Method for detecting antiepileptic drugs in serum based on polarity conversion UPLC-MS / MS

Abstract

The invention relates to a method for detecting antiepileptic drugs in serum based on polarity conversion UPLC-MS / MS. The antiepileptic drugs (AEDs) are respectively levetiracetam, pregabalin, gabapentin, lamotrigine, phenobarbital, oxcarbazepine, phenytoin sodium, carbamazepine, clonazepam, diazepam and sodium valproate. Simplifying sample pretreatment by adopting a methanol (containing formic acid) one-step extraction method; in the chromatographic analysis, C18 is used as a chromatographic column and a pre-column, a 5% methanol aqueous solution (containing formic acid)-95% methanol aqueous solution (containing formic acid) is used as a mobile phase, gradient elution is carried out, and the mass spectrum adopts an MRM mode. The method has the advantages of being high in sensitivity, good in precision and accuracy, easy to operate, free of matrix interference and the like, can be used for measuring serum AEDs of different species, and provides technical support for the prevention and treatment mechanism of epilepsy drugs, clinical AEDs blood concentration monitoring, individualized drug delivery scheme formulation and pharmacokinetic research.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis and pharmaceutical detection, and specifically relates to a method for detecting anti-epileptic drugs in serum based on polarity conversion UPLC-MS / MS, which is suitable for clinical therapeutic drug monitoring and individualized medication guidance. Background Art

[0002] Epilepsy is a common and complex neurological disorder characterized by recurrent epileptic seizures caused by highly synchronized neuronal discharges. Currently, antiepileptic drugs (AEDs) remain the preferred treatment for epilepsy. Because epilepsy is characterized by recurrent seizures, patients with epilepsy require long-term AED use to control seizures, which can lead to drug accumulation in the body and potentially toxic side effects. Some AEDs also have significant toxicity, a narrow therapeutic window, and significant individual variability. Clinically, coadministration of AEDs or concomitant medications or foods can alter AED blood levels, increasing the risk of adverse reactions. Therefore, to ensure safe and effective medication use, clinically monitoring the blood concentrations of one or more AEDs (therapeutic drug monitoring, TDM) is of significant clinical significance.

[0003] Currently, the main TDM detection methods for AEDs include immunoassays, high-performance liquid chromatography (HPLC), and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). Immunoassays are characterized by short cycle times, mature technology, high commercialization, and simple operation. However, they are susceptible to nonspecific interference from the sample matrix, which can affect the accuracy of the measurement results. In addition, there are no commercial immunoassay kits for most AEDs. HPLC is one of the most common analytical methods for measuring blood drug concentrations in the laboratory. It can detect multiple substances simultaneously and can eliminate the interference of endogenous substances to a certain extent. However, this method has low sensitivity, and some drugs even require tedious derivatization for detection. At the same time, as the number of analytes increases, the drawbacks of this method's strict requirements for analyte separation become increasingly apparent. UPLC-MS / MS combines the advantages of high-efficiency separation and sensitive mass spectrometry detection and has become the preferred method for the simultaneous detection of multiple trace or micro-components in complex matrices.

[0004] Currently, a wide variety of AEDs are used clinically. Using different UPLC-MS / MS methods to detect different AEDs would inevitably place a tedious and heavy workload on TDM practitioners. Furthermore, combination therapy is often necessary in clinical practice, and the pharmacodynamic and pharmacokinetic interactions caused by combination therapy increase the difficulty of assessing the relationship between blood drug concentrations and efficacy. Therefore, it is crucial to establish a UPLC-MS / MS method suitable for the determination of multiple AEDs in clinical practice. Currently, several AED detection methods have been reported in the literature, but the types detected differ from the AEDs used in our hospital and cannot fully meet clinical testing needs. Summary of the Invention

[0005] In response to the problems existing in the above-mentioned prior art, the present invention provides a method for detecting antiepileptic drugs in serum based on polarity conversion UPLC-MS / MS. During use, the sample is extracted using simple liquid phase extraction to simultaneously determine the concentrations of 11 antiepileptic drugs in serum, including levetiracetam, pregabalin, gabapentin, lamotrigine, phenobarbital, oxcarbazepine, phenytoin sodium, carbamazepine, clonazepam, diazepam and sodium valproate. The method can be used for both clinical serum determination of epilepsy patients and for basic scientific research to determine the blood concentrations of such drugs in animals (rats, mice, beagles, etc.). This method provides technical support for the drug prevention and treatment mechanisms of related diseases, the monitoring of antiepileptic drug blood concentrations in clinical practice, the formulation of individualized dosing regimens, and the study of drug metabolism and pharmacokinetic studies.

[0006] To achieve the above object, the present invention adopts a method for detecting anti-epileptic drugs in serum based on polarity switching UPLC-MS / MS, wherein the anti-epileptic drugs are levetiracetam, pregabalin, gabapentin, lamotrigine, phenobarbital, oxcarbazepine, phenytoin sodium, carbamazepine, clonazepam, diazepam and sodium valproate;

[0007] A mixed standard reference solution was prepared with anti-epileptic drug reference substances, and a standard curve was prepared for quantification. The above anti-epileptic drugs in the pretreated serum were detected by UPLC-MS / MS method;

[0008] The following steps are involved:

[0009] (1) Setting chromatography-mass spectrometry conditions

[0010] Ultra-high performance liquid chromatography (UHPLC) conditions: liquid chromatography column and pre-column, mobile phases including solution A and solution B, solution A being a 1% to 10% methanol aqueous solution containing formic acid, and solution B being an 80% to 100% methanol aqueous solution containing formic acid;

[0011] Gradient elution was performed according to the following program: 0-0.5 min, 95% A; 0.5-2.5 min, 95%→65% A; 2.5-3.5 min, 65% A; 3.5-4.0 min, 65%→60% A; 4.0-6.0 min, 60% A; 6.0-6.5 min, 60%→40% A; 6.5-8 min, 40% A; 8-8.5 min, 40%→5% A; 8.5-9.0 min, 5% A; 9.0-9.5 min, 5%→95% A; 9.5-10.0 min, 95% A;

[0012] Mass spectrometry conditions: electrospray ionization source; multiple reaction monitoring mode; ionization mode: positive and negative ion switching scan; capillary voltage: +3.5 kV / -2.8 kV; desolvation temperature: 350 °C; nitrogen flow rate: 650 L / h;

[0013] (2) The serum pretreatment process is as follows: add methanol containing formic acid to the serum sample, vortex and sonicate, let it stand and then centrifuge to obtain the supernatant for injection.

[0014] In a preferred embodiment, the serum is any one of rat serum, mouse serum and human serum.

[0015] In a preferred embodiment, the liquid chromatography column has a length of 10 to 100 mm and an inner diameter of 2.1 mm; the pre-column has a length of 2 to 10 mm and an inner diameter of 2.1 mm; the liquid chromatography column and pre-column are both filled with octadecyl bonded silica gel, and the particle size of the octadecyl bonded silica gel is 1.7 to 1.9 μm; and the column temperature of the liquid chromatography column and pre-column is 20 to 60°C. During chromatographic analysis, levetiracetam, pregabalin, gabapentin, lamotrigine, phenobarbital, oxcarbazepine, phenytoin sodium, carbamazepine, clonazepam, diazepam, and sodium valproate have significant differences in polarity and are difficult to separate. The chromatographic column type, whether a pre-column is used, the pre-column type, and different column temperatures have different effects on the separation and analysis of these 11 anti-epileptic drugs. The present invention selects a combination of a chromatographic column and a pre-column, both of which are ACQUITY UPLC BEH C18 columns, to obtain good peak shape and resolution for the aforementioned compounds. The chromatographic column has a length of 50 mm, an inner diameter of 2.1 mm, and a filler particle size of 1.7 μm; the pre-column has a length of 5 mm, an inner diameter of 2.1 mm, and a filler particle size of 1.7 μm; and the column temperature is maintained at 40°C. Under the same chromatographic conditions, using other similar chromatographic columns, such as an Agilent Zorbax Eclipse Plus C18 column (100 mm × 2.1 mm, 1.8 μm), the resulting chromatograms exhibit low peak response values, poor peak shape, tailing, poor resolution, and other issues, resulting in poor separation results.

[0016] In a preferred embodiment, the solution A is a 5% methanol aqueous solution containing 0.05% formic acid, and the solution B is a 95% methanol aqueous solution containing 0.05% formic acid. The flow rate of the mobile phase is 0.2-0.6 mL / min, and the injection volume is 1-5 μL. During the chromatographic analysis process, the type and flow rate of the mobile phase have a significant effect on the chromatographic separation and chromatographic peak shape of the target compound. The liquid chromatography mobile phase of the present invention is: A is a 5% methanol aqueous solution (containing 0.05% formic acid), and B is a 95% methanol aqueous solution (containing 0.05% formic acid);

[0017] Gradient elution program: 0-0.5 min, 95% A; 0.5-2.5 min, 95%→65% A; 2.5-3.5 min, 65% A; 3.5-4.0 min, 65%→60% A; 4.0-6.0 min, 60% A; 6.0-6.5 min, 60%→40% A; 6.5-8 min, 40% A; 8-8.5 min, 40%→5% A; 8.5-9.0 min, 5% A; 9.0-9.5 min, 5%→95% A; 9.5-10.0 min, 95% A. Under these mobile phase conditions, the 11 antiepileptic drugs were well separated and had good peak response.

[0018] In the present invention, the serum samples used for the verification of the above 11 antiepileptic drugs in serum are blank serum, simulated serum samples (blank serum + mixed standard control solution), and real serum samples. The preparation method of the simulated serum samples is as follows:

[0019] Prepare reference substance stock solutions by accurately weighing a certain amount of levetiracetam, pregabalin, gabapentin, lamotrigine, phenobarbital, oxcarbazepine, phenytoin sodium, carbamazepine, clonazepam, diazepam, and sodium valproate reference substances, dissolving them in methanol and adjusting the volume.

[0020] Then accurately measure a certain volume of the stock solution of the above-mentioned reference substances, vortex mix, and prepare mixed standard reference solutions with mass concentrations of 500.0, 250.0, 250.0, 250.0, 650.0, 500.0, 650.0, 250.0, 1.0, 10.0, and 2000.0 μg / mL, respectively. Then dilute with methanol to form a series of mixed standard reference solutions;

[0021] Accurately measure 10 μL of each of the above series of mixed standard control solutions, add 90 μL of blank serum, and mix evenly to obtain standard serum samples of a series of concentrations;

[0022] The same method was used to prepare three quality control serum samples with high, medium and low concentrations, and 6 replicates were prepared for each concentration.

[0023] In a preferred embodiment, the serum pretreatment process in step (2) is as follows: 50 to 150 μL of serum sample is taken into an EP tube, 100 to 500 μL of methanol containing 0.01 to 0.2% formic acid is added, vortexed for 20 to 60 seconds, ultrasonicated for 3 to 10 minutes, allowed to stand for 2 to 10 minutes, centrifuged at 2 to 8°C and 5000 to 15000 rpm for 3 to 10 minutes, and the supernatant is taken for sampling and analysis.

[0024] In the present invention, different extraction agents significantly affect the extraction efficiency of antiepileptic drugs during serum sample pretreatment, necessitating the selection of specific extraction agents to improve the extraction efficiency of the analytes. Experiments have shown that the chromatographic peak responses for 11 antiepileptic drugs are optimal when methanol (containing 0.1% formic acid) is the extraction agent.

[0025] Mass spectrometry detection conditions have a significant impact on the peak response of the chromatographic peak and the sensitivity of the detection method. In a preferred embodiment, the parameters and ionization mode of the multiple reaction monitoring mode of the antiepileptic drug are as follows:

[0026] Levetiracetam, 171.14→126.10, ESI + ; Pregabalin, 160.16→142.50, ESI + ; Gabapentin, 172.16→137.00, ESI + ; Lamotrigine, 255.91→108.94, ESI + ; Phenobarbital, 231.18→84.92, ESI - ; Oxcarbazepine, 252.99→180.04, ESI + ; Phenytoin sodium, 251.19→101.92, ESI - ; Carbamazepine, 237.00→179.00, ESI + ; Clonazepam, 316.01→270.02, ESI + ; Diazepam, 285.04→154.00, ESI + ; Sodium valproate, 143.08→143.08, ESI - .

[0027] The technical solution of the present invention has the following beneficial effects:

[0028] (1) The present invention uses UPLC-MS / MS to simultaneously determine 11 anti-epileptic drugs in serum. Methanol (containing 0.1% formic acid) is used as an extractant to extract serum samples, and better chromatographic peak response values of the 11 anti-epileptic drugs can be obtained simultaneously.

[0029] (2) The present invention uses ACQUITY UPLC BEH C18 (2.1 mm × 50 mm, 1.7 μm) as the chromatographic column, ACQUITY UPLC BEH C 18 A column (2.1×5mm, 1.7μm) was used as a pre-column, and a gradient of 5% methanol in water (containing 0.05% formic acid) to 95% methanol in water (containing 0.05% formic acid) was used as the mobile phase. Eleven antiepileptic drugs in serum were separated, and each component had good peak shape and resolution.

[0030] (3) The present invention has the advantages of high sensitivity, good precision and accuracy, simple operation, and no matrix interference. It can achieve rapid and accurate analysis of serum samples and can be better applied to the clinical detection of blood drug concentrations in patients taking AEDs.

[0031] (4) The present invention is applicable to the detection of human, rat, and mouse serum. It can be used for the detection of rats and mice with different phenotypes in basic scientific research, and can also be used for the determination of 11 anti-epileptic drugs in the serum of patients with different diseases in clinical practice. The present invention can be used for both the determination of serum concentrations of clinical epilepsy patients and the determination of blood drug concentrations of different animals (such as rats, mice, beagles, etc.) in basic scientific research, thereby providing technical support for the research on the prevention and treatment mechanism of related epilepsy drugs, the monitoring of blood drug concentrations of anti-epileptic drugs in clinical practice, the formulation of individualized dosing regimens, and the study of drug metabolism and kinetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 The MRM chromatograms of six AEDs are shown in Figure 1-6, respectively: levetiracetam, pregabalin, gabapentin, lamotrigine, phenobarbital, and oxcarbazepine; A is blank serum, B is simulated serum sample (blank serum + mixed standard control solution), and C is real serum sample.

[0034] Figure 2 These are the MRM chromatograms of five antiepileptic drugs; 7 to 11 in the figure are phenytoin sodium, carbamazepine, clonazepam, diazepam, and sodium valproate, respectively; A is blank serum, B is a simulated serum sample (blank serum + mixed standard control solution), and C is a real serum sample. DETAILED DESCRIPTION

[0035] In order to make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present application are described in detail below through specific embodiments. It should be understood that the embodiments of the present application and the specific features in the embodiments are detailed descriptions of the technical solutions of the present application, rather than limitations on the technical solutions of the present application. Unless there is a conflict, the embodiments of the present application and the technical features in the embodiments can be combined with each other.

[0036] The instruments used in the embodiments of the present invention are:

[0037] Xevo TM TQ-S micro ultra-high performance liquid chromatography-triple quadrupole mass spectrometer (Waters, USA); MX105DU electronic balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); Legend Micro 21R high-speed refrigerated centrifuge (Thermo Fisher Scientific, USA); SB-5200DT ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); XW-80A micro vortex mixer (Shanghai Huxi Analytical Instrument Factory Co., Ltd.).

[0038] Drugs and reagents:

[0039] Gabapentin (batch number L1305026, purity 98%) was purchased from Shanghai Aladdin Technology Co., Ltd.; sodium valproate (batch number DL030141, purity 98%) was purchased from Shanghai Anaiji Co., Ltd.; phenobarbital (batch number 171222-201206, purity 99.9%), clonazepam (batch number 171227-201404, purity 99.7%), and diazepam (batch number 171225-201502, purity 99.8%) were obtained from the China Food and Drug Administration; levetiracetam (batch number C1021-201404, purity 99.7%) was obtained from the China Food and Drug Administration. 0448, purity 97%), pregabalin (batch number C10286539, purity 98%), lamotrigine (batch number C14769829, purity 98%), oxcarbazepine (batch number C11956913, purity 98%), phenytoin sodium (batch number C11997231, purity 98%), carbamazepine (batch number C11880371, purity 99%), and formic acid (batch number C15916452, chromatographic grade) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; chromatographic grade methanol and acetonitrile were purchased from Sigma.

[0040] Blank serum and AEDs serum samples:

[0041] The relevant experiments have been approved by the Ethics Committee of Suining County People's Hospital (LL-2022-43). Blank serum samples were obtained from healthy volunteers with no recent medication history. Real serum samples were obtained from outpatient and inpatient patients who were taking AEDs long-term or had taken AEDs the day before blood collection. Serum was separated from whole blood and stored at -80°C.

[0042] Example 1

[0043] A method for detecting anti-epileptic drugs in serum based on polarity switching UPLC-MS / MS, wherein the anti-epileptic drugs include levetiracetam, pregabalin, gabapentin, lamotrigine, phenobarbital, oxcarbazepine, phenytoin sodium, carbamazepine, clonazepam, diazepam, and sodium valproate;

[0044] The specific steps include:

[0045] 1) Preparation of standard control solution

[0046] Prepare standard stock solutions of a certain concentration by accurately weighing appropriate amounts of levetiracetam, pregabalin, gabapentin, lamotrigine, phenobarbital, oxcarbazepine, phenytoin sodium, carbamazepine, clonazepam, diazepam, and sodium valproate reference substances, dissolving them in methanol and adjusting the volume.

[0047] Then accurately measure a certain volume of the standard stock solution of the above-mentioned reference substances, vortex mix, and prepare mixed standard reference solutions with mass concentrations of 500.0, 250.0, 250.0, 250.0, 650.0, 500.0, 650.0, 250.0, 1.0, 10.0, and 2000.0 μg / mL, respectively. Then dilute them with methanol to form a series of mixed standard reference solutions for use.

[0048] 2) Set chromatographic and mass spectrometry conditions

[0049] 2-1) Chromatographic conditions

[0050] Column: Waters ACQUITY UPLC BEH C 18 Column (2.1×50 mm, 1.7 μm); Pre-column: Waters ACQUITY UPLC BEH C 18 Column (2.1 × 5 mm, 1.7 μm); Mobile phase: A: 5% methanol in water (containing 0.05% formic acid), B: 95% methanol in water (containing 0.05% formic acid);

[0051] Gradient elution program: 0-0.5 min, 95% A; 0.5-2.5 min, 95%→65% A; 2.5-3.5 min, 65% A; 3.5-4.0 min, 65%→60% A; 4.0-6.0 min, 60% A; 6.0-6.5 min, 60%→40% A; 6.5-8 min, 40% A; 8-8.5 min, 40%→5% A; 8.5-9.0 min, 5% A; 9.0-9.5 min, 5%→95% A; 9.5-10.0 min, 95% A; flow rate: 0.3 mL min-1 ; Column temperature: 40℃; Injection volume: 2μL.

[0052] 2-2) Mass spectrometry conditions

[0053] Ion source: electrospray ionization; Scan mode: Multiple reaction monitoring (MRM) mode, simultaneous positive and negative ion scanning; Capillary voltage: +3.5 kV / -2.8 kV; Desolvation temperature: 350°C; Nitrogen flow rate: 650 L / h. The mass spectrometry parameters for the 11 AEDs are shown in Table 1.

[0054] Table 1 Mass spectrometry parameters of 11 AEDs

[0055]

[0056]

[0057] 3) Processing of serum samples

[0058] 100 μL of serum sample was placed in an EP tube, 300 μL of methanol (containing 0.1% formic acid) was added, vortexed for 30 s, sonicated for 5 min, allowed to stand for 3 min, centrifuged at 4°C and 10,000 rpm for 5 min, and the supernatant was taken for sampling and analysis.

[0059] Verification of the method of the present invention:

[0060] (1) Specificity

[0061] Six blank serum samples, simulated serum samples (blank serum + mixed standard control solution), and real serum samples from different sources were pretreated and analyzed according to the method in step 3). Endogenous substances in blank serum did not interfere with the peak positions of 11 AEDs. The results are shown in the attached Figure 1 , Attachment Figure 2 , indicating that the method has good specificity.

[0062] (2) Linear range

[0063] To 90 μL of blank serum, 10 μL of a series of seven mixed standard control solutions of varying concentrations were added and vortexed to prepare standard serum samples. These samples were then processed and analyzed according to the method described in step 3). Linear regression was performed using the least-squares method, with the analyte concentration as the independent variable (x) and the peak area as the dependent variable (y). The regression curve equation and correlation coefficient (r) were calculated. The linear equation is shown in Table 2. The results showed that the 11 AEDs had good linear relationships within their respective linear ranges, with correlation coefficients no less than 0.9965. The linear range for each AED covered the effective plasma concentration range of serum samples treated with this method.

[0064] Table 2 Linear equations and correlation coefficients of 11 AEDs in serum

[0065]

[0066]

[0067] Note: a The concentration unit is ng / mL.

[0068] (3) Accuracy and precision

[0069] Take 90 μL of blank serum sample and add 10 μL of mixed standard solution of different concentrations to prepare three spiked samples (quality control samples, QC samples) with high, medium and low concentrations respectively. Prepare 6 replicates for each concentration and analyze them according to the method in step 3) above to investigate the intra-batch precision and accuracy of the analytical method. Prepare and measure three qualified analytical batches on different days in succession to investigate the inter-batch precision and accuracy of the method.

[0070] Precision was expressed as relative standard deviation (RSD), and accuracy was expressed as relative recovery (measured concentration / theoretical concentration × 100%). The intra-assay and inter-assay accuracies for the 11 AEDs ranged from 85.0% to 105.2% and 92.2% to 106.2%, respectively. The intra-assay RSD was ≤8.7%, and the inter-assay RSD was ≤9.9%. The results are shown in Table 3. These results demonstrate that the accuracy and precision of this method meet the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) requirements for quantitative analysis of biological samples.

[0071] Table 3 Accuracy and precision of 11 AEDs in serum

[0072]

[0073]

[0074] Note: a The concentration unit is ng / mL.

[0075] (4) Matrix effect and extraction recovery

[0076] Take 90 μL of blank serum from 6 batches of different sources, add 10 μL of mixed standard control solution of different concentrations, and prepare three QC samples with high, medium and low concentrations respectively. Prepare 6 replicates of each mass concentration in parallel. After processing according to the method in step 3) above, inject and analyze, record the chromatogram and the peak area (Aa) of each analyte.

[0077] Take 90 μL of blank serum from 6 batches of different sources and process them according to the method in step 3) above to obtain the supernatant. Take 90 μL of the supernatant and add 10 μL of mixed standard control solution of different concentrations to prepare three QC samples with high, medium and low concentrations. Prepare 6 replicates of each mass concentration in parallel, inject and analyze, and record the chromatogram and peak area (A) of each analyte. b ).

[0078] In addition, ultrapure water was used instead of blank serum to prepare three standard control solutions with high, medium and low concentrations. The solutions were injected and analyzed, and the chromatograms and peak areas (Ac) of each analyte were recorded.

[0079] Matrix effect (ME) = A b / A c × 100%, extraction recovery (ER) = A a / A b × 100%. The extraction recoveries of all analytes were ≥75.3%, with RSDs ≤9.9%. The matrix effects ranged from 93.1% to 112.9%, with RSDs ≤9.5%. The results are shown in Table 4, indicating that both the extraction recovery and matrix effects of this method meet the ICH requirements for quantitative analysis of biological samples.

[0080] Table 4 Matrix effects and extraction recovery results of 11 AEDs in serum (n=6, )

[0081]

[0082]

[0083] Note: a The concentration unit is ng / mL.

[0084] (5) Stability

[0085] Short-term room temperature stability: Six high- and low-concentration QC samples were stored at 25°C for 24 hours, with samples collected and analyzed at 0, 2, 4, 8, 12, and 24 hours. The measured values for the 11 AEDs were within ±15% of the labeled concentrations. The results are shown in Table 5, demonstrating that the serum samples for the 11 AEDs were stable within 24 hours of storage at room temperature.

[0086] Long-term frozen storage stability: Six aliquots of high- and low-concentration QC samples were stored at -20°C for 14 days. Samples were collected and analyzed at 0, 1, 3, 6, 10, and 14 days. The results are shown in Table 5. The deviations between the measured values and the labeled concentrations for all 11 AEDs were within ±15%, indicating that the serum samples for all 11 AEDs are stable when stored at -20°C for 14 days.

[0087] Freeze-thaw stability: Six aliquots of high- and low-concentration QC samples were frozen and thawed three times at -80°C, with measurements performed once after each freeze-thaw cycle. The results are shown in Table 5. After the second freeze-thaw cycle, the measurement results of the low-concentration QC samples of levetiracetam, oxcarbazepine, and carbamazepine exhibited deviations exceeding ±15%. However, after three freeze-thaw cycles, the measurement results of the QC samples of the other eight AEDs remained within ±15%. This indicates that the results of serum samples containing levetiracetam, oxcarbazepine, and carbamazepine are stable after a single freeze-thaw cycle, while the results of the other eight AEDs are stable after three freeze-thaw cycles.

[0088] Table 5 Stability results of 11 AEDs in serum under different conditions (n=6)

[0089]

[0090] Note: a The concentration unit is ng / mL.

[0091] Example 2

[0092] Clinical sample testing:

[0093] The detection method established in the present invention was used to analyze the blood drug concentration of patients taking AEDs. If the sample concentration was lower than the lower limit of quantification of the standard curve of this method, it was not detected. If the sample concentration was higher than the upper limit of quantification of the standard curve of this method, blank serum was used for dilution and then measured. The blood drug concentration was calculated using the accompanying standard curve on the day the sample was measured. The results are shown in Table 6.

[0094] Referring to the effective blood drug concentration range recommended in previous reports, the test results showed that the concentration of epilepsy drugs in most patients did not reach the effective concentration. Among them, the blood concentrations of phenytoin sodium, diazepam or sodium valproate in 3 patients were higher than the effective concentration range. In addition, in the serum of some patients, not only the AEDs prescribed by the doctor were detected, but also other AEDs not prescribed by the doctor.

[0095] Table 6 Results of 11 AEDs determination in clinical samples

[0096]

[0097]

[0098] Note: a The concentration unit is ng / mL.

[0099] Although UPLC-MS / MS methods exist for the determination of multiple AEDs in serum or plasma, these methods do not fully cover the AEDs in our clinical samples, and the linear ranges of these methods do not fully cover the effective clinical blood concentration ranges of the selected AEDs. Therefore, the present invention aims to develop a UPLC-MS / MS method suitable for the 11 AEDs clinically used in our hospital and to provide a reference for other researchers. Internal standard methods are commonly used for biological sample analysis. However, due to the large chemical structure differences of the analytes involved in this invention, a single internal standard cannot be suitable for all analytes. Furthermore, considering the cost of clinical testing, the present invention chose an external standard method for quantification.

[0100] Furthermore, those skilled in the art will appreciate that, although some embodiments described herein include certain features included in other embodiments but not other features, combinations of features from different embodiments are also intended to fall within the scope of protection of the present invention and form different embodiments. For example, in the above embodiments, those skilled in the art will be able to use them in combination based on the known technical solutions and the technical problems to be solved by this application.

Claims

1. A method for detecting anti-epileptic drugs in serum based on polarity switching UPLC-MS / MS, characterized in that: The anti-epileptic drugs are levetiracetam, pregabalin, gabapentin, lamotrigine, phenobarbital, oxcarbazepine, phenytoin sodium, carbamazepine, clonazepam, diazepam and sodium valproate; A mixed standard reference solution was prepared with anti-epileptic drug reference substances, and a standard curve was prepared for quantification. The above anti-epileptic drugs in the pretreated serum were detected by UPLC-MS / MS method; The following steps are involved: (1) Setting chromatography-mass spectrometry conditions Ultra-high performance liquid chromatography (UHPLC) conditions: liquid chromatography column and pre-column, mobile phases including solution A and solution B, solution A being a 1% to 10% methanol aqueous solution containing formic acid, and solution B being an 80% to 100% methanol aqueous solution containing formic acid; Gradient elution was performed according to the following program: 0-0.5 min, 95% A; 0.5-2.5 min, 95%→65% A; 2.5-3.5 min, 65% A; 3.5-4.0 min, 65%→60% A; 4.0-6.0 min, 60% A; 6.0-6.5 min, 60%→40% A; 6.5-8 min, 40% A; 8-8.5 min, 40%→5% A; 8.5-9.0 min, 5% A; 9.0-9.5 min, 5%→95% A; 9.5-10.0 min, 95% A; Mass spectrometry conditions: electrospray ionization source; multiple reaction monitoring mode; ionization mode: positive and negative ion switching scan; capillary voltage: +3.5 kV / -2.8 kV; desolvation temperature: 350 °C; nitrogen flow rate: 650 L / h; (2) The serum pretreatment process is as follows: add methanol containing formic acid to the serum sample, vortex and sonicate, let it stand and then centrifuge to obtain the supernatant for injection.

2. The method for detecting antiepileptic drugs in serum based on polarity switching UPLC-MS / MS according to claim 1, characterized in that: The serum is any one of rat serum, mouse serum and human serum.

3. The method for detecting antiepileptic drugs in serum based on polarity switching UPLC-MS / MS according to claim 1, characterized in that: The length of the liquid chromatography column is 10 to 100 mm, and the inner diameter is 2.1 mm; the length of the pre-column is 2 to 10 mm, and the inner diameter is 2.1 mm; the fillers of the liquid chromatography column and the pre-column are both octadecyl bonded silica gel, and the particle size of the octadecyl bonded silica gel is 1.7 to 1.9 μm; the column temperature of the liquid chromatography column and the pre-column is 20 to 60°C.

4. The method for detecting antiepileptic drugs in serum based on polarity switching UPLC-MS / MS according to claim 1, characterized in that: The solution A is a 5% methanol aqueous solution containing 0.05% formic acid, and the solution B is a 95% methanol aqueous solution containing 0.05% formic acid.

5. The method for detecting antiepileptic drugs in serum based on polarity switching UPLC-MS / MS according to claim 4, characterized in that: The flow rate of the mobile phase is 0.2-0.6 mL / min, and the injection volume is 1-5 μL.

6. The method for detecting antiepileptic drugs in serum based on polarity switching UPLC-MS / MS according to claim 1, characterized in that: The preparation of a mixed standard reference solution using an anti-epileptic drug reference substance comprises the following steps: Accurately weigh appropriate amounts of levetiracetam, pregabalin, gabapentin, lamotrigine, phenobarbital, oxcarbazepine, phenytoin sodium, carbamazepine, clonazepam, diazepam, and sodium valproate reference substances, dissolve them in methanol and make up to volume to prepare reference substance stock solutions; Then accurately measure a certain volume of the stock solution of the above-mentioned reference substances, vortex mix, and prepare mixed standard reference solutions with mass concentrations of 500.0, 250.0, 250.0, 250.0, 650.0, 500.0, 650.0, 250.0, 1.0, 10.0, and 2000.0 μg / mL, respectively. Then dilute with methanol to form a series of mixed standard reference solutions; Accurately measure 10 μL of the above series of mixed standard control solutions, add 90 μL of blank serum, and mix well to obtain a series of standard serum samples; The same method was used to prepare three quality control serum samples with high, medium and low concentrations, and 6 replicates were prepared for each concentration.

7. The method for detecting antiepileptic drugs in serum based on polarity switching UPLC-MS / MS according to claim 1, characterized in that: The pretreatment process of the serum in step (2) is as follows: Take 50-150 μL of serum sample into an EP tube, add 100-500 μL of methanol containing 0.01-0.2% formic acid, vortex for 20-60 seconds, sonicate for 3-10 minutes, let it stand for 2-10 minutes, centrifuge at 2-8°C and 5000-15000 rpm for 3-10 minutes, and take the supernatant for sampling and analysis.

8. The method for detecting anti-epileptic drugs in serum based on polarity switching UPLC-MS / MS according to claim 1, characterized in that: The parameters and ionization mode of the multiple reaction monitoring mode of the antiepileptic drug are as follows: Levetiracetam, 171.14→126.10, ESI + ; Pregabalin, 160.16→142.50, ESI + ; Gabapentin, 172.16→137.00, ESI + ; Lamotrigine, 255.91→108.94, ESI + ; Phenobarbital, 231.18→84.92, ESI - ; Oxcarbazepine, 252.99→180.04, ESI + ; Phenytoin sodium, 251.19→101.92, ESI - ; Carbamazepine, 237.00→179.00, ESI + ; Clonazepam, 316.01→270.02, ESI + ; Diazepam, 285.04→154.00, ESI + ; Sodium valproate, 143.08→143.08, ESI - .

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