Method and device for detecting antiepileptic drugs in serum based on liquid chromatography-tandem mass spectrometry

Abstract

The invention discloses a method and a device for detecting antiepileptic drugs in serum based on liquid chromatography-tandem mass spectrometry. The method comprises the following steps: preparing a basic solution; preparing each standard stock solution; respectively diluting all the standard stock solutions into a first mixed solution and a second mixed solution, and respectively diluting again to correspondingly obtain a plurality of calibration product working solutions and quality control product working solutions; preparing a calibration product and a quality control product; preparing an internal standard solution from methanol, eight isotope internal standard standard substances and an internal standard diluent; the method comprises the following steps: respectively pre-treating a serum sample, a calibration product and a quality control product by adopting an internal standard solution to form corresponding to-be-detected solutions; detecting each to-be-detected solution by using liquid chromatography-tandem mass spectrometry, and establishing a standard curve of each analyte according to the detected data to obtain the concentration of the anti-epileptic drug in the serum sample. The method can realize efficient and sensitive detection of 11 antiepileptic drugs, is high in precision and accuracy, ensures a wide linear range and a low sample size, reduces the use variety and use amount of the internal standard, and is low in cost.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of drug detection, and particularly relates to a serum anti-epileptic drug detection method and device based on liquid chromatography tandem mass spectrometry. BACKGROUND

[0002] Common anti-epileptic drugs include levetiracetam (LEV), 10-hydroxycarbamazepine (HCBZ), carbamazepine (CBZ), oxcarbazepine (OXC), phenytoin (PHT), topiramate (TPM), valproic acid (VPA), phenobarbital (PBB), lamotrigine (LMT), clonazepam (CNP), gabapentin (GBP) and the like. By detecting the concentration of anti-epileptic drugs in blood, combined with the principles of pharmacokinetics and pharmacodynamics, it is beneficial to balance the effectiveness and safety of drugs through drug research, and has been practically applied in drug abuse investigation and poisoning case identification. The existing concentration detection of anti-epileptic drugs usually adopts the following scheme: Firstly, the traditional high performance liquid chromatography method is adopted, but the sample pretreatment is complex and the single sample detection time is relatively long. Although the two-dimensional chromatography co-column system can perform multi-component analysis, there are problems of long analysis time, long method switching time and high quantitative limit, which are difficult to meet the needs of pharmacokinetic research. In addition, it is also impossible to detect multiple drugs and metabolites at the same time, which is easy to produce metabolite cross-reaction, high reagent cost and dependent on import.

[0003] Second, liquid chromatography-tandem mass spectrometry (LC-MS / MS) is used. The existing technology discloses a variety of methods for determining the blood drug concentration of antiepileptic drugs by LC-MS / MS. For example, in the paper "Simultaneous determination of 12 antiepileptic drugs in serum by ultra-high performance liquid chromatography-tandem mass spectrometry" by Dai Jing et al. (Reference: Dai Jing, Gao Lehong, Peng Fanda, et al. Simultaneous determination of 12 antiepileptic drugs in serum by ultra-high performance liquid chromatography-tandem mass spectrometry [J]. Chromatography, 2020, 38(08): 900-905.), a method for quantitatively detecting 12 antiepileptic drugs in 9 minutes by LC-MS / MS is proposed. Although 12 drugs are detected, 10-hydroxy carbamazepine (the active metabolite of oxcarbazepine) is not included in the detection project. And this method uses two internal standards, there is a big matrix interference problem, such as lamotrigine, oxcarbazepine and clonazepam, the accuracy of low and medium value is more than 10%; The linear range of the drug is 12.5 μg / mL-2500 μg / mL, which cannot meet the lower limit of quantification, and the sample requirement is large, reaching 200 μL. In the literature Merone G, Tartaglia A, Rossi S, et al. Fast liquid chromatography-tandem mass spectrometry method for the simultaneous determination of phytocannabinoids in oily based preparations. [J]. Journal of pharmaceutical and biomedical analysis, 2021, 203:114174. DOI: 10.1016 / j.jpba.2021.114174., 50 μL of sample is required, and each sample requires 8.5 minutes for detection, the analysis time is long, and the linear range and the concentration of the quality control product do not cover the concentration range and the warning concentration required by the conventional. The concentration of valproic acid also has a significant matrix effect problem, with an accuracy of only 71.3%-72.3%; and 9 isotopic internal standards are used to detect 8 analytes, increasing the cost. The Chinese patent with publication number CN111579680 A discloses a serum antiepileptic drug detection kit and its application, which uses 50 μL of sample to detect 10 antiepileptic drugs in 6 minutes, and uses 10 isotopic internal standards, 7-point calibration, increasing the cost and preparation difficulty, and the concentration of the quality control product does not cover the laboratory critical value; The accuracy of the recovery of valproic acid at medium and high concentrations is only 88.88% and 87.89%. SUMMARY

[0004] The purpose of the present application is to solve the above problems, and provide a serum anti-epileptic drug detection method and device based on liquid chromatography tandem mass spectrometry, which realizes efficient and sensitive detection of 11 kinds of anti-epileptic drugs, has high detection precision and accuracy, good reproducibility and high throughput capacity, reduces sample size on the basis of ensuring wide linear range to realize full coverage, and reduces the types and amount of internal standards used, thereby reducing the cost.

[0005] To achieve the above purpose, the technical solution adopted by the present application is: The serum anti-epileptic drug detection method based on liquid chromatography tandem mass spectrometry provided by the present application comprises the following steps: S1, preparing a basic solution, the basic solution comprising an internal standard diluent and a 50% methanol solution; S2, preparing levetiracetam standard stock solution, oxcarbazepine standard stock solution, carbamazepine standard stock solution, phenytoin standard stock solution, gabapentin standard stock solution, topiramate standard stock solution, 10-hydroxy carbamazepine standard stock solution, lamotrigine standard stock solution, valproic acid standard stock solution, phenobarbital standard stock solution and clonazepam standard stock solution; S3, diluting all the standard stock solutions into first mixed solution and second mixed solution respectively by using 50% methanol solution, and then diluting the first mixed solution and the second mixed solution again by using 50% methanol solution, to obtain a plurality of calibration working solutions and quality control working solutions; S4, preparing calibration and quality control, specifically as follows: S41, adding 3mg / mL-8mg / mL stabilizer and 0.5%-2% lyophilization protective agent to the serum of a large cow, and mixing to form a matrix solution; S42, diluting each calibration working solution and quality control working solution 20 times by using the matrix solution to obtain corresponding calibration and quality control; S5, preparing an internal standard solution by using methanol, eight isotope internal standard standard samples and an internal standard diluent, the eight isotope internal standard standard samples comprising levetiracetam-d6, carbamazepine-d2N15, phenytoin-d10, oxcarbazepine-d4, topiramate-d12, gabapentin-d4, valproic acid-d6 and phenobarbital-d5; S6, pre-treating the serum sample, calibration and quality control by using the internal standard solution to form corresponding to-be-measured solutions; S7, detecting each to-be-measured solution by using liquid chromatography tandem mass spectrometry, and establishing a standard curve of each analyte according to the detection data to obtain the concentration of the anti-epileptic drug in the serum sample.

[0006] Preferably, the internal standard diluent is prepared as follows: 8-10 g of zinc sulfate is weighed, dissolved in 100 mL of ultrapure water, then 900 mL of methanol is added, and 0.15-0.30 g of ammonium acetate is added, and mixed to obtain; the 50% methanol solution is prepared by mixing 20 mL of methanol and 20 mL of ultrapure water.

[0007] Preferably, the concentrations of the levetiracetam standard stock solution, oxcarbazepine standard stock solution, carbamazepine standard stock solution, phenytoin standard stock solution, gabapentin standard stock solution, topiramate standard stock solution, 10-hydroxy carbamazepine standard stock solution, lamotrigine standard stock solution, valproic acid standard stock solution, phenobarbital standard stock solution, and clonazepam standard stock solution are 25 mg / mL, 5 mg / mL, 15 mg / mL, 20 mg / mL, 20 mg / mL, 20 mg / mL, 20 mg / mL, 10 mg / mL, 60 mg / mL, 20 mg / mL, and 3 mg / mL, respectively, and the solvent is methanol.

[0008] Preferably, the concentrations of levetiracetam, oxcarbazepine, carbamazepine, phenytoin, gabapentin, topiramate, 10-hydroxy carbamazepine, lamotrigine, valproic acid, phenobarbital, and clonazepam in the first mixed solution are 1440.00 μg / mL, 480.00 μg / mL, 600.00 μg / mL, 800.00 μg / mL, 640.00 μg / mL, 640.00 μg / mL, 1100.00 μg / mL, 600.00 μg / mL, 3600.00 μg / mL, 1360.00 μg / mL, and 2.56 μg / mL, respectively, and the concentrations of levetiracetam, oxcarbazepine, carbamazepine, phenytoin, gabapentin, topiramate, 10-hydroxy carbamazepine, lamotrigine, valproic acid, phenobarbital, and clonazepam in the second mixed solution are 1120.00 μg / mL, 360.00 μg / mL, 480.00 μg / mL, 600.00 μg / mL, 520.00 μg / mL, 480.00 μg / mL, 840.00 μg / mL, 480.00 μg / mL, 2720.00 μg / mL, 1000.00 μg / mL, and 1.80 μg / mL, respectively.

[0009] Preferably, the calibration working solution includes six kinds, i.e., the first mixed solution is diluted by 1.00 times, 1.44 times, 2.25 times, 4.80 times, 12.00 times, and 32.00 times with the 50% methanol solution; the quality control working solution includes three kinds, i.e., the second mixed solution is diluted by 1.00 times, 2.00 times, and 8.00 times with the 50% methanol solution.

[0010] Preferably, the internal standard solution is prepared by using methanol, eight isotopic internal standard substances, and the internal standard diluent, and the preparation is as follows: S51, dissolve eight isotopic internal standard samples respectively by using methanol, and prepare levetiracetam-d6 internal standard stock solution, carbamazepine-d2N15 internal standard stock solution, phenytoin-d10 internal standard stock solution, oxcarbazepine-d4 internal standard stock solution, topiramate-d12 internal standard stock solution, gabapentin-d4 internal standard stock solution, valproic acid-d6 internal standard stock solution, and phenobarbital-d5 internal standard stock solution with concentrations of 2 mg / mL, 1 mg / mL, 2 mg / mL, 1 mg / mL, 2 mg / mL, 1 mg / mL, 10 mg / mL and 2 mg / mL respectively; S52, dilute levetiracetam-d6 internal standard stock solution, carbamazepine-d2N15 internal standard stock solution, phenytoin-d10 internal standard stock solution, oxcarbazepine-d4 internal standard stock solution, topiramate-d12 internal standard stock solution, gabapentin-d4 internal standard stock solution, valproic acid-d6 internal standard stock solution and phenobarbital-d5 internal standard stock solution respectively by using methanol, and the dilution multiples are 166.67, 250, 250, 250, 500, 125, 166.67 and 50 respectively, to obtain an internal standard mixed solution, and the concentrations of levetiracetam-d6, carbamazepine-d2N15, phenytoin-d10, oxcarbazepine-d4, topiramate-d12, gabapentin-d4, valproic acid-d6 and phenobarbital-d5 in the internal standard mixed solution are 12 μg / mL, 4 μg / mL, 8 μg / mL, 4 μg / mL, 4 μg / mL, 8 μg / mL, 60 μg / mL and 40 μg / mL respectively; S53, dilute the internal standard mixed solution with the internal standard dilution solution by 20 times to prepare an internal standard solution.

[0011] Preferably, the pretreatment is specifically as follows: S61, take 20 μL of the sample to be tested, add it to a 1.5 mL centrifuge tube, and then add 180 μL of the internal standard solution, mix at 1000 rpm~1500 rpm for 5 min, and the sample to be tested is a serum sample or a calibrator or a quality control sample; S62, centrifuge at 5000 rpm for 10 min~15 min to separate the supernatant; S63, take 30 μL~50 μL of the supernatant, dilute with 120 μL~150 μL of ultrapure water, place it in a container, and vortex at 200 rpm~500 rpm for 5 min to form a corresponding test solution.

[0012] Preferably, the base solution further comprises mobile phase A and mobile phase B, and the detection conditions of the liquid chromatography tandem mass spectrometry are as follows: 1) liquid chromatography: The mobile phase A is 0.01‰-0.05‰ ammonia water and 1mmol / L-5mmol / L aqueous solution of ammonium acetate; the mobile phase B is 0.01‰-0.05‰ ammonia water and 1mmol / L-5mmol / L methanol solution of ammonium acetate; the chromatographic column is a C18 chromatographic column with a particle size of 2.6 microns, and the column temperature is 35-55 DEG C; the flow rate is 0.5mL / min, gradient elution is adopted for liquid phase, the elution gradient is 0min0.5min, 10% mobile phase B; 0.5min2.0min, the mobile phase B is increased to 60%; 2.0min2.5min, the mobile phase B is increased to 65%; 2.5min-3.0min, the mobile phase B is maintained at 80%; 3.0min3.8min, the mobile phase B is increased and maintained at 95%, 3.9min-4.5min, the mobile phase B is decreased and maintained at 10%, and the sample collection time of the test solution is 4.5min; the flow rate is 0.3mL / min-0.6mL / min; and the injection amount is 1-10 microliter. 2) Mass spectrometry: The ionization is realized in the detection mode of positive and negative ion switching, wherein the left ethyl acetate, carbamazepine, oxcarbazepine, 10-hydroxy carbamazepine, lamotrigine, phenytoin, gabapentin, topiramate and clonazepam adopt the positive ion mode, and valproic acid and phenobarbital adopt the negative ion mode; the mass spectrometry scanning mode of multiple reaction monitoring is adopted, the ion source voltage is 5500V in the positive ion mode, the ion source temperature is 550 DEG C, the ion source voltage is 4500V in the negative ion mode, the ion source temperature is 450 DEG C, the curtain gas is 25psi, the collision gas is 6psi, the atomization gas is 55psi, and the desolvation gas is 55psi.

[0013] Preferably, the stabilizer is ascorbic acid, and the lyophilization protective agent is a preservative.

[0014] A serum anti-epileptic drug detection device based on liquid chromatography tandem mass spectrometry is used to realize the serum anti-epileptic drug detection method based on liquid chromatography tandem mass spectrometry.

[0015] Compared with the prior art, the beneficial effects of the present application are: The method realizes efficient and sensitive detection of multiple antiepileptic drugs by optimizing sample pretreatment and liquid chromatography tandem mass spectrometry analysis steps, has high detection precision and accuracy, good reproducibility and high throughput capacity, reduces sample size on the basis of ensuring wide linear range to realize full coverage, and reduces the types and amount of internal standards used, thereby reducing the cost. Specifically, 11 kinds of antiepileptic drugs, including levetiracetam, oxcarbazepine, carbamazepine, phenytoin, gabapentin, topiramate, 10-hydroxy carbamazepine, lamotrigine, valproic acid, phenobarbital and clonazepam, including active metabolites of drugs (such as oxcarbazepine), and the method completes sample detection in 4.5 minutes with a sample size of 20 μL, and the internal standard solution is prepared by mixing methanol, internal standard diluent, calibration working solution and quality control working solution, and the sample is pretreated by precipitating protein, so that the sample pretreatment is simply and efficiently completed without any additional sample enrichment operation, the operation is simple, and only 8 kinds of isotopic internal standards are used for detecting 11 kinds of analytes, which saves cost and has no matrix effect, and the accuracy deviation of each analyte is within 10%; the elution gradient, mass spectrometry and liquid chromatography conditions are optimized to improve the detection sensitivity, and the concentrations of the quality control samples are set as low, medium and high values to meet the wide range requirement. Compared with the prior art, the single detection time of the present application is controlled within 4.5 minutes, and only 20 μL of sample size is required to reach the detection lower limit, the relative bias of the quantitative lower limit is within ±8.38%, the relative standard deviation RSD is within 0.76%~13.23%, the within-batch precision is between 1.64%~7.27%, the between-batch precision is between 2.42%~7.51%, and the reproducibility and stability are good. The method is simple and fast, the accuracy of the sample standard addition is between 91.70% and 108.24%, the interference of endogenous impurities can be limited, the quantitative is accurate, a plurality of antiepileptic drugs can be screened at the same time, the linear range and quality control are set to cover the concentration range and warning concentration required in the conventional, and the method is suitable for monitoring of antiepileptic drug blood concentration. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 A flow chart of the serum antiepileptic drug detection method based on liquid chromatography tandem mass spectrometry in embodiment 1 of the present application; Figure 2 A flow chart of step S6 in embodiment 1 of the present application; Figure 3 A total ion chromatogram of 11 kinds of antiepileptic drugs in embodiment 1 of the present application; Figure 4 A standard curve diagram of 11 kinds of antiepileptic drugs in embodiment 1 of the present application. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of the present application.

[0018] It should be noted that, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terms used in the specification of the present application are only for the purpose of describing the specific embodiments and are not intended to limit the present application.

[0019] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) is the "gold standard" for measuring the blood concentration of antiepileptic drugs. It has high selectivity, specificity and anti-interference ability, and provides higher sensitivity and detection efficiency. Compared with traditional immunoassay and luminescence method, the liquid chromatography-tandem mass spectrometry has low sample pretreatment requirements, significantly shortens the analysis time, and thus improves the detection speed and accuracy. The high sensitivity and specificity of liquid chromatography-tandem mass spectrometry for monitoring the concentration of antiepileptic drugs have been practically applied in drug abuse investigation and poisoning case identification, and at the same time, provide method and technical support for pharmacokinetic research and drug interaction evaluation in scientific research and drug development.

[0020] Embodiment 1: As shown in the following table, a serum antiepileptic drug detection method based on liquid chromatography-tandem mass spectrometry includes the following steps: Figures 1-4 S1, preparing a basic solution, the basic solution including an internal standard diluent and a 50% methanol solution.

[0021] In an embodiment, the internal standard diluent is prepared as follows: 8g-10g of zinc sulfate is weighed, dissolved in 100mL of ultrapure water, then 900mL of methanol is added, and 0.15g-0.30g of ammonium acetate is added, and mixed to obtain the internal standard diluent; the 50% methanol solution is prepared by mixing 20mL of methanol and 20mL of ultrapure water.

[0022] S2, preparing a levetiracetam standard stock solution, an oxcarbazepine standard stock solution, a carbamazepine standard stock solution, a phenytoin standard stock solution, a gabapentin standard stock solution, a topiramate standard stock solution, a 10-hydroxy carbamazepine standard stock solution, a lamotrigine standard stock solution, a valproic acid standard stock solution, a phenobarbital standard stock solution, and a clonazepam standard stock solution.

[0023] ​In an embodiment, the concentrations of the levetiracetam standard stock solution, the oxcarbazepine standard stock solution, the carbamazepine standard stock solution, the phenytoin standard stock solution, the gabapentin standard stock solution, the topiramate standard stock solution, the 10-hydroxycarbamazepine standard stock solution, the lamotrigine standard stock solution, the valproic acid standard stock solution, the phenobarbital standard stock solution and the clonazepam standard stock solution are 25 mg / mL, 5 mg / mL, 15 mg / mL, 20 mg / mL, 20 mg / mL, 20 mg / mL, 20 mg / mL, 10 mg / mL, 60 mg / mL, 20 mg / mL and 3 mg / mL, respectively, and the solvent is methanol.

[0024] S3, dilute all the standard stock solutions into a first mixed solution and a second mixed solution with 50% methanol solution, and dilute the first mixed solution and the second mixed solution again with 50% methanol solution to obtain a plurality of calibration working solutions and quality control working solutions.

[0025] In an embodiment, the concentrations of levetiracetam, oxcarbazepine, carbamazepine, phenytoin, gabapentin, topiramate, 10-hydroxycarbamazepine, lamotrigine, valproic acid, phenobarbital and clonazepam in the first mixed solution are 1440.00 μg / mL, 480.00 μg / mL, 600.00 μg / mL, 800.00 μg / mL, 640.00 μg / mL, 640.00 μg / mL, 1100.00 μg / mL, 600.00 μg / mL, 3600.00 μg / mL, 1360.00 μg / mL and 2.56 μg / mL, respectively, and the concentrations of levetiracetam, oxcarbazepine, carbamazepine, phenytoin, gabapentin, topiramate, 10-hydroxycarbamazepine, lamotrigine, valproic acid, phenobarbital and clonazepam in the second mixed solution are 1120.00 μg / mL, 360.00 μg / mL, 480.00 μg / mL, 600.00 μg / mL, 520.00 μg / mL, 480.00 μg / mL, 840.00 μg / mL, 480.00 μg / mL, 2720.00 μg / mL, 1000.00 μg / mL and 1.80 μg / mL, respectively.

[0026] In an embodiment, the calibration working solutions include six kinds, i.e. the first mixed solution is diluted by 1.00 times, 1.44 times, 2.25 times, 4.80 times, 12.00 times and 32.00 times with 50% methanol solution, respectively; and the quality control working solutions include three kinds, i.e. the second mixed solution is diluted by 1.00 times, 2.00 times and 8.00 times with 50% methanol solution, respectively.

[0027] S4, prepare the calibration and quality control, specifically as follows: S41, 3mg / mL~8mg / mL stabilizer and 0.5%~2% lyophilization protective agent are added in the Danqi serum, and mixed to form a matrix solution; S42, each calibration working solution and quality control working solution is diluted 20 times by using the matrix solution to obtain corresponding calibration and quality control.

[0028] In an embodiment, the stabilizer is ascorbic acid, and the lyophilization protective agent is a preservative.

[0029] S5, an internal standard solution is prepared by using methanol, eight isotopic internal standard standards, and internal standard diluent solution. The eight isotopic internal standard standards include levetiracetam-d6, carbamazepine-d2N15, phenytoin-d10, oxcarbazepine-d4, topiramate-d12, gabapentin-d4, valproic acid-d6, and phenobarbital-d5.

[0030] In an embodiment, the internal standard solution is prepared by using methanol, eight isotopic internal standard standards, and internal standard diluent solution, and the preparation is as follows: S51, the eight isotopic internal standard standards are dissolved in methanol respectively to prepare levetiracetam-d6 internal standard stock solution, carbamazepine-d2N15 internal standard stock solution, phenytoin-d10 internal standard stock solution, oxcarbazepine-d4 internal standard stock solution, topiramate-d12 internal standard stock solution, gabapentin-d4 internal standard stock solution, valproic acid-d6 internal standard stock solution, and phenobarbital-d5 internal standard stock solution, and the concentrations are 2mg / mL, 1mg / mL, 2mg / mL, 1mg / mL, 2mg / mL, 1mg / mL, 10mg / mL, and 2mg / mL respectively. S52, the levetiracetam-d6 internal standard stock solution, carbamazepine-d2N15 internal standard stock solution, phenytoin-d10 internal standard stock solution, oxcarbazepine-d4 internal standard stock solution, topiramate-d12 internal standard stock solution, gabapentin-d4 internal standard stock solution, valproic acid-d6 internal standard stock solution, and phenobarbital-d5 internal standard stock solution are diluted in methanol respectively, the dilution multiples are 166.67, 250, 250, 250, 500, 125, 166.67, and 50 respectively, an internal standard mixture is obtained, and the concentrations of levetiracetam-d6, carbamazepine-d2N15, phenytoin-d10, oxcarbazepine-d4, topiramate-d12, gabapentin-d4, valproic acid-d6, and phenobarbital-d5 in the internal standard mixture are 12 μg / mL, 4 μg / mL, 8 μg / mL, 4 μg / mL, 4 μg / mL, 8 μg / mL, 60 μg / mL, and 40 μg / mL respectively. S53, the internal standard mixture is diluted 20 times by using the internal standard diluent solution to prepare the internal standard solution.

[0031] S6, the serum sample, the calibrator, and the quality control are pretreated by using the internal standard solution to form the corresponding test solution.

[0032] In an embodiment, the pretreatment is as follows: S61, 20 μL of the test sample is taken and added to a 1.5 mL centrifuge tube, 180 μL of the internal standard solution is added, and the mixture is mixed at 1000 rpm-1500 rpm for 5 min, the test sample being a serum sample or a calibrator or a quality control; S62, centrifugation is performed at 5000 rpm for 10 min-15 min to separate the supernatant; S63, 30 μL-50 μL of the supernatant is taken, diluted with 120 μL-150 μL of ultrapure water, placed in a container, and vortexed at 200 rpm-500 rpm for 5 min to form the corresponding test solution.

[0033] S7, each test solution is detected by using liquid chromatography tandem mass spectrometry, and a standard curve of each analyte is established according to the detection data to obtain the concentration of the antiepileptic drug in the serum sample.

[0034] In an embodiment, the base solution further includes mobile phase A and mobile phase B, and the detection conditions of the liquid chromatography tandem mass spectrometry are as follows: 1) liquid chromatography: mobile phase A: 0.01‰-0.05‰ ammonia water and 1 mmol / L-5 mmol / L ammonium acetate aqueous solution; mobile phase B: 0.01‰-0.05‰ ammonia water and 1 mmol / L-5 mmol / L ammonium acetate methanol solution; the chromatographic column is a C18 chromatographic column with a particle size of 2.6 μm, and the column temperature is 35℃-55℃; the flow rate is 0.5 mL / min, the liquid phase is eluted by gradient elution, the elution gradient is 0 min-0.5 min, 10% mobile phase B; 0.5 min-2.0 min, the mobile phase B is increased to 60%; 2.0 min-2.5 min, the mobile phase B is increased to 65%; 2.5 min-3.0 min, the mobile phase B is maintained at 80%; 3.0 min-3.8 min, the mobile phase B is increased and maintained at 95%, 3.9 min-4.5 min, the mobile phase B is decreased and maintained at 10%, and the test solution collection time is 4.5 min; the flow rate is 0.3 mL / min-0.6 mL / min; the injection amount is 1 μL-10 μL; 2) mass spectrometry: The ionization was achieved by using the detection mode of electrospray positive-negative ion switching, wherein levetiracetam, carbamazepine, oxcarbazepine, 10-hydroxycarbamazepine, lamotrigine, phenytoin, gabapentin, topiramate and clonazepam were used in the positive ion mode, and valproic acid and phenobarbital were used in the negative ion mode; the mass spectrometry scanning mode of multiple reaction monitoring was used, the ion source voltage was 5500V and the ion source temperature was 550℃ in the positive ion mode, the ion source voltage was 4500V and the ion source temperature was 450℃ in the negative ion mode, the curtain gas was 25psi, the collision gas was 6psi, the atomization gas was 55psi, and the desolvation gas was 55psi.

[0035] The sample to be detected was detected by using liquid chromatography-tandem mass spectrometry to obtain the detection data. The standard curve equation y=ax+b was established by using the concentration (target value, x) of each analyte of the calibration sample and the ratio (y) of the corresponding peak area, y was the ratio of the peak area of the corresponding analyte to the peak area of the internal standard, x was the concentration of the corresponding analyte, a was the slope, and b was the constant, such as noise. Then, the ratio of the peak area of each analyte of the sample to be detected to the peak area of the internal standard was substituted into the corresponding standard curve equation, so that each analyte of the antiepileptic drug in the sample to be detected was quantified.

[0036] The method simultaneously detects 11 commonly used antiepileptic drugs, including active metabolites of the drugs, and the sample detection is completed in 4.5 minutes using only 20 μL of sample amount, and no additional special operation is required for the pretreatment. Only 8 isotopic internal standards are used for detecting 11 analytes, which saves cost and has no matrix effect, and the accuracy deviation of each analyte is within 10%. The linear range of 11 analytes is wide, completely covering the therapeutic window of the drugs, and the concentrations of the quality control samples are set as low, medium and high values, which meet the lower limit of clinical quantification, the medium value of clinical therapeutic window and the laboratory critical value, thereby improving the sensitivity, conventional monitoring accuracy and accuracy. Through optimization of the sample pretreatment and analysis steps, efficient and sensitive detection of various antiepileptic drugs is realized, and good reproducibility and high throughput capacity are achieved.

[0037] For the convenience of understanding, the following will be described in detail through specific embodiments.

[0038] I. Preparation of a basic solution, including: (1) Mobile phase: mobile phase A: 0.05‰ ammonia water + 2 mmol / L ammonium acetate aqueous solution; mobile phase B: 0.05‰ ammonia water + 2 mmol / L ammonium acetate methanol solution; (2) Internal standard diluent: 8 g of zinc sulfate was weighed and dissolved in 100 mL of ultrapure water, then 900 mL of methanol was added, followed by the addition of 0.2 g of ammonium acetate, and the mixture was uniformly mixed to obtain the internal standard diluent; (3) 50% methanol solution: 20 mL of methanol and 20 mL of ultrapure water were uniformly mixed to obtain the 50% methanol solution.

[0039] II. Preparation of standard stock solutions of 11 antiepileptic drugs

[0040] The standards of the 11 analytes were prepared by dissolving in methanol to correspond to the standard stock solutions, wherein the concentrations of the corresponding components in the levetiracetam standard stock solution, oxcarbazepine standard stock solution, carbamazepine standard stock solution, phenytoin standard stock solution, gabapentin standard stock solution, topiramate standard stock solution, 10-hydroxy carbamazepine standard stock solution, lamotrigine standard stock solution, valproic acid standard stock solution, phenobarbital standard stock solution and clonazepam standard stock solution were levetiracetam 25 mg / mL, oxcarbazepine 5 mg / mL, carbamazepine 15 mg / mL, phenytoin 20 mg / mL, gabapentin 20 mg / mL, topiramate 20 mg / mL, 10-hydroxy carbamazepine 20 mg / mL, lamotrigine 10 mg / mL, valproic acid 60 mg / mL, phenobarbital 20 mg / mL, and clonazepam 3 mg / mL, respectively. The concentrations of the standard stock solutions are shown in Table 1.

[0041] Table 1 III. Preparation of calibration and quality control solutions of 11 antiepileptic drugs

[0042] a: According to the concentrations of all standard stock solutions, high-concentration mixed solution 1 (first mixed solution, referred to as mixed standard 1) and mixed solution 2 (second mixed solution, referred to as mixed standard 2) of 11 analytes were prepared, wherein the concentrations of the 11 analytes in the mixed solution 1 and the mixed solution 2 were levetiracetam 1440 μg / mL, 1120 μg / mL, oxcarbazepine 480.00 μg / mL, 360.00 μg / mL, carbamazepine 600.00 μg / mL, 480.00 μg / mL, phenytoin 800.00 μg / mL, 600.00 μg / mL, gabapentin 640.00 μg / mL, 520.00 μg / mL, topiramate 640.00 μg / mL, 480.00 μg / mL, 10-hydroxy carbamazepine 1100.00 μg / mL, 840.00 μg / mL, lamotrigine 600.00 μg / mL, 480.00 μg / mL, valproic acid 3600.00 μg / mL, 2720.00 μg / mL, phenobarbital 1360.00 μg / mL, 1000.00 μg / mL, clonazepam 2.56 μg / mL, 1.80 μg / mL. Specifically, according to the concentrations of all standard stock solutions, high-concentration mixed solution 1 and mixed solution 2 of 11 analytes were prepared, the concentrations of mixed solution 1 and the dilution ratios and concentrations of each analyte are shown in Table 2, and the concentrations of mixed solution 2 and the dilution ratios and concentrations of each analyte are shown in Table 3.

[0043] Table 2 Table 3 b: The mixed solution 1 and the mixed solution 2 are diluted respectively according to the dilution requirements using 50% methanol solution, to obtain the calibration working solution and the quality control working solution. The dilution process is shown in Table 4. Six calibration working solutions are used in this embodiment to ensure a wide linear range, and three quality control working solutions are used to ensure accuracy, covering the required range.

[0044] Table 4 c. The calibration working solution and the quality control working solution are diluted 20 times respectively using the matrix solution formed by adding 5 mg / mL ascorbic acid (stabilizer) and 1% preservative (lyophilization protector such as Proclin 300) to the large bovine serum, to obtain the calibration and quality control of the antiepileptic drugs, and the concentrations are shown in Table 5 (unit: μg / mL). The low quality control, the medium quality control and the high quality control correspond to the dilution liquids of the quality control working solution 1, the quality control working solution 2 and the quality control working solution 3 in Table 4 in turn.

[0045] Table 5 The calibration and quality control prepared by diluting the standard stock solution in a certain proportion can cover the clinical drug concentration range.

[0046] Four, preparation of the internal standard solution of the antiepileptic drugs.

[0047] (1) Internal standard mixture The eight isotopic internal standard samples are dissolved with methanol to prepare an internal standard mixture, and the concentrations in the internal standard mixture are respectively 12 μg / mL of levetiracetam-d6, 4 μg / mL of carbamazepine-d2N15, 8 μg / mL of phenytoin-d10, 4 μg / mL of oxcarbazepine-d4, 4 μg / mL of topiramate-d12, 8 μg / mL of gabapentin-d4, 60 μg / mL of valproic acid-d6 and 40 μg / mL of phenobarbital-d5.

[0048] (2) Internal standard diluent 8 g of zinc sulfate is weighed and dissolved with 100 mL of ultrapure water, 900 mL of methanol is added, and 0.154 g of ammonium acetate is added, and then mixed to obtain an internal standard diluent.

[0049] (3) Internal standard solution The internal standard mixture is diluted 20 times with the internal standard diluent to obtain the internal standard solution. Specifically: a: Internal standard stock solution preparation: 8 isotopic internal standards were dissolved in methanol to prepare internal standard stock solutions. The concentrations of levetiracetam-d6, carbamazepine-d2N15, phenytoin-d10, oxcarbazepine-d4, topiramate-d12, gabapentin-d4, valproic acid-d6, and phenobarbital-d5 internal standard stock solutions were 2 mg / mL, 1 mg / mL, 2 mg / mL, 1 mg / mL, 2 mg / mL, 1 mg / mL, 10 mg / mL, and 2 mg / mL, respectively.

[0050] b: Internal standard mixture solution preparation: The levetiracetam-d6, carbamazepine-d2N15, phenytoin-d10, oxcarbazepine-d4, topiramate-d12, gabapentin-d4, valproic acid-d6, and phenobarbital-d5 internal standard stock solutions were diluted 166.67, 250, 250, 250, 500, 125, 166.67, and 50 times, respectively, with methanol to obtain internal standard mixture solutions with concentrations of 12 μg / mL, 4 μg / mL, 8 μg / mL, 4 μg / mL, 4 μg / mL, 8 μg / mL, 60 μg / mL, and 40 μg / mL, respectively.

[0051] c: Internal standard solution preparation: The internal standard mixture solution was diluted 20 times with internal standard diluent to prepare an internal standard solution for antiepileptic drugs.

[0052] Five, the serum samples, calibrators, and quality controls were pretreated to form corresponding test solutions. Specifically: a: 20 μL of the test sample (serum sample, calibrator, or quality control) was added to a 1.5 mL centrifuge tube, which can be replaced by a 96-well plate (96-well V-shaped sample plate), and the like. Then, 180 μL of the internal standard solution was added, and the mixture was vortexed at 1400 rpm for 5 minutes.

[0053] b: The mixture was centrifuged at 5000 rpm for 10 minutes to separate the supernatant.

[0054] c: 50 μL of the supernatant was diluted with 120 μL of ultrapure water, and the mixture was vortexed at 300 rpm~500 rpm for 5 minutes to form a corresponding test solution, which was subjected to machine detection.

[0055] Six, sample detection.

[0056] The test solution is sent to the sample injector of the high performance liquid chromatograph mass spectrometer for detection. The specific detection conditions are as follows: gradient elution is used for liquid phase, mobile phase A: 0.01‰-0.05‰ ammonia water+1mmol / L-5mmol / L ammonium acetate aqueous solution; mobile phase B: 0.01‰-0.05‰ ammonia water+1mmol / L-5mmol / L ammonium acetate methanol solution; the chromatographic column is a C18 chromatographic column with a particle size of 2.6μm, the column temperature is 35℃-55℃; the flow rate is 0.5mL / min, the elution gradient is 0min0.5min, 10% B liquid (mobile phase B); in 0.5min2.0min, B liquid rises to 60%; 2.0min2.5min, B liquid rises to 65%; 2.5min-3.0min, B liquid maintains 80%; 3.0min3.8min, B liquid rises and maintains at 95%, 3.9min-4.5min, B liquid reduces and maintains at 10%. The test solution collection time is 4.5min; the flow rate is 0.3mL / min-0.6mL / min; the injection volume is 1μL ~10μL.

[0057] The mass spectrometer realizes ionization in the detection mode of positive and negative ion switching, wherein levetiracetam, carbamazepine, oxcarbazepine, 10-hydroxycarbamazepine, lamotrigine, phenytoin, gabapentin, topiramate and clonazepam adopt positive ion mode, valproic acid and phenobarbital adopt negative ion mode. The mass spectrometry scanning mode adopts multiple reaction monitoring, the ion source voltage is 5500V (ESI+) / 4500V (ESI-), the ion source temperature is 550℃ (ESI+) / 450℃ (ESI-), the curtain gas (CUR) is 25psi, the collision gas (CAD) is 6psi, the atomization gas Gas1 is 55psi, the desolvation gas Gas2 is 55psi, ESI+ represents positive ion mode, and ESI- represents negative ion mode. The parent ion / daughter ion pair mass-to-charge ratio and mass spectrometry parameters of each target are shown in Table 6.

[0058] Table 6 The data obtained by the above method are detected. The standard curve equation y=ax+b is established according to the concentration (target value, x) of each analyte of the calibration sample and the ratio (y) of the corresponding peak area, y is the ratio of the peak area of the corresponding analyte to the peak area of the internal standard, x is the concentration of the corresponding analyte, a is the slope, b is the constant, such as noise, and the ratio of the peak area of each analyte of the test sample to the peak area of the internal standard is substituted into the corresponding standard curve equation, so that each analyte of the anti-epileptic drug in the test sample can be quantified. Figure 3 That is, the total ion chromatogram of 11 anti-epileptic drugs, wherein the abscissa represents the detection time (min), and the ordinate represents the detection signal (cps).

[0059] Seven, methodological validation.

[0060] a. Standard curve and quality control verification: Take the prepared calibration and quality control and detect according to the sample pretreatment and liquid chromatography tandem mass spectrometry detection method operation, the results of the standard curve of 11 analytes are shown in Table 7.

[0061] Table 7 The results show that the accuracy of the 11 kinds of calibration and quality control of antiepileptic drugs using the detection method is between 90.32% and 112.50%, and the linear correlation coefficient is greater than 0.9990, showing good linearity.

[0062] b. Linear range verification: The high value sample close to the upper limit of the linear interval and the low value sample close to the lower limit of the linear interval are diluted into at least 5 series gradient concentrations according to the proportion of 0:1, 3:1, 1:1, 1:3 and 1:0, each concentration gradient is tested at least 3 times, and the mean value (yi) of each gradient determination result is calculated. The dilution concentration (xi) is used as the independent variable, and the determination result mean value (yi) is used as the dependent variable to obtain the multiple regression equation (such as using weighted least squares fitting), such as Figure 4 Figures (a) to (k). Calculate the correlation coefficient (R). Substitute the dilution concentration (xi) into the regression equation to calculate the absolute deviation of yi and the estimated value, and the linear range verification results of 11 kinds of analytes are shown in Table 8.

[0063] Table 8 The results show that the correlation coefficient of 11 kinds of analytes of antiepileptic drugs in the linear range of the detection method is greater than 0.9990, and the relative deviation is within ±10.82%, the linearity is good and the linear range is wide, which can well cover the required range.

[0064] c. Lower limit of quantification verification Take 5 low value samples close to the lower limit, test each concentration 5 times, and calculate the mean value, relative standard deviation (RSD%) and relative bias (R%) of each concentration determination result, respectively. The lower limit of quantification verification results of 11 kinds of analytes are shown in Table 9.

[0065] Table 9 The results show that the relative bias of the 11 analytes of the antiepileptic drugs in the range of low value close to the lower limit is within ±8.38%, and the relative standard deviation is 0.76%-13.23%, and the detection of the method is reliable and accurate at low concentration. The quantitative lower limit of the 11 analytes is respectively 1.61 μg / mL for levetiracetam, 0.43 μg / mL for gabapentin, 1.09 μg / mL for valproic acid, 1.43 μg / mL for phenobarbital, 0.34 μg / mL for oxcarbazepine, 0.53 μg / mL for phenytoin, 0.32 μg / mL for carbamazepine, 0.33 μg / mL for topiramate, 0.30 μg / mL for 10-hydroxy carbamazepine, 0.28 μg / mL for lamotrigine, and 1.52 ng / mL for clonazepam.

[0066] d. Accuracy verification The serum sample (human serum sample) is added with a certain amount of high-concentration standard solution (quality control working solution, referred to as standard solution) to prepare low-concentration, medium-concentration and high-concentration samples for detection, and each concentration is detected for 3 times, and the relative standard deviation (RSD%) and accuracy are calculated, and the accuracy verification results of the 11 analytes are shown in Table 10.

[0067] Table 10 The results show that the accuracy of the 11 analytes of the antiepileptic drugs using the detection method is between 91.70-108.24%, and the relative standard deviation is between 0.51%-9.86%, and the accuracy is good.

[0068] e. Precision verification The low-concentration, medium-concentration and high-concentration quality control samples are taken, each concentration is tested for 10 times, and the within-batch precision (relative standard deviation RSD%) of each concentration is calculated, and the precision verification results of the 11 analytes are shown in Table 11. The low-concentration, medium-concentration and high-concentration quality control samples (in turn corresponding to low quality control, medium quality control and high quality control) are detected for 3 consecutive days, each concentration is tested for 10 times, and the inter-day precision (relative standard deviation RSD%) of each concentration is calculated.

[0069] Table 11 The results show that the intra-batch precision of the 11 analytes of the anti-epileptic drugs is between 1.64% and 7.27%, and the inter-batch precision is between 2.42% and 7.51%, which is good.

[0070] Embodiment 2: A serum anti-epileptic drug detection device based on liquid chromatography tandem mass spectrometry is used to realize the serum anti-epileptic drug detection method based on liquid chromatography tandem mass spectrometry as in Embodiment 1, which will not be repeated here.

[0071] The device realizes efficient and sensitive detection of multiple anti-epileptic drugs by optimizing the sample pretreatment and liquid chromatography tandem mass spectrometry analysis steps, has high detection precision and accuracy, good reproducibility and high throughput capacity, reduces the sample size on the basis of ensuring wide linear range, reduces the types and amount of internal standards used, and further reduces the cost. Specifically, it can simultaneously detect 11 anti-epileptic drugs, including levetiracetam, oxcarbazepine, carbamazepine, phenytoin, gabapentin, topiramate, 10-hydroxy carbamazepine, lamotrigine, valproic acid, phenobarbital and clonazepam, including active metabolites of drugs (such as oxcarbazepine), and the method completes sample detection in 4.5 minutes with only 20 μL of sample size, mixes methanol, internal standard diluent, calibration working solution and quality control working solution to prepare an internal standard solution, and performs sample pretreatment by precipitating proteins, thereby simply and efficiently completing sample pretreatment without any additional sample enrichment operations, and the operation is simple. Only 8 isotopic internal standards are used to detect 11 analytes, saving cost and avoiding matrix effect, and the accuracy deviation of each analyte is within 10%; by elution gradient, optimized mass spectrometry and liquid chromatography conditions, the detection sensitivity is improved, the concentrations of the quality control samples are set as low, medium and high values, which meet the wide range requirements. Compared with the existing technology, the detection time of multiple analytes is more than 6 min, the single detection time of the present application is controlled within 4.5 minutes, and only 20 μL of sample size is required to reach the detection lower limit, the relative bias of the quantitative lower limit is within ±8.38%, the relative standard deviation RSD is within 0.76%~13.23%, the intra-batch precision is between 1.64% and 7.27%, and the inter-batch precision is between 2.42% and 7.51%, which has good reproducibility and stability. The method is simple and fast, the accuracy of the detected sample is between 91.70% and 108.24%, which can limit the interference of endogenous impurities, accurately quantify, facilitate simultaneous screening of multiple anti-epileptic drugs, suitable for monitoring of anti-epileptic drug blood concentration, and the linear range and quality control setting cover the required concentration range and warning concentration.

[0072] Any technical features in the above-described embodiments can be combined in any manner, and for the sake of brevity, not all possible combinations are described, however, any combination of the technical features is considered to be within the scope of the present disclosure.

[0073] The above-described embodiments are merely illustrative and exemplary, and should not be considered to limit the scope of the application. It should be noted that, for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the scope of the present application. Therefore, the scope of the present application should be subject to the appended claims.

Claims

1. A method for detecting antiepileptic drugs in serum based on liquid chromatography-tandem mass spectrometry, characterized by: The steps include: S1. Prepare a basic solution, which includes an internal standard diluent and a 50% methanol solution; S2. Prepare levetiracetam standard stock solution, oxcarbazepine standard stock solution, carbamazepine standard stock solution, phenytoin standard stock solution, gabapentin standard stock solution, topiramate standard stock solution, 10-hydroxycarbamazepine standard stock solution, lamotrigine standard stock solution, valproic acid standard stock solution, phenobarbital standard stock solution, and clonazepam standard stock solution; S3. Using 50% methanol solution, dilute all the standard stock solutions into a first mixed solution and a second mixed solution, and again use 50% methanol solution to dilute the first mixed solution and the second mixed solution, respectively, to obtain a plurality of calibrator working solutions and quality control working solutions; S4. Prepare calibrators and quality control materials as follows: S41. Add 3 mg / mL to 8 mg / mL of stabilizer and 0.5% to 2% of lyoprotectant to bovine serum and mix well to form a matrix solution. S42. Dilute each calibrator working solution and quality control working solution 20-fold using the matrix solution to obtain the corresponding calibrator and quality control product; S5. Prepare internal standard solution using methanol, eight isotopic internal standard standards and internal standard diluent. The eight isotopic internal standard standards include levetiracetam-d6, carbamazepine-d2N15, phenytoin-d10, oxcarbazepine-d4, topiramate-d12, gabapentin-d4, valproic acid-d6, and phenobarbital-d5. S6. Pre-treat the serum sample, calibrator, and quality control product using an internal standard solution to form corresponding test solutions; S7. Detect each test solution using liquid chromatography tandem mass spectrometry, and establish a standard curve for each analyte based on the detection data to obtain the concentration of the anti-epileptic drug in the serum sample.

2. The method for detecting antiepileptic drugs in serum based on liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The internal standard dilution solution was prepared as follows: 8 g to 10 g of zinc sulfate was weighed, dissolved in 100 mL of ultrapure water, 900 mL of methanol was added, and 0.15 g to 0.30 g of ammonium acetate was added and mixed. The 50% methanol solution was prepared by mixing 20 mL of methanol and 20 mL of ultrapure water.

3. The method for detecting antiepileptic drugs in serum based on liquid chromatography-tandem mass spectrometry according to claim 1, wherein: The concentrations of the levetiracetam standard stock solution, oxcarbazepine standard stock solution, carbamazepine standard stock solution, phenytoin standard stock solution, gabapentin standard stock solution, topiramate standard stock solution, 10-hydroxycarbamazepine standard stock solution, lamotrigine standard stock solution, valproic acid standard stock solution, phenobarbital standard stock solution and clonazepam standard stock solution are 25 mg / mL, 5 mg / mL, 15 mg / mL, 20 mg / mL, 20 mg / mL, 20 mg / mL, 20 mg / mL, 10 mg / mL, 60 mg / mL, 20 mg / mL and 3 mg / mL, respectively, and the solvent is methanol.

4. The method for detecting antiepileptic drugs in serum based on liquid chromatography-tandem mass spectrometry according to claim 1, wherein: The concentrations of levetiracetam, oxcarbazepine, carbamazepine, phenytoin, gabapentin, topiramate, 10-hydroxycarbamazepine, lamotrigine, valproic acid, phenobarbital, and clonazepam in the first mixed solution are 1440.00 μg / mL, 480.00 μg / mL, 600.00 μg / mL, 800.00 μg / mL, 640.00 μg / mL, 640.00 μg / mL, 1100.00 μg / mL, 600.00 μg / mL, 3600.00 μg / mL, 1360.00 μg / mL, and 2.56 μg / mL, respectively. The concentrations of levetiracetam, oxcarbazepine, carbamazepine, phenytoin, gabapentin, topiramate, 10-hydroxycarbamazepine, lamotrigine, valproic acid, phenobarbital, and clonazepam in the second mixed solution are 1120.00 μg / mL, 360.00 μg / mL, 480.00 μg / mL, 600.00 μg / mL, 520.00 μg / mL, 480.00 μg / mL, 840.00 μg / mL, 480.00 μg / mL, 2720.00 μg / mL, 1000.00 μg / mL, and 1.80 μg / mL, respectively.

5. The method for detecting antiepileptic drugs in serum based on liquid chromatography-tandem mass spectrometry according to claim 4, characterized in that: The calibration working solutions include six types, namely, the first mixed solution is diluted by 50% methanol solution by 1.00 times, 1.44 times, 2.25 times, 4.80 times, 12.00 times, and 32.00 times respectively; the quality control working solutions include three types, namely, the second mixed solution is diluted by 50% methanol solution by 1.00 times, 2.00 times, and 8.00 times respectively.

6. The method for detecting antiepileptic drugs in serum based on liquid chromatography-tandem mass spectrometry according to claim 1, wherein: The internal standard solution is prepared by using methanol, eight isotope internal standard standards and internal standard diluent, as follows: S51. Dissolve eight isotopic internal standard standards separately in methanol to prepare levetiracetam-d6 internal standard stock solution, carbamazepine-d2N15 internal standard stock solution, phenytoin-d10 internal standard stock solution, oxcarbazepine-d4 internal standard stock solution, topiramate-d12 internal standard stock solution, gabapentin-d4 internal standard stock solution, valproic acid-d6 internal standard stock solution, and phenobarbital-d5 internal standard stock solution, with the concentrations of 2 mg / mL, 1 mg / mL, 2 mg / mL, 1 mg / mL, 2 mg / mL, 1 mg / mL, 10 mg / mL, and 2 mg / mL, respectively; S52. Dilute the levetiracetam-d6 internal standard stock solution, carbamazepine-d2N15 internal standard stock solution, phenytoin-d10 internal standard stock solution, oxcarbazepine-d4 internal standard stock solution, topiramate-d12 internal standard stock solution, gabapentin-d4 internal standard stock solution, valproic acid-d6 internal standard stock solution, and phenobarbital-d5 internal standard stock solution with methanol, and the dilution multiples are 166.67, 250, 250, 250, 500, 125, 166.67, and 50, respectively, to obtain an internal standard mixture, wherein the concentrations of levetiracetam-d6, carbamazepine-d2N15, phenytoin-d10, oxcarbazepine-d4, topiramate-d12, gabapentin-d4, valproic acid-d6, and phenobarbital-d5 are 12 μg / mL, 4 μg / mL, and 1 μg / mL, respectively. μg / mL, 8 μg / mL, 4 μg / mL, 4 μg / mL, 8 μg / mL, 60 μg / mL, 40 μg / mL; S53. Dilute the internal standard mixed solution 20 times with the internal standard diluent to prepare an internal standard solution.

7. The method for detecting antiepileptic drugs in serum based on liquid chromatography-tandem mass spectrometry according to claim 1, wherein: The pre-processing is as follows: S61. Take 20 μL of the sample to be tested, add it to a 1.5 mL centrifuge tube, add 180 μL of the internal standard solution, and mix at 1000 rpm to 1500 rpm for 5 min. The sample to be tested is a serum sample, a calibrator, or a quality control product. S62, centrifuge at 5000 rpm for 10 min to 15 min, and separate the supernatant; S63. Take 30 μL to 50 μL of the supernatant, dilute it with 120 μL to 150 μL of ultrapure water, place it in a container, and vortex it at 200 rpm to 500 rpm for 5 minutes to form the corresponding test solution.

8. The method for detecting antiepileptic drugs in serum based on liquid chromatography-tandem mass spectrometry according to claim 1, wherein: The basic solution also includes mobile phase A and mobile phase B. The detection conditions of the liquid chromatography tandem mass spectrometry are as follows: 1) Liquid chromatography: Mobile phase A: 0.01‰-0.05‰ ammonia water and 1mmol / L-5mmol / L ammonium acetate aqueous solution; mobile phase B: 0.01‰-0.05‰ ammonia water and 1mmol / L-5mmol / L ammonium acetate methanol solution; chromatographic column: C18 column, particle size 2.6μm, column temperature 35℃-55℃; flow rate 0.5mL / min, liquid phase gradient elution, elution gradient: 0min0.5min, 10% mobile phase B; 0.5min2.0min, mobile phase B increased to 60%; From 2.0 to 2.5 minutes, mobile phase B was increased to 65%; from 2.5 to 3.0 minutes, mobile phase B was maintained at 80%; from 3.0 to 3.8 minutes, mobile phase B was increased and maintained at 95%; from 3.9 to 4.5 minutes, mobile phase B was decreased and maintained at 10%. The sample collection time was 4.5 minutes; the flow rate was 0.3 mL / min to 0.6 mL / min; the injection volume was 1 μL to 10 μL. 2) Mass spectrometry: Ionization was achieved in an electrospray ionization detection mode with switching between positive and negative ions. Positive ion mode was used for levetiracetam, carbamazepine, oxcarbazepine, 10-hydroxycarbamazepine, lamotrigine, phenytoin, gabapentin, topiramate, and clonazepam, and negative ion mode was used for valproic acid and phenobarbital. Multiple reaction monitoring mass spectrometry scanning mode was used. In the positive ion mode, the ion source voltage was 5500 V and the ion source temperature was 550°C. In the negative ion mode, the ion source voltage was 4500 V and the ion source temperature was 450°C. The curtain gas pressure was 25 psi, the collision gas was 6 psi, the nebulizer gas was 55 psi, and the desolvation gas pressure was 55 psi.

9. The method for detecting antiepileptic drugs in serum based on liquid chromatography-tandem mass spectrometry according to claim 1, wherein: The stabilizer is ascorbic acid, and the freeze-drying protectant is a preservative.

10. A device for detecting anti-epileptic drugs in serum based on liquid chromatography-tandem mass spectrometry, characterized by: Used to implement the method for detecting anti-epileptic drugs in serum based on liquid chromatography tandem mass spectrometry as described in any one of claims 1 to 9.

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