Method for simultaneously determining concentrations of various antiepileptic drugs in plasma

Abstract

The invention relates to the technical field of treatment drug monitoring, in particular to a method for simultaneously determining the concentration of multiple antiepileptic drugs in plasma. Comprising the following steps: S1, preparing a standard working solution; s2, preparing an internal standard solution; s3, mixing the standard working solution with blank plasma, adding an internal standard solution, centrifuging, and detecting to obtain a chromatogram of a standard solution; s4, based on the chromatogram, taking the ratio of the peak area of the anti-epileptic drug to the peak area of the corresponding internal standard substance as a vertical coordinate Y, taking the ratio of the concentration of the anti-epileptic drug to the concentration of the corresponding internal standard substance as a horizontal coordinate X, and fitting to obtain a standard curve equation; s5, preparing a plurality of groups of quality control working solutions, and verifying the standard curve equation obtained in the step S4; and S6, uniformly mixing the blood to be detected with the internal standard solution, centrifuging, detecting the supernate, and calculating the concentration of the antiepileptic drug in the plasma. The method can realize simultaneous, accurate and quantitative detection of multiple antiepileptic drugs in blood.

Description

Technical Field

[0001] This invention relates to the field of therapeutic drug monitoring technology, and more specifically to a method for simultaneously measuring the concentrations of multiple antiepileptic drugs in plasma. Background Technology

[0002] Epilepsy is a common chronic central nervous system disorder, and long-term drug therapy is its core intervention. Perampanel and lacosamide, as novel antiepileptic drugs, are widely used in the treatment of epilepsy in adults and some children due to their unique mechanisms of action and good clinical efficacy. However, the pharmacokinetics of these two drugs vary significantly among individuals, with large fluctuations in blood drug concentrations, which are closely related to clinical efficacy and adverse reactions. Conventional administration often fails to balance efficacy and safety; therefore, reliable therapeutic drug monitoring (TDM) methods are urgently needed in clinical practice to guide individualized medication.

[0003] Currently, clinical methods for detecting the plasma concentrations of perampanel and lacosamide primarily employ immunoassay, high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). However, these methods are mostly limited to single-drug assays and have limitations in sensitivity, throughput, and sample processing efficiency. A simple, rapid, and sensitive method capable of simultaneously detecting the plasma concentrations of perampanel and lacosamide is lacking, making it difficult to meet the needs of individualized epilepsy treatment and efficacy evaluation in clinical practice. Specifically, immunoassay suffers from cross-reactivity and poor specificity. HPLC methods have limited sensitivity and complex sample pretreatment. Existing LC-MS methods mostly focus on single-drug detection and cannot accurately quantify multiple antiepileptic drugs simultaneously, limiting their widespread application in clinical laboratories. Therefore, there is an urgent need for a simple, rapid, and sensitive method capable of simultaneously detecting the plasma concentrations of perampanel and lacosamide to meet the clinical needs for individualized epilepsy treatment and efficacy evaluation. Summary of the Invention

[0004] The purpose of this invention is to provide a method for simultaneously determining the concentration of multiple antiepileptic drugs in plasma, in order to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for simultaneously determining the concentration of multiple antiepileptic drugs in plasma, comprising the following steps: S1. Weigh out various antiepileptic drugs, dissolve them in acetonitrile aqueous solution, and prepare multiple sets of standard working solutions containing different concentrations of each of the aforementioned antiepileptic drugs. S2. Weigh the internal standard corresponding to each of the aforementioned antiepileptic drugs, dissolve it in acetonitrile, and prepare an internal standard solution. The concentration of each of the aforementioned internal standards in the internal standard solution is the same, and is 1-10 ng·mL. -1 Any value in; S3. Take multiple sets of the standard working solutions respectively, mix each set of the standard working solutions with blank plasma to form multiple sets of standard solutions, add the internal standard solution to each standard solution and mix well, centrifuge and take the supernatant for LC-MS / MS detection to obtain the chromatogram of each standard solution; S4. Based on the chromatogram of each standard solution, the ratio of the peak area of ​​each antiepileptic drug to the peak area of ​​its corresponding internal standard is used as the ordinate Y, and the ratio of the concentration of the antiepileptic drug to the concentration of its corresponding internal standard is used as the abscissa X. Linear regression fitting is performed on the data in each group of standard solutions to obtain the standard curve equation for the concentration of each antiepileptic drug. S5. Prepare multiple sets of quality control working solutions according to the method in step S1, and prepare multiple sets of quality control solutions according to the method in step S3. Perform LC-MS / MS detection on each of the quality control solutions to verify each of the standard curve equations obtained in step S4. S6. Centrifuge the blood to be tested to obtain supernatant plasma. Add the internal standard solution to the supernatant plasma and mix well. After centrifugation, take the supernatant for LC-MS / MS detection to obtain the chromatogram of the blood to be tested. Calculate the concentration of each antiepileptic drug in the plasma based on the ratio of the peak area of ​​each antiepileptic drug to the peak area of ​​the corresponding internal standard in the chromatogram of the blood to be tested and the validated standard curve equation of the corresponding antiepileptic drug.

[0006] Furthermore, in step S1, the plurality of antiepileptic drugs include at least perampanel and lacosamide; The standard curve equation for the perampanel obtained in step S4 is: Y1=0.218778X1-0.0891044, and the standard curve equation for the lacosamide is: Y2=62.0117X2-0.135688.

[0007] Further, in step S1, the concentration of perampanel in each of the standard working solutions is 62.5 ng·mL. -1 ~10000ng·mL -1 The concentration of lacosamide is 0.6875 μg·mL for any of the values. -1 ~110μg·mL -1 Any value among them.

[0008] Further, in step S3, the volume ratio of the standard working solution to the blank plasma in the standard solution is any value in the range of 1:(8-10); the volume of the internal standard solution added to the standard solution is 5-8 times the volume of the standard solution.

[0009] Furthermore, in step S5, the concentration of each antiepileptic drug in the multiple sets of quality control working solutions is gradually increased.

[0010] Furthermore, the multiple sets of quality control working solutions include low-concentration quality control working solution, medium-concentration quality control working solution, and high-concentration quality control working solution; In the low-concentration quality control working solution, the concentration of piraminpanel-d5 is 50 ng·mL. -1 ~300ng·mL -1 For any of the values, the lacosamide-d6 has a concentration of 0.5 μg·mL. -1 ~3μg·mL -1 Any value in; In the medium-concentration quality control working solution, the concentration of piraminpanel-d5 is 1000 ng·mL. -1 ~3000ng·mL -1 For any of the values, the lacosamide-d6 has a concentration of 5 μg·mL⁻¹. -1 ~μg·mL -1 Any value in; In the high-concentration quality control working solution, the concentration of piraminpanel-d5 is 5000 ng·mL. -1 ~10000ng·mL -1 For any of the values, the lacosamide-d6 has a concentration of 50 μg·mL. -1 ~110μg·mL -1 Any value in; Furthermore, in step S6, the volume of the internal standard solution added to the supernatant plasma is 5-8 times the volume of the supernatant plasma.

[0011] Furthermore, in the LC-MS / MS detection process, the analytical column used is ACQUITY UPLCHSS T3 C. 18 For chromatographic columns, the injection volume is limited to the range of 2μL-10μL.

[0012] Furthermore, in the LC-MS / MS detection process, the analytical column employs gradient elution. The mobile phase used for elution includes mobile phase A and mobile phase B. Mobile phase A is a 3mM–8mM aqueous solution of ammonium acetate containing 0.05%–0.2% formic acid, and mobile phase B is acetonitrile. The flow rate of the mobile phase is 0.1 mL / min. -1 ~0.5 mL·min -1 Any value among them.

[0013] Furthermore, during the LC-MS / MS detection process, a multiple reaction monitoring (MRM) mode is used for mass spectrometry ion scanning monitoring.

[0014] The beneficial effects of this invention are as follows: The method for simultaneously determining the concentration of multiple antiepileptic drugs in plasma provided in this application is based on liquid chromatography-triple quadrupole tandem mass spectrometry (LC-MS / MS) technology. Combined with the corresponding internal standards for each antiepileptic drug, a standard curve equation is constructed for each drug to calculate its concentration in the blood sample. This allows for the simultaneous and accurate quantification of multiple antiepileptic drugs such as perampanel and lacosamide in the blood sample. This method exhibits good linearity and a low limit of quantitation, enabling accurate detection of low concentrations of antiepileptic drugs in plasma. The detection results are stable and reliable, meeting the standardized requirements for quantitative analysis of antiepileptic drugs in biological samples. This overcomes the limitation of existing detection methods to single-drug analysis, improving the comprehensiveness and throughput of clinical testing. It is particularly suitable for clinical applications involving combination therapy or requiring simultaneous assessment of the blood concentrations of multiple antiepileptic drugs.

[0015] The method provided in this application for simultaneously determining the concentration of multiple antiepileptic drugs in plasma requires only a small amount of plasma sample to complete the detection. The pretreatment of the blood sample to be tested adopts the method of adding internal standard to bind protein precipitation, without the need for complex extraction or multi-step purification operations. The overall operation process is simple and has good repeatability, which can significantly shorten the detection time and reduce human operation error. It is suitable for routine, high-throughput detection in hospital clinical laboratories.

[0016] By introducing an internal standard corresponding to the target antiepileptic drug, the entire detection process can be effectively corrected, which can significantly reduce the impact of matrix effects and sample pretreatment errors on the detection results, improve the accuracy, precision and stability of the method, and enhance the reliability of quantitative analysis of antiepileptic drugs in complex biological matrices.

[0017] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0018] Figure 1 This is a chromatogram of lacosamide in a plasma sample to be tested, as shown in an embodiment of the present invention; Figure 2 This is a chromatogram of perampanel in a plasma sample to be tested, as shown in an embodiment of the present invention. Detailed Implementation

[0019] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0020] A preferred embodiment of this application illustrates a method for simultaneously determining the concentrations of multiple antiepileptic drugs in plasma, comprising the following steps: S1. Weigh out various antiepileptic drugs, dissolve them in acetonitrile aqueous solution, and prepare multiple sets of standard working solutions containing different concentrations of each antiepileptic drug. S2. Weigh the internal standard corresponding to each antiepileptic drug, dissolve it in acetonitrile, and prepare an internal standard solution. The concentration of each internal standard in the internal standard solution is the same, and is 1-10 ng / mL. -1 Any value in; S3. Transfer multiple sets of standard working solutions separately, mix each set of standard working solutions with blank plasma to form multiple sets of standard solutions, add internal standard solution to each standard solution and mix well, centrifuge and take the supernatant for LC-MS / MS detection to obtain the chromatogram of each standard solution. S4. Based on the chromatogram of each standard solution, the ratio of the peak area of ​​each antiepileptic drug to the peak area of ​​its corresponding internal standard is used as the ordinate Y, and the ratio of the concentration of the antiepileptic drug to the concentration of its corresponding internal standard is used as the abscissa X. Linear regression fitting is performed on the data in each group of standard solutions to obtain the standard curve equation for the concentration of each antiepileptic drug. S5. Prepare multiple sets of quality control working solutions according to the method in step S1, and prepare multiple sets of quality control solutions according to the method in step S3. Perform LC-MS / MS detection on each quality control solution to verify each standard curve equation obtained in step S4. S6. Centrifuge the blood to be tested to obtain supernatant plasma. Add internal standard solution to the supernatant plasma and mix well. After centrifugation, take the supernatant for LC-MS / MS detection to obtain the chromatogram of the blood to be tested. Calculate the concentration of each antiepileptic drug in plasma based on the ratio of the peak area of ​​each antiepileptic drug to the peak area of ​​the corresponding internal standard in the chromatogram of the blood to be tested and the validated standard curve equation of the corresponding antiepileptic drug.

[0021] In step S1, multiple accurately weighed antiepileptic drugs can be dissolved in acetonitrile. Using an acetonitrile aqueous solution, multiple sets of standard working solutions containing different concentrations of each antiepileptic drug can be quickly prepared through gradient dilution and other operations to meet the detection needs of different concentration ranges and lay the foundation for the accuracy of subsequent standard curves. In some embodiments, the multiple antiepileptic drugs may also include lamotrigine, levetiracetam, and other drugs.

[0022] In step S2, by using internal standards corresponding to each antiepileptic drug to prepare the internal standard solution, and then combining it with subsequent LC-MS / MS technology, systematic errors generated during sample pretreatment and mass spectrometry analysis can be effectively corrected, enabling simultaneous, rapid, and accurate determination of multiple antiepileptic drugs in plasma. Furthermore, by clearly defining the concentrations of various internal standards in the internal standard solution, the accuracy and repeatability of the detection results can be further improved, enhancing the stability and reliability of the method. This internal standard solution also serves as a protein precipitant for plasma sample pretreatment. By adding the internal standard solution to the plasma to be tested, the internal standards promote protein precipitation in the plasma, eliminating the need for complex extraction or multi-step purification operations on the plasma sample, thereby significantly shortening the detection time and reducing human error, making this method more suitable for routine and high-throughput detection in hospital clinical laboratories. In some embodiments, the internal standard corresponding to perampanel is preferably perampanel-d5, and the internal standard corresponding to lacosamide is preferably lacosamide-d6.

[0023] In step S3, a standard solution is formed by mixing a specific volume of standard working solution with blank plasma, effectively simulating the matrix environment of a real plasma sample. Adding an appropriate amount of internal standard solution to the standard solution maintains a specific and stable ratio between the internal standard and the antiepileptic drug to be tested, thereby correcting deviations in sample processing and detection, and improving the accuracy of quantitative analysis. Centrifugation effectively removes proteins and other impurities. Taking the supernatant and diluting it appropriately before detection helps reduce matrix interference and improve chromatographic peak shape.

[0024] In step S4, based on the chromatogram of each standard solution obtained in step S3, a standard curve equation is established by the peak area ratio and concentration ratio of each antiepileptic drug in the standard solution to its corresponding internal standard. This can accurately characterize the quantitative relationship between the concentration of antiepileptic drugs and the detection signal, thus providing a reliable basis for the accurate calculation of the concentration of antiepileptic drugs in subsequent plasma samples.

[0025] In step S5, by preparing quality control working solutions at different concentration levels and conducting tests, the accuracy and applicability of the standard curve equation established in step S4 can be systematically verified across the entire detection concentration range, thereby ensuring that the detection method has good reliability and stability under different concentration conditions.

[0026] In step S6, by keeping the processing method of the blood sample to be tested basically consistent with that of the standard solution, it is beneficial to ensure the consistency of the sample and the standard in the detection conditions, thereby reducing systematic errors and realizing the accurate calculation of the concentration of antiepileptic drugs in the blood to be tested.

[0027] The method for simultaneously determining the concentration of multiple antiepileptic drugs in plasma provided in this embodiment uses an internal standard as the internal standard and combines it with liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology for plasma detection. This fully leverages the advantages of LC-MS / MS in terms of sensitivity, selectivity, and quantification. This method enables the simultaneous and rapid detection of multiple antiepileptic drugs in plasma, offering advantages such as simple pretreatment and high detection efficiency. It is suitable for the accurate quantitative analysis of trace antiepileptic drugs in complex biological matrices. Furthermore, by introducing the corresponding internal standard for each antiepileptic drug, this method effectively corrects for matrix effects and systematic errors introduced during sample pretreatment, significantly improving the accuracy and repeatability of the detection results. This meets the routine needs of therapeutic drug monitoring (TDM) in clinical practice, providing reliable quantitative evidence for optimizing individualized dosing regimens for epilepsy patients, thereby helping to reduce the risk of drug-related adverse reactions and improve overall treatment efficacy.

[0028] In one embodiment, in step S4, the standard curve equations for perampanel are: Y1 = 0.218778X1 - 0.0891044, and the standard curve equation for lacosamide is: Y2 = 62.0117X2 - 0.135688. These two standard curve equations accurately characterize the relationship between the concentrations of perampanel and lacosamide, these two antiepileptic drugs, in the sample and the detection signal, providing a reliable basis for subsequent concentration calculations of perampanel and lacosamide in the sample.

[0029] In one embodiment, in step S1, the concentration of perampanel in each standard working solution is limited to 62.5 ng / mL. -1 ~10000ng·mL -1 Within the specified range, the concentration of lacosamide is limited to 0.6875 μg·mL. -1 ~110μg·mL -1Within a certain range. By reasonably limiting the concentration of antiepileptic drugs such as perampanel and lacosamide in each standard working solution, the standard curve equation obtained in subsequent steps covers the low to high concentration range that may occur in clinical testing, thereby improving the fitting accuracy and applicability of the standard curve and meeting the detection needs of antiepileptic drug concentrations in different plasma samples. In this embodiment or other embodiments, in step S3, to accurately simulate the actual blood sample environment to be tested, it is preferable to mix the standard working solution and blank plasma at a volume ratio of 1:(8-10) to prepare the standard solution. This mixing ratio can reproduce the actual state of the drug and plasma matrix in the blood to be tested to the greatest extent. The volume of internal standard solution added to the standard solution is 5-8 times the volume of the standard solution to effectively correct systematic and random errors in the detection process, thereby significantly improving the accuracy and repeatability of the detection results.

[0030] In one embodiment, in the multiple sets of quality control working solutions prepared in step S5, the concentration of each antiepileptic drug is gradually increased. By setting this gradually increasing concentration gradient, the accuracy and stability of the fitted standard curve equation can be comprehensively evaluated within different concentration ranges, and potential concentration range deviations can be identified and corrected in a timely manner, thereby ensuring that the entire detection method has high accuracy and reliability, providing a reliable guarantee for subsequent clinical drug monitoring. In this embodiment or other embodiments, at least three sets of quality control working solutions are prepared, namely, a low-concentration quality control working solution, a medium-concentration quality control working solution, and a high-concentration quality control working solution. The low-concentration quality control working solution contains perampanel at a concentration of 50 ng / mL. -1 ~300ng·mL -1 At any given value, the concentration of lacosamide is 0.5 μg·mL. -1 ~3μg·mL -1 Any value is used to evaluate the accuracy and stability of the standard curve equation in the low concentration range, ensuring reliable detection results even at low drug concentrations. The concentration of perampanel in the medium-concentration quality control working solution is 1000 ng / mL. -1 ~3000ng·mL -1 For any given value, the concentration of lacosamide is 5 μg·mL. -1 ~50μg·mL -1 Any value is used to evaluate the performance of the standard curve equation at conventional concentrations, ensuring accurate drug concentration determination in routine testing. The concentration of perampanel in the high-concentration quality control working solution is 5000 ng / mL. -1 ~10000ng·mL -1 At any given value, the concentration of lacosamide is 50 μg·mL. -1 ~110μg·mL -1Any value. When the concentration of each antiepileptic drug in the sample is high, problems such as signal saturation and reagent nonlinearity may be encountered during detection. By setting a high concentration of quality control working solution, the reliability and stability of the standard curve equation under high concentration extreme conditions can be effectively verified. In addition, in some embodiments, in step S6, the volume of internal standard solution added to the supernatant plasma is 5-8 times the volume of the supernatant plasma. This ratio is consistent with the proportion of internal standard solution added during the standard solution processing, thereby helping to ensure the consistency of the blood sample to be tested with the standard in terms of detection conditions, reducing the quantitative error introduced by the difference in the amount of internal standard added, and improving the accuracy and reproducibility of the detection results.

[0031] In one embodiment, during the detection of samples using LC-MS / MS, the analytical column preferably used is an ACQUITY UPLC HSS T3 C. 18 Chromatographic column. This type of column has good separation performance and can effectively separate antiepileptic drugs and their internal standards. During the detection process, the injection volume is limited to the range of 2μL-10μL. By reasonably limiting the injection volume, it helps to ensure the stability and accuracy of the detection and avoid the impact of excessive or insufficient injection volume on the detection results. In this embodiment and other embodiments, during the detection of samples using LC-MS / MS, the analytical column employs gradient elution. Gradient elution can achieve better separation results based on the polarity differences of different substances. The mobile phase used for elution includes mobile phase A and mobile phase B. Mobile phase A is preferably a 3mM-8mM aqueous solution of ammonium acetate containing 0.05%-0.2% formic acid, and mobile phase B is preferably acetonitrile. Using these specific mobile phase compositions helps to improve the resolution and sensitivity of antiepileptic drugs and their internal standards. Specifically, the proportion of the mobile phase in the gradient elution process can be set as follows: (1) 0 min to 1 min, 5% mobile phase B; (2) 1 min to 3.5 min, linearly increasing from 5% mobile phase B to 95% mobile phase B; (3) 3.5 min to 4.5 min, 95% mobile phase B; (4) 4.5 min to 4.6 min, linearly decreasing from 95% mobile phase B to 5% mobile phase B; (5) 4.6 min to 5 min, 5% mobile phase B. Through this gradient elution method, the sample is initially eluted with a low proportion of mobile phase B, and then the proportion of mobile phase B is gradually increased to achieve effective elution and separation of the target compound. The column is then rapidly restored to its initial conditions for rebalancing, thereby improving analytical efficiency and shortening the overall detection time while ensuring separation performance. Furthermore, the flow rate of the mobile phase is preferably limited to 0.1 mL / min. -1 ~0.5 mL·min -1 Within a certain range; so as to ensure the stability and efficiency of the elution process by reasonably limiting the flow rate.

[0032] In one embodiment, during the detection of samples using LC-MS / MS, multiple reaction monitoring (MRM) mode is preferably employed for mass spectrometry ion scanning. By using MRM mode, which possesses high selectivity and high sensitivity, characteristic ions of antiepileptic drugs and their internal standards in the sample can be accurately detected, reducing interference from other factors and significantly improving the accuracy and reliability of the detection results. In this embodiment and other embodiments, the specific quantitative ion pairs used in MRM mode for mass spectrometry ion scanning are shown in Table 1.

[0033] Table 1. Quantitative ion pairs of various antiepileptic drugs and their corresponding internal standards Example 1 S1. First, weigh 10.20 mg of perampanel standard (98% purity) and place it in a 10 mL volumetric flask. Dissolve it in acetonitrile and dilute to the mark to obtain a perampanel standard stock solution with a concentration of 1.00 mg / mL. Dilute this perampanel standard stock solution 10 times with an acetonitrile / water solution at a volume ratio of 1:1 to obtain a perampanel standard intermediate solution with a concentration of 100 μg / mL. Similarly, weigh 11.18 mg of lacosamide standard (98.35% purity) and place it in a 10 mL volumetric flask. Dissolve it in acetonitrile and dilute to the mark to obtain a lacosamide standard stock solution with a concentration of 11.0 mg / mL. Dilute this lacosamide standard stock solution 10 times with an acetonitrile / water solution at a volume ratio of 1:1 to obtain a lacosamide standard intermediate solution with a concentration of 1100 μg / mL. Then, 2000 μL of perampanel standard intermediate solution and 2000 μL of lacosamide standard intermediate solution were pipetted into a 5 mL graduated test tube and diluted to 5 mL with acetonitrile / water solution. The diluted solution contained 40 μg / mL perampanel and 440 μg / mL lacosamide. Seven sets of standard curve working solutions (STD1 to STD7) were prepared by serial dilution with acetonitrile / water solution at a volume ratio of 1:1. The concentrations of perampanel and lacosamide in the seven standard working solutions are shown in Table 2.

[0034] Table 2. Concentrations of perampanel and lacosamide in seven sets of standard working solutions S2. First, weigh 1.02 mg of perampanel-d5 standard and place it in a 10 mL volumetric flask. Add approximately 4 mL of acetonitrile to the flask, shake to completely dissolve the perampanel-d5 standard, and then dilute to the mark with acetonitrile. After mixing, a perampanel-d5 stock solution with a concentration of 100 μg / mL is obtained. Weigh 1.014 mg of lacosamide-d6 standard and prepare a lacosamide-d6 stock solution with a concentration of 100 μg / mL using the same method. Next, transfer 500 μL of the perampanel-d5 stock solution and 500 μL of the lacosamide-d6 stock solution to the same 10 mL volumetric flask, dilute with acetonitrile, and dilute to the mark. After mixing, an internal standard intermediate solution is obtained. Subsequently, 100 μL of the internal standard intermediate solution was measured and placed in another 10 mL volumetric flask, diluted to the mark with acetonitrile, and mixed thoroughly to obtain a concentration of 5 ng / mL for both perampanel-d5 and lacosamide-d6. -1 The internal standard solution was prepared and immediately dispensed into portions, then stored at -80°C for later use.

[0035] S3. Using a pipette, transfer 10 μL of each of the seven standard working solutions into a 1.5 mL centrifuge tube. Add 90 μL of blank plasma to each of the transferred standard working solutions and mix thoroughly to form seven standard solutions. Next, add 600 μL of internal standard solution to each standard solution and vortex to precipitate the protein. Then, centrifuge at 18000 r / min for 5 min and take 100 μL of the supernatant. Dilute with acetonitrile at a volume ratio of supernatant to diluent of 1:9. Analyze the diluted supernatant using LC-MS / MS with a triple quadrupole mass spectrometer to obtain the chromatograms of the seven standard solutions. The mass spectrometer parameters for LC-MS / MS detection are shown in Table 3. During the detection process, the multiple reaction monitoring parameters for the high performance liquid chromatography triple quadrupole tandem mass spectrometry detection are shown in Table 4; the mobile phase gradient elution program parameters for the high performance liquid chromatography triple quadrupole tandem mass spectrometry detection are shown in Table 5.

[0036] Table 3. Mass spectrometer parameters for high performance liquid chromatography-triple quadrupole tandem mass spectrometry detection.

[0037] Table 4. Multiple Reaction Monitoring Parameters Table 5. Gradient elution program parameters S4. Based on the chromatograms of each group of standard solutions, the ratio of the peak area of ​​perampanel in each group of standard solutions to the peak area of ​​its corresponding perampanel-d5 is used as the ordinate Y1, and the ratio of the concentration of perampanel to the concentration of its corresponding perampanel-d5 is used as the abscissa X1. Linear regression is performed on the data of perampanel in the seven groups of standard solutions to obtain the standard curve equation for calculating the concentration of perampanel. The equation is: Y1=0.218778X1-0.0891044. Similarly, using the ratio of the peak area of ​​lacosamide to the peak area of ​​its corresponding internal standard lacosamide-d6 as the ordinate Y2, and the ratio of the concentration of lacosamide to the concentration of its corresponding internal standard lacosamide-d6 as the abscissa X2, linear regression was performed on the detection data of the seven sets of standard solutions to obtain the standard curve equation for lacosamide: Y2=62.0117X2-0.135688; and the correlation coefficient (r), detection range and limit of quantitation (LOQ) of perampanel and lacosamide are shown in Table 6.

[0038] Table 6. Linear relationship and limit of quantitation of perampanel and lacosamide As shown in Table 6, both antiepileptic drugs exhibit good linear correlation within their respective set concentration ranges, with correlation coefficients greater than 0.99, and both have low limits of quantitation, which can meet the requirements for quantitative analysis of biological samples.

[0039] S5. Prepare three sets of quality control working solutions according to the method in step S1: low-concentration quality control working solution (LQC), medium-concentration quality control working solution (MQC), and high-concentration quality control working solution (HQC). The concentrations of perampanel and lacosamide in each set of quality control working solutions are shown in Table 7. Furthermore, prepare three sets of quality control solutions according to the method in step S3, and perform LC-MS / MS analysis on each set of quality control solutions to verify the precision and accuracy of the two standard curve equations obtained in step S4. Specifically, the three sets of quality control working solutions prepared within the same batch and the three sets prepared between different batches were measured three times each. The precision of the method was evaluated using the relative standard deviation (RSD, %), and the results are shown in Table 8.

[0040] Table 7. Concentrations of perampanel and lacosamide in the three sets of quality control working solutions Table 8. Precision results of perampanel and lacosamide As shown in Table 8, this method exhibits good intra- and inter-batch precision at different concentration levels, meeting the precision requirements for quantitative analysis of antiepileptic drugs in plasma.

[0041] Meanwhile, low-concentration quality control working solution (LQC) and high-concentration quality control working solution (HQC) were selected for spiking recovery experiments to verify the accuracy of the standard curve equation. The determination was repeated 3 times according to the method provided in step S3 of this embodiment, and the calculated spiking recovery rates are shown in Table 9.

[0042] Table 9. Recovery rates of perampanel and lacosamide As shown in Table 9, the recovery rates of both antiepileptic drugs at different concentration levels were close to 100%, indicating that the method has high accuracy.

[0043] S6. Take 3 mL of blood to be tested and centrifuge at 3000 rpm for 5 min to separate the supernatant plasma. Store the supernatant plasma at -80℃ for later use. Transfer 100 μL of the supernatant plasma to a 1.5 mL centrifuge tube, add 600 μL of the above internal standard solution to the supernatant plasma, and vortex to mix to form the test solution. Then centrifuge the test solution at 18000 rpm for 5 min, take 100 μL of the supernatant, and dilute it at a supernatant to acetonitrile volume ratio of 1:9. Then, perform LC-MS / MS analysis on the diluted supernatant. Figure 1 and Figure 2 As shown, the chromatogram of the blood sample was obtained. (From...) Figure 1 and Figure 2 It was found that the retention time of lacosamide was 3.16 min, and the retention time of perampanel was 4.20 min. Both target antiepileptic drugs showed clear characteristic peaks at their respective retention times, with no obvious interfering peaks. This indicates that the method used in this embodiment has good accuracy and specificity in identifying the target antiepileptic drugs. Furthermore, this method has a short analysis cycle, minimal matrix interference, and is suitable for rapid detection in plasma samples. The concentrations of perampanel-d5 and lacosamide-d6 in the internal standard solution are known. The concentrations of perampanel and lacosamide in the blood sample can be calculated by using the ratio of the peak area of ​​perampanel and lacosamide to the peak area of ​​the corresponding internal standard in the chromatogram, combined with the standard curve equations of perampanel and lacosamide obtained above.

[0044] Therefore, based on the methodological validation results regarding linearity, limit of quantitation, precision, and recovery rate, the method provided in this embodiment for simultaneously determining the concentrations of multiple antiepileptic drugs in plasma exhibits good linearity, a low limit of quantitation, a high recovery rate, and excellent precision and reproducibility when used for the simultaneous determination of perampanel and lacosamide in plasma. This method can meet the needs for accurate quantitative analysis of antiepileptic drugs in complex biological matrices. The method is simple to operate, highly stable, and suitable for clinical drug monitoring and optimization of individualized medication regimens for epilepsy patients, improving the effectiveness and safety of epilepsy treatment, reducing the risk of adverse reactions, and promoting the implementation of precision medicine and individualized medication strategies. Furthermore, this method can be widely applied in new drug clinical trials, pharmacokinetic studies, and drug interaction studies, demonstrating good versatility and promotional value.

[0045] 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.

[0046] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope 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 the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for simultaneously determining the concentration of multiple antiepileptic drugs in plasma, characterized in that, Includes the following steps: S1. Weigh out various antiepileptic drugs, dissolve them in acetonitrile aqueous solution, and prepare multiple sets of standard working solutions containing different concentrations of each of the aforementioned antiepileptic drugs. S2. Weigh the internal standard corresponding to each of the aforementioned antiepileptic drugs, dissolve it in acetonitrile, and prepare an internal standard solution. The concentration of each of the aforementioned internal standards in the internal standard solution is the same, and is 1-10 ng·mL. -1 Any value in; S3. Take multiple sets of the standard working solutions respectively, mix each set of the standard working solutions with blank plasma to form multiple sets of standard solutions, add the internal standard solution to each standard solution and mix well, centrifuge and take the supernatant for LC-MS / MS detection to obtain the chromatogram of each standard solution; S4. Based on the chromatogram of each standard solution, the ratio of the peak area of ​​each antiepileptic drug to the peak area of ​​its corresponding internal standard is used as the ordinate Y, and the ratio of the concentration of the antiepileptic drug to the concentration of its corresponding internal standard is used as the abscissa X. Linear regression fitting is performed on the data in each group of standard solutions to obtain the standard curve equation for the concentration of each antiepileptic drug. S5. Prepare multiple sets of quality control working solutions according to the method in step S1, and prepare multiple sets of quality control solutions according to the method in step S3. Perform LC-MS / MS detection on each of the quality control solutions to verify each of the standard curve equations obtained in step S4. S6. Centrifuge the blood to be tested to obtain supernatant plasma. Add the internal standard solution to the supernatant plasma and mix well. After centrifugation, take the supernatant for LC-MS / MS detection to obtain the chromatogram of the blood to be tested. Calculate the concentration of each antiepileptic drug in the plasma based on the ratio of the peak area of ​​each antiepileptic drug to the peak area of ​​the corresponding internal standard in the chromatogram of the blood to be tested and the validated standard curve equation of the corresponding antiepileptic drug.

2. The method as described in claim 1, characterized in that, In step S1, the plurality of antiepileptic drugs include at least perampanel and lacosamide; The standard curve equation for the perampanel obtained in step S4 is: Y1=0.218778X1-0.0891044, and the standard curve equation for the lacosamide is: Y2=62.0117X2-0.135688.

3. The method as described in claim 2, characterized in that, In step S1, the concentration of perampanel in each of the standard working solutions is 62.5 ng·mL. -1 ~10000ng·mL -1 The concentration of lacosamide is 0.6875 μg·mL for any of the values. -1 ~110μg·mL -1 Any value among them.

4. The method as described in claim 2, characterized in that, In step S3, the volume ratio of the standard working solution to the blank plasma in the standard solution is any value of 1:(8-10); the volume of the internal standard solution added to the standard solution is 5-8 times the volume of the standard solution.

5. The method as described in claim 2, characterized in that, In step S5, the concentration of each antiepileptic drug in the multiple sets of quality control working solutions is gradually increased.

6. The method as described in claim 5, characterized in that, The multiple sets of quality control working solutions include low-concentration quality control working solution, medium-concentration quality control working solution and high-concentration quality control working solution; In the low-concentration quality control working solution, the concentration of piraminpanel-d5 is 50 ng·mL. -1 ~300ng·mL -1 For any of the values, the lacosamide-d6 has a concentration of 0.5 μg·mL. -1 ~3μg·mL -1 Any value in; In the medium-concentration quality control working solution, the concentration of piraminpanel-d5 is 1000 ng·mL. -1 ~3000ng·mL -1 For any of the values, the lacosamide-d6 has a concentration of 5 μg·mL⁻¹. -1 ~μg·mL -1 Any value among them; In the high-concentration quality control working solution, the concentration of piraminpanel-d5 is 5000 ng·mL. -1 ~10000ng·mL -1 For any of the values, the lacosamide-d6 has a concentration of 50 μg·mL. -1 ~110μg·mL -1 Any value among them.

7. The method as described in claim 2, characterized in that, In step S6, the volume of the internal standard solution added to the supernatant plasma is 5-8 times the volume of the supernatant plasma.

8. The method as described in claim 2, characterized in that, The analytical column used in the LC-MS / MS detection process was an ACQUITY UPLC HSS T3 C. 18 For chromatographic columns, the injection volume is limited to the range of 2μL-10μL.

9. The method as described in claim 8, characterized in that, In the LC-MS / MS detection process, the analytical column employs gradient elution. The elution mobile phase includes mobile phase A and mobile phase B. Mobile phase A is a 3mM–8mM aqueous solution of ammonium acetate containing 0.05%–0.2% formic acid, and mobile phase B is acetonitrile. The flow rate of the mobile phase is 0.1 mL / min. -1 ~0.5 mL·min -1 Any value among them.

10. The method as described in claim 2, characterized in that, During the LC-MS / MS detection process, a multiple reaction monitoring (MRM) mode is used for mass spectrometry ion scanning monitoring.

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