Method for simultaneously determining serum concentration of antiepileptic drug and active metabolite thereof based on LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry) method

Abstract

The invention relates to the technical field of biological detection, and particularly discloses a method for simultaneously determining serum concentrations of an anti-epileptic drug and active metabolites thereof based on an LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) method. According to the method, the C18 chromatographic column with the filler particle size of 3.0 microns is selected for the first time, efficient separation of lamotrigine, levetiracetam, oxcarbazepine and active metabolites thereof is achieved, and the separation efficiency is equivalent to that of a small-particle-size column; a pretreatment process is simplified by precipitating protein through a single organic solvent, and accurate quantification of four components is completed by only using two isotope internal standard oxcarbazep-d4 and levetiracetam-d6, so that the cost is reduced. In the aspect of the detection range, the lower quantification limit of oxcarbazepine is broken through to 0.04 [mu] g.mL <-1 >, and the technical contradiction between oxcarbazepine and the upper quantification limit (as high as 50 [mu] g.mL <-1 >) of LEV across three orders of magnitude is overcome. According to the scheme, hardware requirements and reagent cost are reduced, and meanwhile, high sensitivity and a wide linear range are achieved.

Description

Technical Field

[0001] This application relates to the field of biological detection technology, and particularly to a method for simultaneously determining the serum concentrations of antiepileptic drugs and their active metabolites based on LC-MS / MS method. Background Art

[0002] In the field of therapeutic drug monitoring (TDM) of antiepileptic drugs, liquid chromatography-tandem mass spectrometry (LC-MS / MS) has become a powerful tool for research and clinical monitoring due to its high sensitivity, high specificity, and the ability to simultaneously detect multiple targets. Epileptic patients often need to take multiple antiepileptic drugs for a long time, such as lamotrigine (LTG), levetiracetam (LEV), oxcarbazepine (OXC), and their active metabolite 10-hydroxy-10,11-dihydro-5H-dibenzo[b,f]azepine -5-carboxamide (MHD). The blood drug concentrations of these drugs are crucial for evaluating efficacy and toxicity.

[0003] However, traditional detection methods such as immunoassay are limited by problems such as non-specific interference, complex operation, and high cost, while LC-MS / MS technology provides an effective alternative. Nevertheless, existing LC-MS / MS methods still have certain limitations in aspects such as sample pretreatment, chromatographic column selection, and internal standard application, such as complex operation, high cost, long analysis time, and limited detection linear range for OXC, which cannot meet the detection requirements of all clinical samples. These factors affect the detection efficiency and cost-effectiveness.

[0004] First, in terms of the selection of pretreatment methods, current TDM pretreatment methods based on HPLC or LC-MS / MS are mostly liquid-liquid extraction, solid-phase extraction, or protein precipitation methods. These methods differ in terms of operational simplicity, time consumption, cost, and analysis efficiency. For example, Chinese Patent CN 115060816 B discloses a "headspace solid-phase microextraction tandem gas chromatography-mass spectrometry detection method for fluoramine ketone metabolites in urine", and this pretreatment method is relatively cumbersome. The protein precipitation method is more suitable for hospital TDM projects due to its simple operation and short time consumption, but the selection of different precipitants will affect the accuracy and stability of the detection of analytes. The existing technology generally uses a mixed precipitant system, which can achieve protein precipitation, but requires precise volume ratio and is relatively complex to operate.

[0005] Secondly, the selection of the chromatographic column has a significant impact on the separation efficiency and analysis time. Existing research tends to use chromatographic columns with a particle size less than 3μm to improve the separation efficiency, but this often comes with increased column pressure, risk of leakage, and increased cost.

[0006] In addition, the selection of internal standards is crucial for improving the accuracy of quantification. However, existing external standard methods and non-isotope internal standards may not be able to completely eliminate matrix effects, which can affect accuracy. For example, Chinese Patent CN106168610B discloses a "method for determining the concentration of clozapine in plasma by high performance liquid chromatography-mass spectrometry", which provides a method using high performance liquid chromatography-mass spectrometry and using risperidone as an internal standard to detect clozapine psychotropic drugs. It is a method specifically for a specific psychotropic drug - clozapine drug and is not suitable for the simultaneous measurement of multiple psychotropic drugs.

[0007] In addition, the existing OXC range is limited and difficult to meet clinical needs. Existing studies usually set the lower limit of quantification of OXC at 0.2 μg·mL -1 and above. Some scholars have tried to reduce the lower limit of quantification of OXC to 0.01 μg·mL -1 in their research, but its upper limit is correspondingly reduced to 1.0 μg·mL -1 which cannot meet the detection requirements of some samples. It can be seen that the existing detection linear range of OXC has limited reference significance.

[0008] The above problems limit the wide application of LC-MS / MS technology in TDM, especially in clinical settings with limited resources. Therefore, developing a simple, economical, accurate, and efficient LC-MS / MS method has important clinical value for the individualized treatment of epilepsy patients. Summary of the Invention

[0009] The purpose of this application is to overcome the defects in the prior art such as complex pretreatment methods, over-reliance on small particle size (<3 μm) of chromatographic columns, and limited detection linear range, and to provide a method for simultaneously determining the serum concentrations of antiepileptic drugs and their active metabolites based on LC-MS / MS method. This method significantly improves the operation efficiency and economy while ensuring the detection accuracy.

[0010] To achieve the above purpose, the technical solutions adopted in this application are as follows:

[0011] Provide a method for simultaneously determining the serum concentrations of antiepileptic drugs and their active metabolites based on LC-MS / MS method, including the following steps:

[0012] Add the serum sample to be tested containing antiepileptic drugs and their active metabolites to a mixed internal standard solution containing isotope internal standards for one-step protein precipitation, dilute it with a mixed solution of methanol and water, and perform detection and analysis through an LC-MS / MS system;

[0013] Among them, the mixed internal standard solution containing isotope internal standards includes a methanol solution of oxcarbazepine-d4 (OXC-D4) and levetiracetam-d6 (LEV-D6);

[0014] The LC-MS / MS system uses a C18 chromatographic column with a packing particle size of 3.0 μm.

[0015] Aiming at the problems of complex operation, time-consuming and high cost in sample pretreatment methods such as liquid-liquid extraction method and solid-phase extraction method in the prior art, this application has developed a simpler, faster and more cost-effective sample pretreatment method to improve the efficiency and economy of the overall detection process. In the technical solution of this application, a single methanol solvent is selected to replace the traditional mixed precipitant for protein precipitation, simplifying the pretreatment process; moreover, aiming at the defect of high column pressure caused by the dependence on small particle size chromatographic columns (<3 μm) in the prior art, this application for the first time selects a C18 chromatographic column with a packing particle size of 3.0 μm, and under the condition of maintaining the column pressure ≤ 15 MPa, the high-efficiency separation of lamotrigine, levetiracetam, oxcarbazepine and their active metabolites (10-hydroxy-10,11-dihydro-5H-dibenzo[b,f]azepine -5-formamide) has been successfully achieved, and the detection efficiency is equivalent to that of the conventional small particle size chromatographic column technology; only two isotope internal standards, OXC-D4 and LEV-D6, are used to complete the accurate quantification of the four substances, effectively reducing the cost and having more promotion value; the detection linear range of oxcarbazepine has been expanded, and the technical contradiction of the concentration span of three orders of magnitude between the lower limit of quantification of oxcarbazepine (as low as 0.04 μg·mL -1 ) and the upper limit of quantification of LEV (as high as 50 μg·mL -1 ) has been successfully overcome, solving the problem of insufficient sensitivity or limited detection range of the traditional method.

[0016] Preferably, the volume ratio of methanol to water is 1:1.

[0017] As a preferred embodiment of the method for simultaneously determining the serum concentrations of antiepileptic drugs and their active metabolites based on the LC-MS / MS method described in this application, the antiepileptic drugs include at least one of lamotrigine (LTG), levetiracetam (LEV) and oxcarbazepine (OXC);

[0018] The active metabolites of antiepileptic drugs include 10-hydroxy-10,11-dihydro-5H-dibenzo[b,f]azepine -5-formamide (MHD).

[0019] As a preferred embodiment of the method for simultaneously determining the serum concentrations of antiepileptic drugs and their active metabolites based on the LC-MS / MS method described in this application, the limit of quantification of oxcarbazepine is 0.04 - 5 μg·mL -1 .

[0020] This application optimizes the lower limit of quantification of OXC to 0.04 μg·mL -1 , and the upper limit is maintained at 5 μg·mL -1, significantly expanding the linear range of OXC detection. Analysis of 145 clinical samples showed (including 20 samples for single detection of LTG, 62 samples for LEV, 38 samples for OXC and its metabolite MHD, and 24 samples for simultaneous detection of more than two blood drug concentration items (OXC and its metabolite MHD are regarded as a single item). Except for the OXC concentration of 1 sample being lower than the lowest detection limit, the detection results of the remaining 144 samples were within the linear range of the standard curve, that is, the steady-state trough concentration fluctuation ranges of LTG, LEV, OXC, and MHD were 1.14 - 24.47, 0.77 - 41.65, 0.05 - 2.37, and 4.35 - 34.87 μg·mL -1 , verifying that this method is applicable to the routine TDM of the above four substances. Therefore, the setting of the OXC linear range in the present application scheme is more reasonable.

[0021] As a preferred embodiment of the method for simultaneously determining the serum concentrations of antiepileptic drugs and their active metabolites based on LC-MS / MS in the present application, the linear range of the mass concentration of lamotrigine is 0.2 - 25 μg·mL -1 ; the linear range of the mass concentration of levetiracetam is 0.39 - 50 μg·mL -1 ; the linear range of oxcarbazepine is 0.04 - 5 μg·mL -1 ; the linear range of the mass concentration of 10-hydroxy-10,11-dihydro-5H-dibenzo[b,f]azepine -5-carboxamide is 0.31 - 40 μg·mL -1 .

[0022] As a preferred embodiment of the method for simultaneously determining the serum concentrations of antiepileptic drugs and their active metabolites based on LC-MS / MS in the present application, the mass concentration of lamotrigine is one of 0.20 μg·mL -1 , 0.46 μg·mL -1 , 2.78 μg·mL -1 , 20 μg·mL -1 ; the mass concentration of levetiracetam is one of 0.39 μg·mL -1 , 0.93 μg·mL -1 , 5.56 μg·mL -1 , 40 μg·mL -1 ; the mass concentration of oxcarbazepine is one of 0.04 μg·mL -1 , 0.09 μg·mL -1 , 0.56 μg·mL -1 , 4 μg·mL -1 ; the 10-hydroxy-10,11-dihydro-5H-dibenzo[b,f]azepine - The mass concentration of 5-formamide is 0.31 μg·mL -1 、0.74 μg·mL -1 、4.44 μg·mL -1 、32 μg·mL -1 among the following concentrations.

[0023] When the mass concentrations of lamotrigine, levetiracetam, oxcarbazepine and 10-hydroxy-10,11-dihydro-5H-dibenz[b,f]azepine-5-carboxamide are as above, the technical solution of this application can better and effectively separate the above four components.

[0024] As a preferred embodiment of the method for simultaneously determining the serum concentrations of antiepileptic drugs and their active metabolites based on LC-MS / MS method described in this application, the chromatographic conditions in the LC-MS / MS method include:

[0025] A C18 chromatographic column with a packing particle size of 3.0 μm, column temperature of 35 - 45 °C; flow rate of 0.6 - 1.0 mL·min -1 ; injection volume of 0.5 - 2.0 μL; injector temperature of 2 - 8 °C;

[0026] Gradient elution is adopted, mobile phase A is a formic acid aqueous solution containing ammonium acetate, and mobile phase B is methanol;

[0027] The steps of gradient elution are: 0 - 0.5 min, 60% B; 0.5 - 4 min, 60% → 98% B; 4 - 5 min, 98% B; 5 - 5.1 min, 98% → 60% B; 5.1 - 6 min, 60% B.

[0028] This application improves the applicability and economy of the chromatographic column. Aiming at the problems of high column pressure, leakage risk and cost caused by the chromatographic column with a particle size less than 3 μm used in the prior art, this application is committed to developing or optimizing chromatographic conditions. By using a C18 chromatographic column with a packing particle size of 3.0 μm, the effective separation of LTG, LEV, OXC and MHD is successfully achieved while keeping the column pressure not exceeding 15 Mpa, effectively prolonging the service life of the chromatographic column, and completing the analysis within 6 min; compared with the prior art, the detection time is not significantly extended.

[0029] As a preferred embodiment of the method for simultaneously determining the serum concentrations of antiepileptic drugs and their active metabolites based on LC-MS / MS method described in this application, the mass concentration of formic acid in the formic acid aqueous solution is 0.1%; the mass concentration of ammonium acetate is 5 mmol·L -1 .

[0030] As a preferred embodiment of the method for simultaneously determining the serum concentrations of antiepileptic drugs and their active metabolites based on the LC-MS / MS method described in the present application, the C18 chromatographic column includes a Phenomenex C18 chromatographic column (100 mm × 4.6 mm, 3.0 μm).

[0031] The present application uses a Phenomenex C18 chromatographic column (100 mm × 4.6 mm, 3.0 μm), which can better and effectively separate antiepileptic drugs and their active metabolites, significantly reduce the column pressure, reduce the risk of leakage, and extend the service life of the chromatographic column.

[0032] As a preferred embodiment of the method for simultaneously determining the serum concentrations of antiepileptic drugs and their active metabolites based on the LC-MS / MS method described in the present application, the volume ratio of methanol to water in the methanol and water mixture is 1:1.

[0033] As a preferred embodiment of the method for simultaneously determining the serum concentrations of antiepileptic drugs and their active metabolites based on the LC-MS / MS method described in the present application, the mass spectrometry conditions in the LC-MS / MS method include:

[0034] An electrospray ionization source is used for positive ion multiple reaction monitoring mode scanning; the curtain gas pressure is 20 psi; the ion source spray voltage is 5500 V; the ion source temperature is 650 °C; the nebulizing gas pressure is 60 psi; the auxiliary heater pressure is 60 psi; the ion pairs used for quantitative analysis are respectively:

[0035] Lamotrigine: m / z 256.0 → 211.0;

[0036] Levetiracetam: m / z 171.0 → 126.2;

[0037] Oxcarbazepine: m / z 253.0 → 180.2;

[0038] 10-Hydroxy-10,11-dihydro-5H-dibenzo[b,f]azepine -5-carboxamide: m / z 255.0 → 194.1;

[0039] Levetiracetam-d6: m / z 177.0 → 132.2;

[0040] Oxcarbazepine-d4: m / z 257.0 → 184.2;

[0041] Lamotrigine, levetiracetam, oxcarbazepine, 10-hydroxy-10,11-dihydro-5H-dibenzo[b,f]azepine The declustering voltages of 5-formamide, levetiracetam-d6, and oxcarbazepine-d4 are 80.0, 47.0, 105.0, 70.0, 40.8, and 105.0 V respectively, and the collision voltages are 37.90, 42.00, 38.76, 50.00, 22.01, and 38.76 V respectively.

[0042] As a preferred embodiment of the method for simultaneously determining the serum concentrations of antiepileptic drugs and their active metabolites based on LC-MS / MS described in this application, the mass concentrations of oxcarbazepine-d4 and levetiracetam-d6 solutions in the mixed internal standard solution are both 5 - 15 ng·mL -1 , preferably 10 ng·mL -1 .

[0043] Compared with the prior art, this application has the following beneficial effects:

[0044] This application provides a method for simultaneously determining the serum concentrations of antiepileptic drugs and their active metabolites based on LC-MS / MS. This application simplifies the pretreatment process, selects an organic solvent with a single component, and simplifies the operation; moreover, aiming at the defect of the prior art that relies on small-particle-size chromatographic columns (<3 μm) resulting in high column pressure, this application for the first time selects a C18 chromatographic column with a packing particle size of 3.0 μm, and successfully realizes the efficient separation of LTG, LEV, OXC, and their active metabolite MHD under the condition of maintaining the column pressure ≤ 15 MPa, and the detection efficiency is equivalent to that of the conventional small-particle-size chromatographic column technology; only using two isotope internal standards OXC-D4 and LEV-D6 can accurately quantify four substances, effectively reducing the cost; expanding the detection linear range of oxcarbazepine, and successfully overcoming the technical contradiction that the lower limit of quantification of oxcarbazepine (as low as 0.04 μg·mL -1 ) and the upper limit of quantification of LEV (as high as 50 μg·mL -1 ) have a concentration span of three orders of magnitude, solving the problem of insufficient sensitivity or limited detection range of traditional methods. Finally, this solution has both high sensitivity (the lower limit of quantification of oxcarbazepine is significantly lower than that of the prior art) and a wide linear range while reducing the hardware requirements and reagent costs, providing an efficient, economical, and accurate solution for clinical blood drug concentration monitoring. Description of the Drawings

[0045] Figure 1 They are the total ion scan mass spectra of LTG (A), LEV (B), OXC (C), MHD (D), LEV-D6 (E), and OXC-D4 (F);

[0046] Figure 2Representative chromatograms of the analyte (A: blank serum; B: blank serum + drugs to be measured + internal standards; C: blank serum + internal standards). Detailed implementation manners

[0047] To better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0048] In the following embodiments, unless otherwise specified, the experimental methods used are all conventional methods. The materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels, and the component raw materials used in each parallel experiment are of the same kind.

[0049] The materials used in the following embodiments include:

[0050] Instrument: Jasper TM type high performance liquid chromatography system and AB Sciex Thriple Quad TM 4500MD type triple quadrupole tandem mass spectrometer (AB Sciex Company, USA); CPA2250D type analytical balance (Sartorius Scientific Instruments Beijing Co., Ltd.); H1650 medical centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.); JOANLAB 7K type vortex mixer (Qunan Experimental Instrument Co., Ltd.), etc.

[0051] Drugs and reagents: OXC standard (batch number 100657 - 201102), LTG standard (batch number 100775 - 201902) were purchased from the National Institutes for Food and Drug Control; MHD standard (batch number T10O10F99575) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; LEV standard (batch number 102767 - 28 - 2) was purchased from Beijing Solarbio Science & Technology Co., Ltd.; OXC-D4 standard (batch number 6-NAV-171-1), LEV-D6 standard (batch number 4-MGG-38-2) were used as internal standards and were all purchased from TRC Company, Canada. The purity of all standards is >98%.

[0052] The reagents are all chromatographically pure reagents. Formic acid (batch number C11723085) was purchased from Shanghai Macklin Biochemical Co., Ltd.; methanol (batch number F23NBD201), ammonium acetate (batch number 191008) were all purchased from Thermo Fisher Scientific Company, USA; the water is pure water.

[0053] Serum: The blank serum comes from the laboratory department of Guangzhou First People's Hospital.

[0054] Example 1. A method for simultaneously determining the serum concentrations of antiepileptic drugs and their active metabolites by LC-MS / MS

[0055] 1. Chromatographic conditions: Phenomenex C18 chromatographic column (100 mm × 4.6 mm, 3.0 μm), column temperature 40 °C;

[0056] Using an aqueous solution of 0.1% formic acid containing 5 mmol·L -1 ammonium acetate (A) - methanol (B) as the mobile phase for gradient elution (0 - 0.5 min, 60% B; 0.5 - 4 min, 60% → 98% B; 4 - 5 min, 98% B; 5 - 5.1 min, 98% → 60% B; 5.1 - 6 min, 60% B); flow rate 0.6 mL·min -1 ; injection volume 1 μL; injector temperature 4 °C.

[0057] 2. Mass spectrometry conditions: Electrospray ionization source was used, and positive ion multiple reaction monitoring mode scanning was carried out; curtain gas pressure 20 psi; ion source spray voltage 5500 V; ion source temperature 650 °C; nebulizing gas pressure 60 psi; auxiliary heater pressure 60 psi;

[0058] The ion pairs used for quantitative analysis were m / z 256.0 → 211.0 (LTG), m / z171.0 → 126.2 (LEV), m / z 253.0 → 180.2 (OXC), m / z 255.0 → 194.1 (MHD), m / z177.0 → 132.2 (LEV-D6), m / z 257.0 → 184.2 (OXC-D4), and the declustering voltages of LTG, LEV, OXC, MHD, LEV-D6, OXC-D4 were 80.0, 47.0, 105.0, 70.0, 40.8, 105.0 V respectively, and the collision voltages were 37.90, 42.00, 38.76, 50.00, 22.01, 38.76 V respectively. The full ion scan mass spectrometry diagram is shown in Figure 1 .

[0059] 3. Preparation of reference working solution: Accurately weigh the reference standards of LTG, LEV, OXC, and MHD, dissolve them with methanol, and prepare reference stock solutions with mass concentrations of 2.5, 10, 1.0, and 0.5 mg·mL -1 respectively.

[0060] Mix the above reference stock solutions in a certain proportion and gradually dilute them with methanol to obtain LTG with mass concentrations of 250, 125, 62.50, 31.25, 15.63, 7.81, 3.91, 1.95 μg·mL -1, the working solutions of the mixed standard curves with LEV mass concentrations of 500, 250, 125, 62.50, 31.25, 15.63, 7.81, 3.91 μg·mL -1 , the working solutions of the mixed standard curves with OXC mass concentrations of 50, 25, 12.50, 6.25, 3.13, 1.56, 0.78, 0.39 μg·mL -1 , the working solutions of the mixed standard curves with MHD mass concentrations of 400, 200, 100, 50, 25, 12.50, 6.25, 3.13 μg·mL -1 , and the working quality control solutions with LTG mass concentrations of 200, 27.78, 4.63 μg·mL -1 , and LEV mass concentrations of 400, 55.56, 9.26 μg·mL -1 , and OXC mass concentrations of 40, 5.56, 0.93 μg·mL -1 , and MHD mass concentrations of 320, 44.44, 7.41 μg·mL -1 ; stored in the dark at -20°C.

[0061] 4. Preparation of internal standard solution: Weigh accurately 1 mg of each of LEV-D6 and OXC-D4, dissolve them in methanol, and prepare stock internal standard solutions with LEV-D6 and OXC-D4 mass concentrations of 100 μg·mL -1 respectively. Take an appropriate amount of the stock internal standard solution and dilute it with methanol to obtain a mixed internal standard solution with the above component mass concentrations of 10 ng·mL -1 ; stored in the dark at -20°C.

[0062] 5. Preparation of serum samples containing drugs: Take 5 μL of each of the mixed working solutions with different concentrations, add 45 μL of blank serum (without the analyte to be measured), mix well to prepare serum standard curve samples with LTG mass concentrations of 25, 12.50, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20 μg·mL -1 , and LEV mass concentrations of 50, 25, 12.50, 6.25, 3.13, 1.56, 0.78, 0.39 μg·mL -1 , and OXC mass concentrations of 5, 2.50, 1.25, 0.63, 0.31, 0.16, 0.08, 0.04 μg·mL -1 , and MHD mass concentrations of 40, 20, 10, 5, 2.50, 1.25, 0.63, 0.31 μg·mL -1 , as well as serum samples with LTG mass concentrations of 0.20, 0.46, 2.78, 20 μg·mL -1 , and LEV mass concentrations of 0.39, 0.93, 5.56, 40 μg·mL-1 , the quality concentrations of OXC are 0.04, 0.09, 0.56, 4 μg·mL -1 , the quality concentrations of MHD are 0.31, 0.74, 4.44, 32 μg·mL -1 for the lower limit of quantification, low, medium, and high concentration serum quality control samples.

[0063] 6. Serum sample treatment: Precisely pipette 50 μL of the serum sample to be tested (containing the analyte), add 800 μL of the mixed internal standard solution, vortex for 1 min, centrifuge at 14000 r / min for 10 min, take 100 μL of the supernatant, add 800 μL of methanol:water (50:50) solution, mix well and then inject for analysis.

[0064] Results:

[0065] 1. Specificity: The peak shapes of the chromatographic peaks of LTG, LEV, OXC, MHD, LEV-D6, and OXC-D4 are good, and the retention times are 1.87, 2.27, 4.36, 4.09, 2.26, and 4.34 min respectively. Endogenous substances do not interfere with the determination of the analyte and the internal standard, indicating that the method has good specificity. See Figure 2 .

[0066] 2. Standard curve and lower limit of quantification: The linear equation of LTG (with LEV-D6 as the internal standard) is Y = 0.99456X + 0.02077 (r = 0.9992), the linear equation of LEV (with LEV-D6 as the internal standard) is Y = 0.55478X + 0.01023 (r = 0.9994), the linear equation of OXC (with OXC-D4 as the internal standard) is Y = 4.35228X + 0.04063 (r = 0.9989), the linear equation of MHD (with OXC-D4 as the internal standard) is Y = 0.56057X + 0.00653 (r = 0.9987). LTG, LEV, OXC, and MHD have good linearity in the ranges of 0.20 - 25, 0.39 - 50, 0.04 - 5, and 0.31 - 40 μg·mL -1 respectively, and the lower limits of quantification are 0.20, 0.39, 0.04, and 0.31 μg·mL -1 , which can cover the clinically recommended concentration ranges of LTG, LEV, and MHD, namely 2.5 - 15, 12 - 46, and 3 - 35 μg·mL -1 , and at the same time, the linear range of OXC is significantly extended.

[0067] 3. Accuracy and precision: The accuracy and within-batch and between-batch precisions of LTG, LEV, OXC, and MHD are good (both RE and RSD are < 15.0%), meeting the relevant requirements of the "Guidelines for the Validation of Bioanalytical Methods" in the Chinese Pharmacopoeia. The results are shown in Table 1.

[0068] Table 1 Accuracy and Precision (n = 6)

[0069]

[0070] 4. Extraction Recovery and Matrix Effect: The extraction recoveries of each analyte at different concentrations ranged from 88.56% to 112.18%. The matrix did not interfere with the determination of LTG, LEV, OXC, and MHD. See Table 2 for details.

[0071] Table 2 Experimental Results of Extraction Recovery and Matrix Effect (n = 6)

[0072]

[0073]

[0074] 5. Stability: Serum quality control samples at various mass concentrations remained stable under the conditions of storage at -20°C for 14 days, repeated freezing and thawing 3 times (-20°C to room temperature), room temperature for 3 hours, and placement in the injector (4°C) for 24 hours after treatment. See Table 3 for details.

[0075] Table 3 Experimental Results of Stability (n = 6)

[0076]

[0077] 6. Residual Assessment: Through analysis, no obvious chromatographic peaks were observed in the blank serum at the retention times corresponding to the analytes and their internal standards. In addition, the ratio of the noise in the blank serum to the peak area of the corresponding chromatographic peak in the lower limit of quantification sample was less than 0.10%, indicating no obvious residual effect.

[0078] 7. Application of the Method: In this study, the blood drug concentrations of LTG, LEV, OXC, and MHD in 145 clinical serum samples were detected (including 20 samples with only LTG detected, 62 samples with LEV detected, 38 samples with OXC and its metabolite MHD detected, and 24 samples with two or more blood drug concentration items detected simultaneously (OXC and its metabolite MHD were regarded as a single item)). Except for the OXC concentration in 1 sample being lower than the lowest detection limit, the detection results of the remaining 144 samples were within the linear range of the standard curve, that is, the steady-state trough concentration fluctuation ranges of LTG, LEV, OXC, and MHD were 1.14 - 24.47, 0.77 - 41.65, 0.05 - 2.37, and 4.35 - 34.87 μg·mL -1 , which confirmed that the method is applicable to the routine TDM of the above four substances; therefore, the setting of the linear range of OXC in the proposed application scheme is more reasonable.

[0079] Table 4 Determination Results of Blood Drug Concentrations

[0080]

[0081] Comparative Example 1

[0082] In terms of the selection of the pretreatment method, currently, the pretreatment methods for TDM based on HPLC or LC-MS / MS are mostly liquid-liquid extraction, solid-phase extraction, or protein precipitation methods. These methods differ in terms of operational simplicity, time consumption, cost, and analysis efficiency.

[0083] Comparative Example 1 is a method disclosed in Chinese Patent CN 115060816 B: "A headspace solid-phase microextraction tandem gas chromatography-mass spectrometry detection method for fluoramine-ketone metabolites in urine". This pretreatment method is relatively cumbersome. The protein precipitation method is more suitable for hospital TDM projects due to its simple operation and short time consumption.

[0084] Comparative Example 2

[0085] The selection of different precipitants will affect the accuracy and stability of the detection. Compared with Example 1, Comparative Example 2 (i.e., Document 1) uses a mixed precipitant of methanol-acetonitrile (4:1). Although it can achieve protein precipitation, it has significant defects: 1) The mixed solvent requires precise ratioing, increasing the operation steps and error risk; 2) Acetonitrile is highly toxic (LD50 = 2.7 g / kg), and its cost is 2.3 times that of methanol, resulting in an increase in the single-sample processing cost and complex waste liquid treatment; 3) Experimental data shows that the coefficient of variation of the matrix effect of LEV is as high as 9.36% (3.75% in Example 1), and the coefficient of variation of the matrix effect of MHD reaches 5.81% (4.06% in Example 1), affecting the detection stability; 4) Acetonitrile is prone to crystallization at low temperatures, limiting the environmental adaptability of the method.

[0086] Example 1 of the present application is optimized with a single methanol solvent (LD50 = 5.6 g / kg), which simplifies the operation, reduces the cost, and improves the stability while ensuring the precipitation efficiency, breaking through the technical bottleneck of the mixed solvent system.

[0087] Comparative Example 3

[0088] Compared with Example 1, in the method of Comparative Example 3, 3,5-diamino-6-(2-methoxyphenyl)-1,2,4-triazine (Document 2) and diphenhydramine (Document 3) are used to replace LEV-D6 and OXC-D4.

[0089] However, these methods may not be able to completely eliminate the matrix effect. Comparative Example 3 (Document 4) uses three isotope internal standards to quantify three substances, LTG, LEV, and MHD, respectively, improving the accuracy, but at a higher cost. This study only uses two isotope internal standards, OXC-D4 and LEV-D6, to achieve the accurate quantification of four substances, effectively reducing the cost and having more promotional value.

[0090] Comparative Example 4

[0091] Compared with Example 1, when Comparative Example 4 (i.e., Document 2) uses an ultra-high performance liquid chromatography system (UHPLC) combined with a Waters BEH C18 chromatographic column with a particle size of 1.7 μm (50 mm × 2.1 mm), although rapid analysis in 6 minutes is achieved, its technical solution has systematic defects: First, the pressure of the UHPLC system is significantly higher than that of a conventional HPLC system (the column pressure in Example 1 ≤ 15 MPa), significantly increasing the risk of equipment loss; Second, the procurement cost of small particle size chromatographic columns is usually higher than that of conventional HPLC columns, and their service life is shortened under high pressure, increasing equipment maintenance costs; In addition, this solution requires a 0.22 μm filter membrane for sample filtration (Example 1 does not require filter membrane filtration), increasing sample processing costs and being cumbersome to operate.

[0092] In this application, a chromatographic column with a packing particle size of 3.0 μm is selected for the first time. While maintaining the analysis efficiency of 6 minutes, it effectively realizes the simultaneous detection of lamotrigine, levetiracetam, oxcarbazepine and their active metabolites by a conventional HPLC system. By reducing the system pressure dependence and consumable specification requirements, the comprehensive hardware cost is reduced, providing a solution with both high efficiency and economy for blood drug concentration monitoring.

[0093] Comparative Example 5

[0094] Compared with Example 1, the lower limit of quantification of OXC in Comparative Example 5 (Document 2) is 0.2 μg·mL -1 , however, through in-depth study of 145 clinical samples in this application, the OXC concentration in about 44.9% of the clinical samples is lower than this lower limit, indicating that the lower limit of quantification of 0.2 μg·mL -1 cannot meet the clinical needs.

[0095] In this application, the lower limit of quantification of OXC is breakthroughly reduced to 0.04 μg·mL -1 , the sensitivity is increased by 5 times, and the clinical sample coverage rate is increased to 97.9%, effectively solving the problem of inaccurate monitoring of low-concentration OXC in the prior art.

[0096] Comparative Example 6

[0097] Comparative Example 6 (Document 5) attempts to reduce the lower limit of quantification of OXC to 0.01 μg·mL -1 , however, its upper limit of 1.0 μg·mL -1 cannot meet the detection requirements of some samples, and the OXC concentration in about 10.2% of the clinical samples in this application exceeds this upper limit.

[0098] In this application, the upper limit of quantification of OXC is maintained at 5 μg·mL -1, the upper detection limit is extended by 5 times, enabling accurate quantification within a wide detection range and improving clinical applicability.

[0099] Reference 1: Ma Yinghua, Zhao Yile, Jiang Xijuan, et al. Simultaneous determination of the drug concentrations of levetiracetam, the active metabolite of oxcarbazepine, and lamotrigine in the plasma of children with epilepsy by isotope dilution-HPLC-ESI-MS / MS [J]. Chinese Journal of Hospital Pharmacy, 2022, 42(16): 1647-1652.

[0100] Reference 2: JULIEN D, NATALIA D, SéPHORA B, et al. Simultaneous determination of four antiepileptic drugs in human plasma samples using an ultra-high-performance liquid chromatography tandem mass spectrometry method and its application in therapeutic drug monitoring [J]. BIOMEDICAL CHROMATOGRAPHY, 2016, 30(12).

[0101] Reference 3: LEI Y, TINGTING W, MEIYUN S, et al. Simultaneous determination of ten antiepileptic drugs in human plasma by liquid chromatography and tandem mass spectrometry with positive / negative ion-switching electrospray ionization and its application in therapeutic drug monitoring [J]. JOURNAL OF SEPARATION SCIENCE, 2016, 39(5): 964-972.

[0102] Reference 4: Palte MJ, Basu SS, Dahlin JL, et al. Development and Validation of an Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for the Concurrent Measurement of Gabapentin, Lamotrigine, Levetiracetam, Monohydroxy Derivative of Oxcarbazepine, and Zonisamide Concentrations in Serum in a Clinical Setting[J]. Ther Drug Monit, 2018, 40(4): 469-476.

[0103] Reference 5: Yang Juanjuan, Wang Chenxiang, Zhou Ziye, et al. Determination of the Concentrations of Oxcarbazepine and Its Active Metabolite 10-Hydroxycarbamazepine in Human Serum by LC-MS / MS and Its Clinical Application[J]. Chin J Mod Appl Pharm, 2019, 36(22): 2823-2827.

[0104] Through continuous exploration, the lower limit of quantification of OXC in this application was finally optimized to 0.04 μg·mL -1 , and the upper limit was maintained at 5 μg·mL -1 , significantly expanding the linear range of OXC detection. Analysis of 145 clinical samples showed that the setting of the linear range of OXC in this study was more reasonable.

[0105] This application simplifies the pretreatment process, selects an organic solvent with a single component, and simplifies the operation; moreover, aiming at the defect of high column pressure caused by the dependence on small particle size chromatographic columns (<3 μm) in the prior art, this application first selects a C18 chromatographic column with a packing particle size of 3.0 μm, and successfully realizes the high-efficiency separation of LTG, LEV, OXC and its active metabolite MHD under the condition of maintaining the column pressure ≤ 15 MPa, and the detection efficiency is comparable to that of the conventional small particle size chromatographic column technology; only two isotope internal standards, OXC-D4 and LEV-D6, are used to complete the accurate quantification of the four substances, effectively reducing the cost; expanding the detection linear range of OXC, successfully overcoming the lower limit of quantification of oxcarbazepine (as low as 0.04 μg·mL -1 ) and the upper limit of quantification of LEV (as high as 50 μg·mL -1)The technical contradiction with a concentration span of three orders of magnitude solves the problems of insufficient sensitivity or limited detection range in traditional methods. Finally, while reducing the hardware requirements and reagent costs, this solution has both high sensitivity (the lower limit of quantification of oxcarbazepine is significantly reduced compared with the prior art) and a wide linear range, providing an efficient, economical and accurate solution for clinical blood drug concentration monitoring.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application rather than to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A method for simultaneously determining the serum concentrations of antiepileptic drugs and their active metabolites based on LC-MS / MS method, characterized in that, It includes the following steps: Adding the serum sample to be tested containing antiepileptic drugs and their active metabolites into a mixed internal standard solution containing isotope internal standards for one-step protein precipitation, diluting with a mixed solution of methanol and water, and detecting and analyzing through an LC-MS / MS system; Among them, the mixed internal standard solution containing isotope internal standards includes a methanol solution of oxcarbazepine-d4 and levetiracetam-d6.

2. The method according to claim 1, wherein The antiepileptic drugs include at least one of lamotrigine, levetiracetam, and oxcarbazepine; The active metabolites of the antiepileptic drug include 10-hydroxy-10,11-dihydro-5H-dibenzo[b,f]azepine -5-carboxamide.

3. The method according to claim 2, wherein The lower limit of quantification of oxcarbazepine is 0.04 μg·mL -1 , and the upper limit of quantification of oxcarbazepine is 5 μg·mL -1 .

4. The method according to claim 2, wherein The linear range of the mass concentration of lamotrigine is 0.2 - 25 μg·mL -1 ; The linear range of the mass concentration of levetiracetam is 0.39 - 50 μg·mL -1 ; The linear range of the mass concentration of oxcarbazepine is 0.04 - 5 μg·mL -1 ; The linear range of the mass concentration of 10-hydroxy-10,11-dihydro-5H-dibenzo[b,f]azepine -5-carboxamide is 0.31 - 40 μg·mL -1 .

5. The method according to claim 1, wherein The chromatographic conditions in the LC-MS / MS method include: A C18 chromatographic column with a packing particle size of 3.0 μm, column temperature of 35 - 45 °C; flow rate of 0.6 - 1.0 mL·min -1 ; injection volume of 0.5 - 2.0 μL; injector temperature of 2 - 8 °C; Gradient elution is adopted. Mobile phase A is an aqueous formic acid solution containing ammonium acetate, and mobile phase B is methanol; The steps of gradient elution are: 0 - 0.5 min, 60% B; 0.5 - 4 min, 60% → 98% B; 4 - 5 min, 98% B; 5 - 5.1 min, 98% → 60% B; 5.1 - 6 min, 60% B.

6. The method according to claim 5, wherein The mass concentration of formic acid in the aqueous formic acid solution is 0.1%; the mass concentration of ammonium acetate is 5 mmol·L -1 .

7. The method according to claim 5, wherein The C18 chromatographic column includes a Phenomenex C18 chromatographic column.

8. The method according to claim 1, wherein In the mixed solution of methanol and water, the volume ratio of methanol to water is 1:

1.

9. The method according to claim 1, wherein The mass spectrometric conditions in the LC-MS / MS method include: An electrospray ionization source is adopted for positive ion multiple reaction monitoring mode scanning; the curtain gas pressure is 20 psi; the ion source spray voltage is 5500 V; the ion source temperature is 650 °C; the nebulizing gas pressure is 60 psi; the auxiliary heater pressure is 60 psi; the ion pairs for quantitative analysis are respectively: Lamotrigine: m / z 256.0 → 211.0; Levetiracetam: m / z 171.0 → 126.2; Oxcarbazepine: m / z 253.0 → 180.2; 10-Hydroxy-10,11-dihydro-5H-dibenzo[b,f]azepine -5-carboxamide: m / z 255.0→194.1; Levetiracetam-d6: m / z 177.0 → 132.2; Oxcarbazepine-d4: m / z 257.0 → 184.2; Lamotrigine, Levetiracetam, Oxcarbazepine, 10-Hydroxy-10,11-dihydro-5H-dibenzo[b,f]azepine -5-carboxamide, Levetiracetam-d6, Oxcarbazepine-d4 declustering voltages were 80.0, 47.0, 105.0, 70.0, 40.8, 105.0 V respectively, and collision voltages were 37.90, 42.00, 38.76, 50.00, 22.01, 38.76 V respectively.

10. The method according to claim 1, characterized in that The mass concentrations of oxcarbazepine-d4 and levetiracetam-d6 solutions in the mixed internal standard solution are both 5 to 15 ng·mL -1 .

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