as the ordinate. At this time, the matrix effect and instrument fluctuation can be corrected by the internal standard method, thereby improving the accuracy and repeatability of quantitative detection of antiepileptic drugs.

[0139] In some examples, the steps of detecting the peak intensity of the test solution using ion mobility spectrometry and detecting the standard working solution using ion mobility spectrometry each independently satisfy the following conditions:

[0140] (1) The ion source voltage is 1800V~2000V.

[0141] (2) The migration tube voltage is 8400V~9000V.

[0142] (3) The air inlet temperature is 170℃~190℃.

[0143] (4) The temperature of the migration tube is 170℃~190℃.

[0144] (5) The ion gate voltage is 45V~55V.

[0145] (6) The gate voltage pulse width is 90μs~110μs.

[0146] (7) The migration airflow velocity is 0.9L / min~1.2L / min.

[0147] (8) The exhaust gas flow rate is 0.9L / min~1.2L / min.

[0148] (9) The injection flow rate is 1.5 μL / min to 3 μL / min.

[0149] (10) The sampling frequency is 180000s -1 ~220000 s -1 .

[0150] The ionization mode of ion mobility spectra is determined by the acidity or basicity of the functional groups in the molecular structure of antiepileptic drugs (such as whether they contain carboxyl or amino groups that are prone to gaining or losing protons) and their ionization stability.

[0151] For example, the antiepileptic drug is valproic acid, and the ionization mode of the ion mobility spectrometry is negative ion mode. Further, the antiepileptic drug is valproic acid, and in the analytical ion mobility spectrometry, the drift time of valproic acid is 10 ms to 11 ms. The drift time of the internal standard is 11 ms to 12 ms. Even further, the antiepileptic drug is valproic acid, and in the analytical ion mobility spectrometry, the drift time of valproic acid is 10.583 ms; the drift time of the internal standard is 11.188 ms.

[0152] For example, the antiepileptic drug is lamotrigine, and the ionization mode of the ion mobility spectrometry is positive ion mode. Further, the antiepileptic drug is lamotrigine, and in the analytical ion mobility spectrometry, the drift time of lamotrigine is 10 ms to 11 ms. The drift time of the internal standard is 11.5 ms to 12.5 ms. Even further, the antiepileptic drug is lamotrigine, and in the analytical ion mobility spectrometry, the drift time of lamotrigine is 10.441 ms. The drift time of the internal standard is 11.751 ms.

[0153] For example, the antiepileptic drug is phenytoin, and the ionization mode of the ion mobility spectrometry is negative ion mode. Further, the antiepileptic drug is phenytoin, and in the analytical ion mobility spectrometry, the drift time of phenytoin is 12.9 ms to 13.5 ms. The drift time of the internal standard is 13.5 ms to 14.5 ms. Even further, the antiepileptic drug is phenytoin, and in the analytical ion mobility spectrometry, the drift time of phenytoin is 13.066 ms. The drift time of the internal standard is 13.726 ms.

[0154] For example, the antiepileptic drug is carbamazepine, and the ionization mode of the ion mobility spectrometry is positive ion mode. Further, the antiepileptic drug is carbamazepine, and in the analytical ion mobility spectrometry, the drift time of carbamazepine is 11 ms–12.5 ms; the drift time of the internal standard is 12.6 ms–13.5 ms. Even further, the antiepileptic drug is carbamazepine, and in the analytical ion mobility spectrometry, the drift time of carbamazepine is 11.615 ms; the drift time of the internal standard is 12.844 ms.

[0155] In some examples, the detection method for antiepileptic drugs also includes a step of testing a quality control solution. Testing the quality control solution allows for monitoring the precision and accuracy of the antiepileptic drug detection process, ensuring the reliability and repeatability of the test results.

[0156] Understandably, the parameters for detecting the quality control solution using ion mobility spectrometry are similar to the process parameters for detecting the test solution using ion mobility spectrometry and for detecting the standard working solution containing the internal standard using ion mobility spectrometry, so they will not be repeated here.

[0157] In some of these examples, the quality control solution includes three concentrations: low-concentration, medium-concentration, and high-concentration.

[0158] For example, the antiepileptic drug is valproic acid, and the concentration of the antiepileptic drug in the low-concentration quality control solution is 55 μg / mL to 65 μg / mL; the concentration in the medium-concentration quality control solution is 95 μg / mL to 105 μg / mL; and the concentration in the high-concentration quality control solution is 155 μg / mL to 165 μg / mL. Further, for example, the antiepileptic drug is valproic acid, and the concentration in the low-concentration quality control solution is 60 μg / mL; the concentration in the medium-concentration quality control solution is 100 μg / mL; and the concentration in the high-concentration quality control solution is 160 μg / mL.

[0159] For example, the antiepileptic drug is lamotrigine, and the concentration of the antiepileptic drug in the low-concentration quality control solution is 2.5 μg / mL to 3.5 μg / mL; the concentration in the medium-concentration quality control solution is 18 μg / mL to 22 μg / mL; and the concentration in the high-concentration quality control solution is 28 μg / mL to 32 μg / mL. Further, for example, the antiepileptic drug is lamotrigine, and the concentration in the low-concentration quality control solution is 3 μg / mL; the concentration in the medium-concentration quality control solution is 20 μg / mL; and the concentration in the high-concentration quality control solution is 30 μg / mL.

[0160] For example, the antiepileptic drug is phenytoin, and the concentration of the antiepileptic drug in the low-concentration quality control solution is 8.5 μg / mL to 9.5 μg / mL; the concentration in the medium-concentration quality control solution is 28 μg / mL to 32 μg / mL; and the concentration in the high-concentration quality control solution is 40 μg / mL to 44 μg / mL. Further, for example, the antiepileptic drug is phenytoin, and the concentration in the low-concentration quality control solution is 9 μg / mL; the concentration in the medium-concentration quality control solution is 30 μg / mL; and the concentration in the high-concentration quality control solution is 42 μg / mL.

[0161] For example, the antiepileptic drug is carbamazepine, and the concentration of the antiepileptic drug in the low-concentration quality control solution is 8 μg / mL to 12 μg / mL; the concentration in the medium-concentration quality control solution is 14 μg / mL to 18 μg / mL; and the concentration in the high-concentration quality control solution is 24 μg / mL to 26 μg / mL. Further, for example, the antiepileptic drug is carbamazepine, and the concentration in the low-concentration quality control solution is 10 μg / mL; the concentration in the medium-concentration quality control solution is 16 μg / mL; and the concentration in the high-concentration quality control solution is 25 μg / mL.

[0162] In some of these examples, the preparation method of the quality control solution includes: (1) diluting the antiepileptic drug with methanol to prepare standard stock solutions of different concentrations; and (2) taking bovine serum albumin, adding standard stock solutions of different concentrations, and preparing quality control solutions.

[0163] In some examples, the pretreatment step of the quality control solution includes treating the quality control sample using the pretreatment method described in the first aspect of this application, the main difference being that the test sample is replaced with the quality control sample. Understandably, the pretreatment steps for the quality control sample are the same as those for the test sample, and the internal standard solution added is also the same. Therefore, it will not be described in detail here.

[0164] A fourth aspect of this application provides a kit for detecting antiepileptic drugs, comprising a pretreatment kit for antiepileptic drug detection, a standard working solution, and an internal standard solution. Alternatively, the kit for detecting antiepileptic drugs comprises a pretreatment kit for antiepileptic drug detection, a standard stock solution, and an internal standard solution. Understandably, the standard stock solution has a high concentration and can be used to prepare a standard working solution or a quality control solution after dilution.

[0165] Understandably, the pretreatment kit for antiepileptic drug detection is the same as that in the second aspect of this application. For example, the pretreatment kit for antiepileptic drug detection includes: magnetic beads, activation solution, eluent, and elution solution.

[0166] Understandably, the internal standard solution included in the antiepileptic drug detection kit is the same as the internal standard solution in the pretreatment method of the first aspect of this application. For example, the internal standard solution includes an internal standard and an internal standard solvent. The internal standard solvent includes an aqueous formic acid solution with a volume concentration of 0.05%–0.5%, an aqueous acetic acid solution with a volume concentration of 0.05%–0.5%, an aqueous phosphoric acid solution with a volume concentration of 0.05%–0.5%, an ammonium acetate solution with a molar concentration of 10 mmol / L–50 mmol / L, an ammonium formate solution with a molar concentration of 10 mmol / L–50 mmol / L, or an ammonia solution with a volume concentration of 0.08%–0.15%. The internal standard in the internal standard solution has a mass-volume concentration of 8 μg / mL–12 μg / mL. The internal standard is the same as in the pretreatment method of the first aspect of this application, so it will not be described again here.

[0167] The above-mentioned antiepileptic drug detection kit is compatible with the steps for detecting antiepileptic drug blood concentration based on ion mobility spectrometry. (1) The pretreatment kit for antiepileptic drug detection is compatible with the sample pretreatment steps for antiepileptic drug detection. It overcomes the limitations of traditional pretreatment methods, such as complex procedures and long processing times, and shortens the pretreatment time of the sample to be tested to 1 / 3 of the conventional method. (2) The detection cycle is controlled within 20 to 30 seconds, which is several to tens of times more efficient than traditional techniques. Combining the advantages of convenient operation, fast detection, and controllable cost of the detection method, the above-mentioned antiepileptic drug detection kit is particularly suitable for the deployment and implementation of automated detection platforms. It can provide accurate data support for the formulation of individualized clinical medication plans, effectively reduce the incidence of adverse reactions such as neurotoxicity caused by drug overdose, and achieve precise control of blood drug concentration within the therapeutic window, thereby improving the efficacy / risk ratio of epilepsy treatment.

[0168] In some examples, when the kit includes a standard working solution, the kit for detecting antiepileptic drugs also includes a quality control solution. Understandably, as previously stated, the quality control solution includes three concentrations: low-concentration, medium-concentration, and high-concentration. The correspondence between the concentrations of the low-concentration, medium-concentration, and high-concentration quality control solutions and the types of antiepileptic drugs is as described in the third aspect of this application and will not be repeated here.

[0169] The present application will be further described in detail below with reference to specific embodiments.

[0170] Example 1

[0171] The antiepileptic drug is valproic acid.

[0172] The sample pretreatment method for detecting antiepileptic drugs includes the following steps: (1) Take PLS magnetic beads (matrix Fe3O4@SiO2), mix PLS magnetic beads with activation solution (50% isopropanol aqueous solution) to prepare magnetic bead suspension (magnetic bead concentration 50mg / mL). (2) Take the magnetic bead suspension (containing 1mg of magnetic beads), 50μL of serum sample to be tested containing valproic acid, and 450μL of internal standard solution (internal standard is 2-propylhexanoic acid, internal standard solvent is 0.1% formic acid aqueous solution, and the mass-volume concentration of internal standard in the internal standard solution is 10μg / mL), and perform magnetic adsorption treatment at 2000rpm~5000rpm for 1min to separate the supernatant and magnetic bead complex. (3) Add 1000μL of rinsing solution (5% methanol aqueous solution) to the magnetic bead complex, and perform rinsing treatment at 2000rpm~5000rpm for 1min, and collect the magnetic bead-target complex. (4) Add 1000 μL of elution buffer (methanol-ammonia mixture, with a methanol volume concentration of 99.9% and an ammonia volume concentration of 0.1%) to the magnetic bead-target complex, and perform elution at 2000 rpm to 5000 rpm for 1 min. Take 30 μL of the supernatant for detection.

[0173] The pretreatment kit for antiepileptic drug detection includes: magnetic beads, activation buffer, eluent, and elution buffer. The magnetic beads are PLS magnetic beads. The activation buffer is a 50% (v / v) isopropanol aqueous solution. The eluent is a 5% (v / v) methanol aqueous solution. The elution buffer is a methanol-ammonia mixture, with a methanol concentration of 99.9% and an ammonia concentration of 0.1%.

[0174] The detection method for antiepileptic drugs includes the following steps: After pretreating the serum sample using the pretreatment method of this embodiment, 30 μL of the supernatant is taken as the test solution. The peak intensity of the test solution is detected using ion mobility spectrometry. The detection result is substituted into a standard curve reflecting the relationship between peak intensity and concentration to obtain the concentration of the antiepileptic drug in the test solution. The test showed that the mass-volume concentration of the antiepileptic drug in the test solution was 19 μg / mL.

[0175] The steps for constructing the above standard curve include:

[0176] Standard stock solutions of valproic acid at different concentrations were prepared (using methanol as the solvent). These stock solutions were diluted 50-fold with serum albumin to obtain standard working solutions. The valproic acid concentrations in the standard working solutions of different concentrations of antiepileptic drugs were 20 μg / mL, 30 μg / mL, 50 μg / mL, 90 μg / mL, 150 μg / mL, or 200 μg / mL, respectively. The standard working solutions were pretreated using a method similar to the sample pretreatment method for antiepileptic drug detection in Example 1; the main difference was that the serum sample to be tested was replaced with the standard working solution. Ion mobility spectrometry was used to detect the pretreated standard working solutions. A standard curve for valproic acid was constructed by plotting the ratio of the peak area of ​​the characteristic peak of valproic acid to the peak area of ​​the internal standard on the ordinate and the mass-volume concentration of the standard working solution containing valproic acid on the abscissa.

[0177] The parameters of the ion mobility spectrometry in the above detection methods are shown in Table 1.

[0178] Table 1 Parameters of ion mobility spectra

[0179]

[0180] The kit for detecting antiepileptic drugs includes: a pretreatment kit, standard working solutions, internal standard solutions, and quality control solutions. The standard working solutions contain valproic acid at concentrations of 20 μg / mL, 30 μg / mL, 50 μg / mL, 90 μg / mL, 150 μg / mL, or 200 μg / mL. The internal standard solutions include an internal standard and an internal standard solvent. The internal standard is 2-propylhexanoic acid, and the internal standard solvent is a 0.1% (v / v) aqueous solution of formic acid. The internal standard concentration in the internal standard solution is 10 μg / mL. The quality control solutions include low-concentration, medium-concentration, and high-concentration solutions. The low-concentration quality control solution contains 60 μg / mL of valproic acid; the medium-concentration solution contains 100 μg / mL; and the high-concentration solution contains 160 μg / mL.

[0181] Methodological Validation

[0182] The above detection method was validated as follows:

[0183] (1) Accuracy: Standard stock solutions were diluted with human serum at different ratios to prepare samples with a valproic acid mass-volume concentration of 20 μg / mL (LLOQ), and three concentrations (L, M, and H, respectively) were used as accuracy samples for the accuracy experiment. Five samples at each concentration level were continuously measured for 3 days, and the accuracy of the test results was calculated. The preparation, pretreatment, and detection steps of the accuracy samples were basically the same as those of the standard working solution. The main difference was the different concentrations of valproic acid and the different matrix. The accuracy calculation formula was: Accuracy = Measured concentration / Theoretical concentration × 100%. The measured concentration is the concentration calculated by the ratio of the standard curve and the measured peak area. The corresponding test results are shown in Table 2.

[0184] Table 2 Accuracy Data

[0185]

[0186] Accuracy verification results: The accuracy test results are shown in Table 2. The above accuracy is between 80% and 115%, which meets the testing requirements.

[0187] (2) Precision: Intra-batch precision: The standard stock solution was diluted with mixed human serum to prepare a sample with a valproic acid mass-volume concentration of 20 μg / mL (LLOQ), and three concentrations (L, M, and H, respectively) were used as precision samples for precision experiments. The preparation, pretreatment, and detection steps of the precision samples were basically the same as those of the standard working solution. The main difference was the different concentrations of valproic acid and the different matrices. Five samples at each concentration level were processed in parallel, that is, five sets of data were obtained for each concentration level, and the intra-batch precision of the detection results was calculated. Inter-batch precision: The standard stock solution was diluted with mixed human serum to prepare a sample with a valproic acid mass-volume concentration of 20 μg / mL (LLOQ), and three concentrations (L, M, and H, respectively) were used as precision samples for inter-batch precision experiments. The samples at each concentration level were measured continuously for 3 days, and the inter-batch precision of the detection results was calculated. The corresponding test results are shown in Table 3.

[0188] Table 3 Precision Data

[0189]

[0190] Precision verification results: The precision experimental verification results are shown in Table 3. The intra-batch precision and inter-batch precision are both less than 15%, which meets the testing requirements.

[0191] (3) Linearity Validation: This linearity validation experiment was conducted on valproic acid antiepileptic drugs. The mass-volume concentrations of valproic acid in the standard working solutions of different concentrations of antiepileptic drugs were 20 μg / mL, 30 μg / mL, 50 μg / mL, 90 μg / mL, 150 μg / mL, or 200 μg / mL, and the standard working solutions contained internal standard solutions. The above standard working solutions were tested in parallel three times, and the average value of the same concentration was calculated daily for three consecutive days. A standard curve was plotted using the ratio of the peak area of ​​the characteristic peak of valproic acid in the standard working solution to the peak area of ​​the internal standard, and the measured value of the standard working solution. R was calculated. 2 ;R 2 A value of 0.98 is met, thus satisfying the requirement. The corresponding test results are shown in Table 4.

[0192] Table 4 Linear Validation Data

[0193]

[0194] (4) Matrix effect verification: The L and H samples for matrix effect were different serum samples to be tested, with L concentration of 60 µg / mL and H concentration of 160 µg / mL; L / H1-L / H6 represent 6 different serum samples to be tested, and L1-1~L1-3 indicate that the sample was measured in parallel three times. By measuring each of the 6 different serum samples to be tested (L concentration 60 µg / mL, H concentration 160 µg / mL) in parallel three times, the coefficient of variation (CV) was ≤5%, indicating that the detection method had good precision.

[0195] Table 5 Low Concentration Matrix Effect

[0196]

[0197] Table 6 High Concentration Matrix Effect

[0198]

[0199] (5) Spike Recovery Validation: LLOQ solution (spiking concentration of 20 μg / mL, matrix: human serum), low, medium, and high-level quality control solutions (spiking concentrations of 60 μg / mL, 100 μg / mL, and 160 μg / mL, matrix: human serum) were added to the test solution (base concentration of 19 μg / mL), and the results were measured by repeating the same steps. Three parallel treatments were performed for each group, and the recovery rate of the spiked samples was calculated. The corresponding test results are shown in Table 7. In Table 7, the theoretical concentration is the sum of the base concentration and the spiked concentration. R-LLOQ represents the mixture of the test solution and LLOQ solution; RL represents the mixture of the test solution and the low-concentration quality control solution; RM represents the mixture of the test solution and the medium-concentration quality control solution; and RH represents the mixture of the test solution and the high-concentration quality control solution.

[0200] Table 7 Spike Recovery Validation Data

[0201]

[0202] As shown in Table 7, the spiked recoveries of valproic acid ranged from 91.6% to 108.3%, meeting the requirement of 100% ± 20%, indicating that the method was accurate.

[0203] (6) Dilution consistency verification: A 400 µg / mL human serum sample was used as the pre-dilution sample. The pre-dilution sample was diluted to the theoretical concentration of 80 µg / mL using 5% bovine serum albumin solution as a dilution solvent. Six parallel determinations were performed, and the detection concentration was calculated based on the peak area ratio to obtain the accuracy and precision. The corresponding test results are shown in Table 8.

[0204] Table 8 Dilution Consistency Data

[0205]

[0206] As shown in Table 8, the above samples, even after dilution, still maintain good linear response and good result accuracy.

[0207] (7) Standard curve and quality control verification:

[0208] Samples of valproic acid with mass-volume concentrations of 20 μg / mL, 30 μg / mL, 50 μg / mL, 90 μg / mL, 150 μg / mL, or 200 μg / mL were taken from the standard working solution, along with low-concentration, medium-concentration, and high-concentration quality control solutions. The ratio of the characteristic peak area to the internal standard peak area obtained from the above sample tests was substituted into the standard curve to obtain the measured concentration. The ratio of the measured concentration to the theoretical concentration was calculated to obtain the accuracy. The corresponding test results are shown in Table 9.

[0209] Table 9 Standard Curve and Quality Control Validation Data

[0210]

[0211] As shown in Table 9, the accuracy of the standard solution and the quality control solution is 89.9%~111.2%, and the linear correlation coefficient r=0.9987, which indicates that there is a strong correlation between the concentration of valproic acid and the ratio of the characteristic peak of the compound to the internal standard peak area.

[0212] As a further example, taking a standard working solution with a mass / volume of 90 μg / mL as an example, the characteristic peaks of valproic acid and its internal standard (2-propylhexanoic acid) in the measured ion mobility spectrum are as follows: Figure 2 As shown. By Figure 2It can be seen that the characteristic peak at a drift time of 10.583 ms is the valproic acid peak, and the characteristic peak at a drift time of 11.188 ms is the characteristic peak of the valproic acid internal standard. The two peaks have good shapes and good separation.

[0213] Example 2

[0214] The antiepileptic drug is lamotrigine.

[0215] The sample pretreatment method for detecting antiepileptic drugs includes the following steps: (1) Take PLS magnetic beads (matrix Fe3O4@SiO2), mix the PLS magnetic beads with activation solution (50% isopropanol aqueous solution) to prepare magnetic bead suspension (magnetic bead concentration 50mg / mL). (2) Take the magnetic bead suspension (containing 1mg of magnetic beads), 200μL of serum sample to be tested containing lamotrigine, and 400μL of internal standard solution (internal standard is benzotriamine). The internal standard solvent was a 0.1% formic acid aqueous solution (with a mass-volume concentration of 10 μg / mL for the internal standard). The supernatant and the magnetic bead complex were separated by magnetic adsorption at 2000 rpm to 5000 rpm for 1 min. (3) 1000 μL of elution buffer (5% methanol aqueous solution) was added to the magnetic bead complex. After elution at 2000 rpm to 5000 rpm for 1 min, the magnetic bead-target complex was collected. (4) 1000 μL of elution buffer (methanol-formic acid mixture, with a methanol volume concentration of 99.8% and a formic acid volume concentration of 0.2%) was added to the magnetic bead-target complex. After elution at 2000 rpm to 5000 rpm for 1 min, 30 μL of the supernatant was taken for detection.

[0216] The pretreatment kit for antiepileptic drug detection includes: magnetic beads, activation buffer, eluent, and elution buffer. The magnetic beads are PLS magnetic beads. The activation buffer is a 50% (v / v) isopropanol aqueous solution. The eluent is a 5% (v / v) methanol aqueous solution. The elution buffer is a methanol-formic acid mixture, with a methanol volume concentration of 99.8% and a formic acid volume concentration of 0.2%.

[0217] The detection method for antiepileptic drugs includes the following steps: After pretreating the serum sample to be tested using the pretreatment method of this embodiment, 30 μL of the supernatant is taken as the test solution. The peak intensity of the test solution is detected by ion mobility spectrometry, and the detection result is substituted into a standard curve reflecting the relationship between peak intensity and concentration to obtain the concentration of antiepileptic drugs in the test solution.

[0218] The steps for constructing the above standard curve include:

[0219] Lamotrigine standard stock solutions of different concentrations (using methanol as solvent) were prepared. These stock solutions were diluted 50-fold with serum albumin to obtain standard working solutions. The mass-volume concentrations of lamotrigine in the standard working solutions of different concentrations of antiepileptic drugs were 2 μg / mL, 2.5 μg / mL, 10 μg / mL, 18 μg / mL, 32 μg / mL, or 40 μg / mL, respectively. The pretreatment of the standard working solutions was performed using a method similar to that used for antiepileptic drug detection in Example 2; the main difference was that the serum sample to be tested was replaced with the standard working solution. Ion mobility spectrometry was used to detect the standard working solutions. A standard curve for lamotrigine was constructed by plotting the ratio of the peak area of ​​the lamotrigine characteristic peak to the peak area of ​​the internal standard on the ordinate and the mass-volume concentration of the standard working solution containing lamotrigine on the abscissa.

[0220] The parameters of the ion mobility spectrometry in the above detection methods are shown in Table 10.

[0221] Table 10 Ion mobility spectrum parameters

[0222]

[0223] The kit for detecting antiepileptic drugs includes: a pretreatment kit, standard working solutions, internal standard solutions, and quality control solutions. The standard working solutions contain lamotrigine at concentrations of 2 μg / mL, 2.5 μg / mL, 10 μg / mL, 18 μg / mL, 32 μg / mL, or 40 μg / mL. The internal standard solutions include an internal standard and an internal standard solvent; the internal standard is benzotricyanate. The internal standard solvent was a 0.1% (v / v) formic acid aqueous solution, with a mass-volume concentration of 10 μg / mL for the internal standard. The quality control solutions included three concentrations: low, medium, and high. The mass-volume concentration of lamotrigine in the low-concentration quality control solution was 3 μg / mL; in the medium-concentration solution, it was 20 μg / mL; and in the high-concentration solution, it was 30 μg / mL.

[0224] Methodological validation is exemplified by using standard curves and quality control validation:

[0225] Samples with lamotrigine concentrations of 2 μg / mL, 2.5 μg / mL, 10 μg / mL, 18 μg / mL, 32 μg / mL, or 40 μg / mL were prepared from the standard working solution, along with low-concentration, medium-concentration, and high-concentration quality control solutions. The preparation, pretreatment, and detection steps for the quality control solutions were the same as those for the standard working solutions; the main difference was the concentration of lamotrigine. The ratio of the characteristic peak area to the internal standard peak area obtained from the above sample tests was substituted into the standard curve to obtain the measured concentration. The ratio of the measured concentration to the theoretical concentration was calculated to obtain the accuracy. The corresponding test results are shown in Table 11.

[0226] Table 11 Standard Curve and Quality Control Validation Data

[0227]

[0228] As shown in Table 11, the accuracy of the standard solution and the quality control solution ranged from 92.6% to 108.34%, and the linear correlation coefficient r = 0.9979. This indicates that there is a strong correlation between the concentration of lamotrigine and the ratio of the characteristic peak area of ​​the compound to the internal standard peak area.

[0229] As a further example, taking a standard working solution with a mass / volume of 40 μg / mL as an example, the characteristic peaks of lamotrigine and the lamotrigine internal standard in its measured ion mobility spectrum are as follows: Figure 3 As shown. By Figure 3 It can be seen that the characteristic peak at a drift time of 10.441 ms is the lamotrigine peak, and the characteristic peak at a drift time of 11.751 ms is the lamotrigine internal standard peak. The two peaks have good shapes and good separation.

[0230] Example 3

[0231] The antiepileptic drug is phenytoin.

[0232] The sample pretreatment method for detecting antiepileptic drugs includes the following steps: (1) Take C18 magnetic beads (matrix Fe3O4@SiO2), mix C18 magnetic beads with activation solution (50% isopropanol aqueous solution) to prepare magnetic bead suspension (magnetic bead concentration 50mg / mL). (2) Take the magnetic bead suspension (containing 2mg of magnetic beads), 100μL of serum sample containing phenytoin to be tested, and 400μL of internal standard solution (internal standard is 5-(4-methylphenyl)5-phenylhydantoin). The internal standard solvent was a 0.1% (v / v) phosphoric acid aqueous solution, and the mass-volume concentration of the internal standard in the internal standard solution was 6.25 μg / mL. Magnetic adsorption was performed at 2000 rpm to 5000 rpm for 1 min to separate the supernatant and the magnetic bead complex. (3) 1000 μL of elution buffer (5% (v / v) methanol aqueous solution) was added to the magnetic bead complex, and elution was performed at 2000 rpm to 5000 rpm for 1 min. The magnetic bead-target complex was then collected. (4) 1000 μL of elution buffer (methanol-ammonia water mixture, methanol volume concentration of 99%, ammonia water volume concentration of 1%) was added to the magnetic bead-target complex, and elution was performed at 2000 rpm to 5000 rpm for 1 min. 30 μL of the supernatant was taken for detection.

[0233] The pretreatment kit for antiepileptic drug detection includes: magnetic beads, activation buffer, eluent, and elution buffer. The magnetic beads are C18 magnetic beads. The activation buffer is a 50% (v / v) isopropanol aqueous solution. The eluent is a 5% (v / v) methanol aqueous solution. The elution buffer is a methanol-ammonia mixture, with a methanol volume concentration of 99% and an ammonia volume concentration of 1%.

[0234] The detection method for antiepileptic drugs includes the following steps: After pretreating the serum sample to be tested using the pretreatment method of this embodiment, 30 μL of the supernatant is taken as the test solution. The peak intensity of the test solution is detected by ion mobility spectrometry, and the detection result is substituted into a standard curve reflecting the relationship between peak intensity and concentration to obtain the concentration of antiepileptic drugs in the test solution.

[0235] The steps for constructing the above standard curve include:

[0236] Standard stock solutions of phenytoin at different concentrations were prepared (using methanol as the solvent). These stock solutions were diluted 50-fold with serum albumin to obtain standard working solutions. The phenytoin concentrations in the standard working solutions of different concentrations of antiepileptic drugs were 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 40 μg / mL, or 50 μg / mL. The standard working solutions were pretreated using a method similar to the sample pretreatment method for antiepileptic drug detection in Example 2; the main difference was that the serum sample to be tested was replaced with the standard working solution. Ion mobility spectrometry was used to detect the pretreated standard working solutions. A standard curve for phenytoin was constructed by plotting the ratio of the peak area of ​​the phenytoin characteristic peak to the peak area of ​​the internal standard on the ordinate and the mass-volume concentration of the standard working solution containing phenytoin on the abscissa.

[0237] The parameters of the ion mobility spectrometry in the above detection methods are shown in Table 12.

[0238] Table 12 Ion mobility spectrum parameters

[0239]

[0240] The kit for detecting antiepileptic drugs includes: a pretreatment kit, standard working solutions, internal standard solutions, and quality control solutions. The standard working solutions contain phenytoin at concentrations of 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 40 μg / mL, or 50 μg / mL. The internal standard solutions include an internal standard and an internal standard solvent. The internal standard is 5-(4-methylphenyl)5-phenylhydantoin, and the internal standard solvent is a 0.1% (v / v) aqueous solution of phosphoric acid. The concentration of the internal standard in the internal standard solution is 6.25 μg / mL. The quality control solutions include low-concentration, medium-concentration, and high-concentration solutions. The low-concentration quality control solution contains 9 μg / mL of phenytoin; the medium-concentration solution contains 30 μg / mL of phenytoin; and the high-concentration solution contains 42 μg / mL of phenytoin.

[0241] Methodological validation is exemplified by using standard curves and quality control validation:

[0242] Samples with phenytoin concentrations of 4 μg / mL, 8 μg / mL, 16 μg / mL, 32 μg / mL, 40 μg / mL, or 50 μg / mL were prepared in the standard working solution, along with low-concentration, medium-concentration, and high-concentration quality control solutions. The preparation, pretreatment, and detection steps for the quality control solutions were the same as those for the standard working solutions; the main difference was the phenytoin concentration. The ratio of the characteristic peak area to the internal standard peak area obtained from the above sample tests was substituted into the standard curve to obtain the measured concentration. The ratio of the measured concentration to the theoretical concentration was calculated to obtain the accuracy. The corresponding test results are shown in Table 13.

[0243] Table 13 Standard Curve and Quality Control Validation Data

[0244]

[0245] As shown in Table 13, the accuracy of the standard solution and the quality control solution is 92.2%~104.6%, and the linear correlation coefficient r=0.9997, which indicates that there is a strong correlation between the phenytoin concentration and the ratio of the characteristic peak of the compound to the internal standard peak area.

[0246] As a further example, taking a standard working solution with a mass-volume concentration of 32 μg / mL as an example, the characteristic peaks of phenytoin and phenytoin internal standard in its measured ion mobility spectrum are as follows: Figure 4 As shown. By Figure 4It can be seen that the characteristic peak at a drift time of 13.066 ms is the phenytoin peak, and the characteristic peak at a drift time of 13.726 ms is the characteristic peak of the phenytoin internal standard. The two peaks have good shapes and good separation.

[0247] Example 4

[0248] The antiepileptic drug is carbamazepine.

[0249] The sample pretreatment method for detecting antiepileptic drugs includes the following steps: (1) Take PLS magnetic beads (matrix Fe3O4@SiO2), mix the PLS magnetic beads with activation solution (50% isopropanol aqueous solution) to prepare magnetic bead suspension (magnetic bead concentration 50mg / mL). (2) Take the magnetic bead suspension (containing 0.5mg of magnetic beads), 100μL of serum sample to be tested containing carbamazepine, and 400μL of internal standard solution (internal standard is oxcarbazepine impurity). The internal standard solvent was a 0.1% formic acid aqueous solution (with a mass-volume concentration of 0.4 μg / mL for the internal standard). The supernatant and the magnetic bead complex were separated by magnetic adsorption at 2000 rpm to 5000 rpm for 1 min. (3) 1000 μL of elution buffer (15% methanol aqueous solution) was added to the magnetic bead complex. After elution at 2000 rpm to 5000 rpm for 1 min, the magnetic bead-target complex was collected. (4) 250 μL of elution buffer (methanol-formic acid mixture, with a methanol volume concentration of 99.9% and a formic acid volume concentration of 0.1%) was added to the magnetic bead-target complex. After elution at 2000 rpm to 5000 rpm for 1 min, 30 μL of the supernatant was taken for detection.

[0250] The pretreatment kit for antiepileptic drug detection includes: magnetic beads, activation buffer, eluent, and elution buffer. The magnetic beads are PLS magnetic beads. The activation buffer is a 50% (v / v) isopropanol aqueous solution. The eluent is a 15% (v / v) methanol aqueous solution. The elution buffer is a methanol-formic acid mixture, with a methanol volume concentration of 99.9% and a formic acid volume concentration of 0.1%.

[0251] The detection method for antiepileptic drugs includes the following steps: After pretreating the serum sample to be tested using the pretreatment method of this embodiment, 30 μL of the supernatant is taken as the test solution. The peak intensity of the test solution is detected by ion mobility spectrometry, and the detection result is substituted into a standard curve reflecting the relationship between peak intensity and concentration to obtain the concentration of antiepileptic drugs in the test solution.

[0252] The steps for constructing the above standard curve include:

[0253] Standard stock solutions of carbamazepine at different concentrations were prepared (using methanol as the solvent). These stock solutions were diluted 10-fold with serum albumin to obtain standard working solutions. The carbamazepine concentrations in the standard working solutions of different concentrations of antiepileptic drugs were 2 μg / mL, 5 μg / mL, 8 μg / mL, 15 μg / mL, 24 μg / mL, or 32 μg / mL, respectively. The standard working solutions were pretreated using a method similar to the sample pretreatment method for antiepileptic drug detection in this embodiment; the main difference was that the serum sample to be tested was replaced with the standard working solution. Ion mobility spectrometry was used to detect the pretreated standard working solutions. A standard curve for carbamazepine was constructed by plotting the ratio of the peak area of ​​the carbamazepine characteristic peak to the peak area of ​​the internal standard on the ordinate and the mass-volume concentration of the standard working solution containing carbamazepine on the abscissa.

[0254] The parameters of the ion mobility spectrometry in the above detection methods are shown in Table 14.

[0255] Table 14 Ion mobility spectrum parameters

[0256]

[0257] The kit for detecting antiepileptic drugs includes: a pretreatment kit, standard working solutions, internal standard solutions, and quality control solutions. The standard working solutions contain carbamazepine at concentrations of 2 μg / mL, 5 μg / mL, 8 μg / mL, 15 μg / mL, 24 μg / mL, or 32 μg / mL. The internal standard solutions include an internal standard and an internal standard solvent; the internal standard is an impurity of oxcarbazepine. The internal standard solvent was a 0.1% (v / v) formic acid aqueous solution, and the mass-volume concentration of the internal standard in the internal standard solution was 0.4 μg / mL. The quality control solutions included three concentrations: low-concentration, medium-concentration, and high-concentration. The mass-volume concentration of carbamazepine in the low-concentration quality control solution was 10 μg / mL; in the medium-concentration solution, it was 16 μg / mL; and in the high-concentration solution, it was 25 μg / mL.

[0258] Methodological validation is exemplified by using standard curves and quality control validation:

[0259] Samples of carbamazepine at concentrations of 2 μg / mL, 5 μg / mL, 8 μg / mL, 15 μg / mL, 24 μg / mL, or 32 μg / mL were prepared in the standard working solution, along with low-concentration, medium-concentration, and high-concentration quality control solutions. The preparation, pretreatment, and detection steps for the quality control solutions were the same as those for the standard working solutions; the main difference was the concentration of lamotrigine. The ratio of the characteristic peak area to the internal standard peak area obtained from the above sample tests was substituted into the standard curve to obtain the measured concentration. The ratio of the measured concentration to the theoretical concentration was calculated to obtain the accuracy. The corresponding test results are shown in Table 15.

[0260] Table 15 Standard Curve and Quality Control Validation Data

[0261]

[0262] As shown in Table 15, the accuracy of the standard solution and the quality control solution ranged from 92.2% to 111.9%, indicating a strong correlation between the carbamazepine concentration and the ratio of the characteristic peak area of ​​the compound to the internal standard peak area.

[0263] As a further example, taking a standard working solution with a mass-volume concentration of 32 μg / mL as an example, the characteristic peaks of carbamazepine and its internal standard in the measured ion mobility spectrum are as follows: Figure 5 As shown. By Figure 5 It can be seen that the characteristic peak at a drift time of 11.615 ms is the carbamazepine peak, and the characteristic peak at a drift time of 12.844 ms is the characteristic peak of the carbamazepine internal standard. The two peaks have good shapes and good separation.

[0264] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0265] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. It should be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for detecting antiepileptic drugs, characterized in that, The antiepileptic drugs include one or more of valproic acid, carbamazepine, phenytoin, and lamotrigine; the detection method includes the following steps: The sample pretreatment method for detecting antiepileptic drugs is used to pretreat the sample, and the collected supernatant is used as the test solution. The peak intensity of the test solution is detected by ion mobility spectrometry, and the detection result is substituted into the standard curve reflecting the relationship between peak intensity and concentration to obtain the concentration of the antiepileptic drug in the test solution. The sample pretreatment method includes the following steps: mixing magnetic beads with an activation solution to prepare a magnetic bead suspension; the mass-volume concentration of the magnetic beads in the magnetic bead suspension is 40 mg / mL to 60 mg / mL; the sample pretreatment method does not include the step of equilibrating the magnetic beads with water; The sample to be tested, the magnetic bead suspension and the internal standard solution are mixed, and after magnetic adsorption treatment, the supernatant and the magnetic bead complex are separated. Eluent was added to the magnetic bead complex, and after rinsing, the magnetic bead-target complex was collected. An eluent was added to the magnetic bead-target complex, and after elution, the supernatant was collected for detection. In the step of detecting the peak intensity of the test solution using ion mobility spectrometry, the ion mobility spectrometry conditions include: ion source voltage of 1800V~2000V, migration tube voltage of 8400V~9000V, inlet temperature of 170℃~190℃, migration tube temperature of 170℃~190℃, ion gate voltage of 45V~55V, gate voltage pulse width of 90μs~110μs, migration gas flow rate of 0.9L / min~1.2L / min, exhaust gas flow rate of 0.9L / min~1.2L / min, injection flow rate of 1.5μL / min~3μL / min, and acquisition frequency of 180000s. -1 ~220000 s -1 ; The correspondence between the ionization mode of the ion mobility spectrum and the types of antiepileptic drugs included in the test sample has one or more of the following characteristics: (1) The antiepileptic drug is valproic acid, and the ionization mode of the ion mobility spectrum is the negative ion mode; (2) The antiepileptic drug is lamotrigine, and the ionization mode of the ion mobility spectrum is positive ion mode; (3) The antiepileptic drug is phenytoin, and the ionization mode of the ion mobility spectrum is negative ion mode; (4) The antiepileptic drug is carbamazepine, and the ionization mode of the ion mobility spectrum is positive ion mode.

2. The method for detecting antiepileptic drugs according to claim 1, characterized in that, The surface of the magnetic beads has modifying groups, which include one or more of aromatic groups, pyrrolidone groups, and octadecyl groups.

3. The method for detecting antiepileptic drugs according to claim 1, characterized in that, The internal standard solution includes an internal standard and an internal standard solvent; the correspondence between the internal standard and the types of antiepileptic drugs included in the test sample has one or more of the following characteristics: (1) The antiepileptic drug is valproic acid, and the internal standard is a homologue of valproic acid; (2) The antiepileptic drug is carbamazepine, and the internal standard is an impurity of oxcarbazepine. ; (3) The antiepileptic drug is phenytoin, and the internal standard is a homologue of phenytoin; (4) The antiepileptic drug is lamotrigine, and the internal standard is benzotriamine. .

4. The method for detecting antiepileptic drugs according to claim 1, characterized in that, The activation solution is an isopropanol aqueous solution with a volume concentration of 40% to 60%; And / or, the rinsing solution includes one or more of water, a methanol aqueous solution with a volume concentration of 5% to 50%, an acetonitrile aqueous solution with a volume concentration of 5% to 50%, and an isopropanol aqueous solution with a volume concentration of 5% to 50%. And / or, the eluent comprises one or more of methanol, acetonitrile, a methanol-formic acid mixture, a methanol-ammonia mixture, an acetonitrile-formic acid mixture, and an acetonitrile-ammonia mixture; wherein, the methanol-formic acid mixture comprises methanol with a volume concentration of 99%~99.95% and formic acid with a volume concentration of 0.05%~1%; the methanol-ammonia mixture comprises methanol with a volume concentration of 99%~99.95% and ammonia with a volume concentration of 0.05%~1%; the acetonitrile-formic acid mixture comprises acetonitrile with a volume concentration of 99%~99.95% and formic acid with a volume concentration of 0.05%~1%; and the acetonitrile-ammonia mixture comprises acetonitrile with a volume concentration of 99%~99.95% and ammonia with a volume concentration of 0.05%~1%.

5. The method for detecting antiepileptic drugs according to any one of claims 1 to 4, characterized in that, The preprocessing method has one or more of the following features: (1) The sample to be tested is a serum sample; (2) The volume-to-mass ratio of the sample to be tested and the magnetic beads included in the magnetic bead suspension is (10~500) μL : (0.25~0.5) mg; (3) The volume ratio of the sample to be tested to the internal standard solution is (10~500) μL: (100~500) μL.

6. The method for detecting antiepileptic drugs according to any one of claims 1 to 4, characterized in that, The process parameters for magnetic attraction include: oscillation at 2000 rpm to 5000 rpm; and / or, The process parameters for the rinsing treatment include: oscillation at 2000 rpm to 5000 rpm; and / or, The elution process parameters include oscillation at 2000 rpm to 5000 rpm.

7. The method for detecting antiepileptic drugs according to any one of claims 1 to 4, characterized in that, The steps for constructing a standard curve reflecting the relationship between peak intensity and concentration include: Standard working solutions of antiepileptic drugs at different concentrations were prepared using standard stock solutions. Ion mobility spectrometry was used to detect the standard working solutions, and a standard curve of the antiepileptic drugs was constructed based on the peak area and concentration.

8. The method for detecting antiepileptic drugs according to claim 7, characterized in that, In the procedure of detecting standard working solutions using ion mobility spectrometry, the ion mobility spectrometry conditions each independently possess one or more of the following characteristics: (1) The ion source voltage is 1800V~2000V; (2) The migration tube voltage is 8400V~9000V; (3) The air inlet temperature is 170℃~190℃; (4) The temperature of the migration tube is 170℃~190℃; (5) The ion gate voltage is 45V~55V; (6) The gate voltage pulse width is 90μs~110μs; (7) The migration airflow velocity is 0.9 L / min to 1.2 L / min; (8) The exhaust gas flow rate is 0.9 L / min to 1.2 L / min; (9) The injection flow rate is 1.5 μL / min to 3 μL / min; (10) The sampling frequency is 180000s -1 ~220000 s -1 .

9. The method for detecting antiepileptic drugs according to claim 1, characterized in that, In standard working solutions of antiepileptic drugs of different concentrations, the mass-volume concentration of the antiepileptic drugs varies in a gradient.

10. A kit for detecting antiepileptic drugs, characterized in that, The kit is used in the detection method of antiepileptic drugs according to any one of claims 1 to 8, and the kit includes a pretreatment kit for antiepileptic drug detection, a standard working solution, and an internal standard solution; Alternatively, it may include pretreatment kits, standard stock solutions, and internal standard solutions for the detection of antiepileptic drugs; The internal standard solution includes an internal standard and an internal standard solvent; The pretreatment kit for detecting antiepileptic drugs has one or more of the following characteristics: (1) The surface of the magnetic beads has a modifying group, which includes one or more of aromatic group, pyrrolidone group and octadecyl group; (2) The activation solution is an isopropanol aqueous solution with a volume concentration of 40%~60%; (3) The rinsing solution includes one or more of the following: water, methanol aqueous solution with a volume concentration of 5% to 50%, acetonitrile aqueous solution with a volume concentration of 5% to 50%, and isopropanol aqueous solution with a volume concentration of 5% to 50%. (4) The eluent includes one or more of methanol, acetonitrile, methanol-formic acid mixture, methanol-ammonia mixture, acetonitrile-formic acid mixture, and acetonitrile-ammonia mixture; wherein the methanol-formic acid mixture includes methanol with a volume concentration of 99%~99.95% and formic acid with a volume concentration of 0.05%~1%; the methanol-ammonia mixture includes methanol with a volume concentration of 99%~99.95% and ammonia with a volume concentration of 0.05%~1%; the acetonitrile-formic acid mixture includes acetonitrile with a volume concentration of 99%~99.95% and formic acid with a volume concentration of 0.05%~1%; and the acetonitrile-ammonia mixture includes acetonitrile with a volume concentration of 99%~99.95% and ammonia with a volume concentration of 0.05%~1%.

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