CN115639306B - Method for rapidly detecting concentration of antiepileptic drug in clinical sample - Google Patents
Method for rapidly detecting concentration of antiepileptic drug in clinical sample Download PDFInfo
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Abstract
The invention relates to the field of antiepileptic drug detection, in particular to a method for rapidly detecting the concentration of an antiepileptic drug in a clinical sample. The antiepileptic drug comprises one or more of levetiracetam, carbamazepine, lamotrigine, gabapentin, zonisamide, pamidone, clonazepam, lacosamide, valproic acid, phenytoin, pirampanel, carbamazepine-10,11-epoxide, and licarbazepine, and the method comprises: s1, establishing a calibration curve by adopting a stable isotope internal standard quantitative method; s2, detecting the processed clinical sample by ultra high performance liquid chromatography tandem mass spectrometry; and S3, calculating and obtaining the concentration of the antiepileptic drug in the clinical sample by using the calibration curve.
Description
Technical Field
The invention relates to the field of detection of antiepileptic drugs, in particular to a method for rapidly detecting the concentration of antiepileptic drugs in a clinical sample, wherein the antiepileptic drugs comprise one or more of Levetiracetam (Levetiracetam), carbamazepine (CBZ), lamotrigine (LTG), gabapentin (GBP), zonisamide (ZNS), primidone (PMD), clonazepam (CLN), lacosamide (LAC), valproic acid (VPA), phenytoin (PHYTOIN, PHT), pyripyropene (PER), carbamazepine metabolite-10, 11-epoxide (Carbamazepine-8978-epoxyde, 8978-epoxyde) and oxcarbazepine-10, 11-hydroxy-5 (LC).
Background
Epilepsy is a chronic neurological disease with cerebral dysfunction caused by highly synchronized abnormal discharge of cerebral neurons, and is clinically characterized by paroxysmal, transient, recurrent and stereotypical characteristics. The pathogenesis of epilepsy is complex and diverse, including abnormalities in the brain structure (external injury, tumors, infection, etc.), genetic variations, abnormalities in the immune and metabolic systems, and other unknown causes. Epileptic seizures can cause severe damage to the brain of a patient, affect the intelligence development of children, and cause the memory decline and cognitive impairment of the patient. Vomiting or asphyxia is easily caused in the process of epileptic seizure, and the life of a patient is threatened. The long-term seizures of epilepsy can also have negative effects on the psychology and character of patients, and affect normal life.
At present, the treatment of epilepsy is mainly based on drug therapy, and more than 20 anti-epilepsy drugs are applied to clinic. The number of epileptic patients in China is over ten million, about 40 million new epileptic patients are added every year, and about 25 percent of patients can not receive individualized treatment to cause poor control of epilepsy or generate toxic and side effects and complications. In addition, since epileptic patients require long-term medication, their "compliance" is also an important factor affecting the efficacy of treatment. In adult patients with epilepsy, about 25% to 50% of patients have poor compliance; of newly diagnosed pediatric epileptic patients, up to over 60% of patients have varying degrees of poor compliance within the first 6 months of starting to take the medication. Therefore, it is the key of anti-epileptic treatment to make and adjust individualized medication strategies according to the type and course of the patient, and to know and intervene in time the patient's medication compliance.
Some first-generation antiepileptic drugs which are common in clinic have narrow therapeutic window, have nonlinear pharmacokinetic characteristics and are easy to cause toxic and side effects. For example, effective plasma concentrations of phenytoin are 10-20 μ g/mL, and when blood concentrations are higher than 20 μ g/mL, phenytoin users develop toxic symptoms such as dizziness, nystagmus, nausea, vomiting, headache, confusion, and the like. In addition, most antiepileptic drugs are metabolized by hepatic CYP450 enzymes, and are also enzymatic inducers or inhibitors of CYP450 (e.g., phenytoin, carbamazepine, valproic acid, etc.). For intractable epilepsy patients which cannot be controlled by a single medicine, combined medicines are often needed, and the risk of medicine-medicine interaction exists, so that the treatment effect is influenced. In addition, in children, pregnant women, the elderly, patients with liver and kidney dysfunction, and other special groups, the metabolism and the clearance capacity of the medicine can be changed to a certain extent, so that the concentration of the medicine in the body is abnormal, and the risk of poisoning or insufficient treatment exists. Research shows that the safety of the second generation antiepileptic drugs (such as lamotrigine, oxcarbazepine, levetiracetam and the like) is improved to a certain extent, but the blood concentration of the drugs can still be influenced by factors such as age, renal function and pregnancy of patients.
Therefore, performing Therapeutic Drug Monitoring (TDM) in epileptic patients (especially in the above-mentioned special groups), timely obtaining the concentration of antiepileptic drugs in patients' bodies, and evaluating drug-drug interactions and drug compliance are of great significance for achieving accurate treatment of epilepsy.
Chinese patent CN111812216a discloses a method for detecting 10 antiepileptic drugs in pretreated serum by using an ultra-high performance liquid chromatography tandem mass spectrometry technology, which not only covers a few types of antiepileptic drugs, but also has a high lower limit of quantitation, and cannot meet the requirement for detecting antiepileptic drugs for various types of epileptic patients (e.g., common epileptic patients, epileptic patients who adopt a complicated medication scheme, epileptic patients who have poor medication compliance, children, the elderly, liver and kidney insufficiency, pregnant women and other special groups). Chinese patent CN115326960a discloses an analytical method for simultaneously detecting concentrations of 8 antiepileptic drugs and 1 active metabolite in human plasma, which not only covers a few types of antiepileptic drugs, but also has a high upper limit of quantitation, which is almost the upper limit of a therapeutic concentration range, and thus cannot accurately detect the concentration of drugs in vivo of patients who take antiepileptic drugs in excess or are affected by drug interaction. Moreover, the technical solution of CN115326960a in chinese patent has a situation that multiple analytes share the same internal standard substance, and a difference in chromatographic or mass spectrometry behavior may occur in actual measurement, which affects the actual measurement result. In addition, the technical scheme disclosed in chinese patent CN115326960a has a long analysis time (greater than 10 min), and the required plasma sample volume is large (100 μ L), which also fails to meet the requirement of detecting antiepileptic drugs for various types of epileptic patients.
Disclosure of Invention
The invention provides a method for rapidly detecting the concentration of an antiepileptic drug in a clinical sample, wherein the antiepileptic drug comprises one or more of levetiracetam, carbamazepine, lamotrigine, gabapentin, zonisamide, pamidone, clonazepam, lacosamide, valproic acid, phenytoin, piranphane, carbamazepine-10,11-epoxide and licarbazepine, and is characterized by comprising the following steps of:
s1, a stable isotope internal standard quantitative method is adopted, the concentration ratio of the standard substance of the antiepileptic drug to the internal standard substance of the antiepileptic drug is used as an X axis, the peak area ratio of the standard substance of the antiepileptic drug to the internal standard substance of the antiepileptic drug is used as a Y axis, and a calibration curve is established.
The invention provides a method for simply and rapidly determining common clinical antiepileptic drugs and active metabolites in biological matrixes (such as blood plasma and saliva), which can detect the first-generation to third-generation antiepileptic drugs and specifically comprises the following steps:
first-generation antiepileptic drugs: carbamazepine, phenytoin, valproic acid, oxcarbazepine, primidone, and clonazepam;
the second generation antiepileptic drugs: zonisamide, gabapentin, lamotrigine and levetiracetam;
the third generation of antiepileptic drugs: lacosamide, and perampanel.
Because oxcarbazepine is almost completely converted into its active metabolite 10, 11-dihydro-10-hydroxy-carbamazepine (also called licarbazepine) in vivo to exert pharmacological effects after being taken, the present invention uses licarbazepine as a substitute test substance for oxcarbazepine. The carbamazepine generates an active metabolite, namely carbamazepine-10,11-epoxide in vivo, and the carbamazepine jointly play a pharmacological role in resisting epilepsy, but the content of the carbamazepine-10,11-epoxide in vivo is lower than that of the parent carbamazepine, so the invention detects and analyzes the carbamazepine and the parent carbamazepine.
In some embodiments, the internal standard corresponding to the antiepileptic drug comprises: levetiracetam-d 6 (LEV-d 6), carbamazepine-d 10 (CBZ-d 10, corresponding to CBZ and CBZO), lamotrigine- 13 C, 15 N 4 (LTG-C13), gabapentin-d 6 (GBP-d 6), zonisamide-d 4 (ZNS-d 4), pamidone-d 5 (PMD-d 5), clonazepam-d 4 (CLN-d 4), lacosamide-d 3 (LAC-d 3), valproic acid-d 6 (VPA-d 6), phenytoin-d 10 (PHT-d 10), pyrinepalene-d 5 (PER-d 5) and licarbazepine-d 4 (LC-d 4).
The stable isotope labeled derivative is used as an internal standard substance, and each target analyte (namely the antiepileptic drug) and the corresponding internal standard substance have similar chromatographic behavior and mass spectrum cracking rule, so that the influence of matrix effect in actual detection can be effectively reduced, and the method provided by the invention has good reproducibility and accuracy.
S2, detecting the processed clinical sample by ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), wherein the clinical sample comprises plasma or saliva, and the chromatographic conditions comprise: performing gradient elution by using an aqueous solution containing 5mM ammonium acetate and 0.1% formic acid as a mobile phase A and acetonitrile as a mobile phase B, wherein the volume ratio of the mobile phase A to the mobile phase B in the initial mobile phase of the gradient elution is 90; the processed clinical sample is the clinical sample after being diluted by 5 times by a diluent, and the diluent is the mobile phase A; the conditions of the mass spectrum include: adopting a detection mode of positive and negative ion switching by electrospray, wherein the levetiracetam, the carbamazepine, the lamotrigine, the gabapentin, the primidone, the clonazepam, the lacosamide, the phenytoin, the perampanel, the carbamazepine-10,11-epoxide and the licarbazepine adopt a positive ion mode, and the corresponding collision voltages are 45V, 60V, 25V, 32V, 22V, 25V, 34V, 38V, 35V, 30V and 28V respectively; the zonisamide and the valproic acid adopt a negative ion mode, and the corresponding collision voltages are-12V and-11V respectively.
And S3, calculating and obtaining the concentration of the antiepileptic drug in the clinical sample by using the calibration curve.
The ionization efficiency of the mass spectrum positive ion mode is improved by 0.1% formic acid in the mobile phase; while 5mM ammonium acetate improves the response of valproic acid in negative ion mode, which helps to improve the sensitivity in detecting valproic acid.
The initial mobile phase of the gradient elution is that the volume ratio of the mobile phase A to the mobile phase B is 90.
In some embodiments, the method for pretreating comprises mixing the clinical sample and a protein precipitant containing the internal standard substance uniformly, and centrifuging to obtain a supernatant, wherein the supernatant is the pretreated clinical sample.
In some embodiments, the volume ratio of the clinical sample to the protein precipitant comprising the internal standard is 1:4.
In some embodiments, the protein precipitating agent comprises acetonitrile.
In some embodiments, the manner of centrifugation comprises centrifugation at 13000 rpm for 10 minutes.
The invention adopts 4 times of diluent to dilute the pretreated clinical sample to obtain the treated clinical sample. The diluent is an aqueous solution containing 5mM ammonium acetate and 0.1% formic acid (v/v), mobile phase a. The pre-treated clinical sample is diluted by the diluent to comprise about 16% of organic phase prior to injection, similar to the initial mobile phase (which comprises about 10% of organic phase). The organic phase ratio of the clinical sample after pretreatment is about 80% after the clinical sample is subjected to protein precipitation by the protein precipitator and centrifugation. The dilution treatment not only reduces the pollution to the instrument and optimizes the chromatographic peak shape of the analyte with larger polarity, but also avoids the signal saturation of a high-concentration sample, effectively reduces the solvent effect, avoids the phenomena of chromatographic peak front edge and the like, and ensures that each target analyte can keep a good peak shape and is easy to quantify.
In some embodiments, the conditions of the mass spectrum further comprise: the levetiracetam-d 6, the carbamazepine-d 10, the lamotrigine- 13 C, 15 N 4 The gabapentin-d 6, the paminone-d 5, the clonazepam-d 4, the lacosamide-d 3, the phenytoin-d 10, the perampanel-d 5 and the licarbazepine-d 4 adopt a positive ion mode, and the corresponding collision voltages are 42V, 58V, 36V, 25V, 16V, 25V, 36V, 30V, 35V and 30V, respectively; the zonisamide-d 4 and the valproic acid-d 6 adopt a negative ion mode, and the corresponding collision voltages are-20V and-11V respectively.
In some embodiments, the procedure of gradient elution comprises: at 0-0.5 min, the flow rate of the mobile phase is 0.3 mL/min, and the volume ratio of the mobile phase A to the mobile phase B is 90; 1.50 At min, the flow rate of the mobile phase is 0.3 mL/min, and the volume ratio of the mobile phase A to the mobile phase B is 55; 2.50 At min, the flow rate of the mobile phase is 0.3 mL/min, and the volume ratio of the mobile phase A to the mobile phase B is 45; 3.00-3.50 min, the flow rate of the mobile phase is 0.3 mL/min, and the volume ratio of the mobile phase A to the mobile phase B is 20; at 3.60-4.50 min, the flow rate of the mobile phase is 0.5 mL/min, and the volume ratio of the mobile phase A to the mobile phase B is 5;4.60 At min, the flow rate of the mobile phase is 0.3 mL/min, and the volume ratio of the mobile phase A to the mobile phase B is 90; 5.5 And at min, the flow rate of the mobile phase is 0.3 mL/min, and the sample injection is stopped.
In some embodiments, the conditions of the mass spectrum further comprise a mass spectrometry scan mode using multiple reaction monitoring, with capillary voltages of 5500V (ESI +)/4500V (ESI-), and ion source temperatures of 500 ℃ (ESI +)/600 ℃ (ESI-).
In some embodiments, the chromatographic conditions further comprise the use of ACQUITY TM An ultra-high performance liquid chromatograph and an ACQUITY UPLC BEH C18 chromatographic column, wherein the column temperature of the chromatographic column is 40 ℃.
In some embodiments, the before and after the detection comprises a step of washing the injection needle with a multiple needle wash solution comprising a methanol-acetonitrile-isopropanol-water-formic acid mixed solution (25.
After each sample injection, the invention adopts the multiple needle washing liquid to clean the sample injection needle, and sets the high flow rate and the high proportion of organic phase to carry out chromatographic column elution after the peak discharge is finished, so as to effectively reduce the residue and the pollution of the antiepileptic drug in the UPLC-MS/MS system. The preparation proportion of the multi-element needle washing liquid is preferably as follows: strong needle wash (methanol: acetonitrile: isopropanol: water: formic acid =25, 25), weak needle wash (acetonitrile: water: formic acid =20, 1 (v/v)) to avoid retention of target analytes of different polarity at the injection needle. Meanwhile, after all target analytes generate peaks, the chromatographic column is washed by using a high-proportion organic phase (95% of mobile phase B) and a high flow rate (0.5 mL/min), so that the elution of fat-soluble substances in the chromatographic column by a liquid phase system is enhanced, and the detection noise is reduced.
Preferably, the present invention selects a channel with a high signal-to-noise ratio and a small residual effect among a plurality of ion channels for each analyte to monitor.
Preferably, the mass spectrum energy parameter and the monitoring channel which are similar to those of the corresponding analytes are selected for each isotope-labeled internal standard substance.
Compared with the prior art, the invention has the beneficial effects that:
the antiepileptic drug detection method in the prior art can detect a few types of antiepileptic drugs, and cannot evaluate the complete medication condition of patients who come from different medical institutions and receive different treatment schemes through one-time detection, so that the applicability is not strong. In addition, the detection method of the antiepileptic drug in the prior art usually consumes long time and has low flux, and the requirements of clinical rapid and high-flux detection are difficult to meet.
The invention provides a method for simultaneously determining the concentrations of 13 antiepileptic drugs (11 antiepileptic drugs and 2 active metabolites) in a clinical sample based on a UPLC-MS/MS method, which at least has the following advantages:
a) Epilepsy is a chronic disease, and its medication cycle may be at least 3-5 years. During the medical treatment of epilepsy, adjustments in dosage and dosing regimens are often involved. In particular, for patients with drug refractory epilepsy, it may also involve the use of a combination regimen. Just as epileptics require long-term administration and even dynamic adjustments to the regimen, epileptics may experience problems of poor compliance, such as concealing actual medication or missed medication (including unauthorized use of other types of antiepileptic drugs, increased medication doses, unauthorized withdrawal, etc.). This not only can bring the hindrance to the doctor to formulate and adjust the individualized dosing strategy, but also can make the epileptic seizure difficult to control effectively, and especially can also produce the huge hidden danger such as drug interaction, overdose in pregnant women, children and the special population who has other basic diseases, etc. and bring the life danger to the patient. The method provided by the invention can detect analytes covering a large variety of antiepileptic drugs, including the first generation of antiepileptic drugs (carbamazepine, phenytoin, valproic acid, oxcarbazepine, prometrone and clonazepam) and the second generation of antiepileptic drugs (zonisamide, gabapentin, lamotrigine and levetiracetam) which are more common in clinic and the third generation of antiepileptic drugs (lacosamide and pirampanel), and is suitable for monitoring the treatment drugs of various types and various medication schemes of epileptic patients by different medical institutions. The method provided by the invention can detect the trace content of the medicine in the body of the subject, and is particularly suitable for judging the medicine taking history of the epileptic and improving the medicine taking normalization of the epileptic.
b) The method provided by the invention can enable each analyte (namely the antiepileptic drug) to show good linearity and sensitivity. Valproic acid, one of the antiepileptic drugs, is a small molecular compound (the molecular weight is only 144), which is difficult to crack in mass spectrum, and the sensitivity is lower than that of other antiepileptic drugs, so that the prior art does not dilute a sample before sample injection detection, thereby improving the sensitivity of detecting valproic acid. Most other antiepileptic drugs are easy to be cracked in mass spectrum, and have high content in the body of a subject (often reaching the order of tens of micrograms and even tens of micrograms), so that the antiepileptic drugs have strong response in mass spectrum; even high concentration points in the calibration curve can exhibit "saturation" phenomena that affect the linearity and measurement of the calibration curve. The prior art usually adopts a means of diluting a sample in a large proportion to avoid the high-concentration point saturation phenomenon of the antiepileptic drugs. And when the valproic acid and the alkaline peroxide are measured simultaneously, the detection sensitivity of the valproic acid is reduced by large-proportion dilution. The method provided by the invention selects a collision voltage which is not 'optimal' (even if the analyte achieves the highest response in the mass spectrum), but can enable a part of the easily responsive analyte (for example, most antiepileptic drugs except valproic acid) to have a proper response at a high concentration point, and simultaneously ensures that a low concentration point has a sufficient signal-to-noise ratio, thereby avoiding the overhigh response of most antiepileptic drugs at the high concentration point and avoiding the reduction of the sensitivity of the valproic acid caused by diluting a sample by a large proportion.
In addition, the invention adds a dilution step with a proper proportion, not only optimizes the response of a chromatographic peak pattern and a mass spectrum, but also reduces biological matrix components entering a liquid system, and reduces the influence of matrix effect and the pollution to the liquid system.
c) The method provided by the invention reduces the lower limit of the quantitative determination of the detected antiepileptic drug and has higher detection sensitivity. For example, compared with the prior art, the method provided by the invention can reduce the lower limit of the quantification of levetiracetam and phenytoin to 0.05 μ g/mL and 0.04 μ g/mL respectively, reduce the lower limit of the quantification of carbamazepine to 0.02 μ g/mL, reduce the lower limit of the quantification of carbamazepine-10,11-epoxide to 0.01 μ g/mL, reduce the lower limit of the quantification of valproic acid from 0.5 μ g/mL to 0.25 μ g/mL, and the like.
d) The invention is particularly suitable for monitoring the treatment medicine of a special epileptic. For example, the method provided by the invention can not only realize therapeutic drug monitoring on common epileptics with a single medication scheme, but also realize therapeutic drug monitoring on epileptics with complex conditions (such as children, the elderly, patients with liver and kidney insufficiency, pregnant women and other special populations) following complex medication schemes (such as patients who need to take multiple anti-epileptic drugs and patients who need dynamic dose adjustment). Even under the influence of different physiology, pathology and drug interaction, the drug concentration range detectable by the method provided by the invention can still cover the drug concentration range in clinical samples from the epileptic patients, and the requirement of carrying out comprehensive therapeutic drug monitoring on the population is met.
e) The method provided by the invention is not only suitable for detecting plasma samples, but also suitable for saliva samples. For some special patient groups (such as pregnant women, children, patients with liver and kidney damage, etc.) needing to be closely monitored with therapeutic drugs, plasma samples of patients are often required to be collected for multiple times, which further reduces the acceptance and the matching degree of children, old people, epileptic patients with blood drawing difficulty, etc., and is not beneficial to carrying out close monitoring on the therapeutic drugs for the epileptic patients. A series of experiments prove that the method provided by the invention not only can accurately detect the concentration of the antiepileptic drug in the plasma sample, but also can accurately detect the concentration of the antiepileptic drug in the saliva sample which is easier to obtain, and the concentration of the antiepileptic drug in the two sample types can reflect the level and the fluctuation of the free antiepileptic drug in the body of a patient. Therefore, the method provided by the invention not only can accurately reflect the level of the free antiepileptic drug in the body of the patient, but also improves the acceptance and the matching degree of the patient to a certain extent, and is beneficial to evaluating the medication condition of the patient more conveniently and for a long time.
In conclusion, the invention provides a simple, rapid, reliable and high-applicability antiepileptic drug quantification method based on UPLC-MS/MS, which is beneficial to realizing the therapeutic drug monitoring of the drug use condition of various epileptics. The method provided by the invention has a wider detection range, and reduces the difficulty degree of monitoring the treatment medicines for various epileptic patients to a certain extent.
As used herein, the term "epilepsy" refers to a clinical phenomenon in which an individual has two or more (more than 24 hours apart) non-evoked seizures.
As used herein, the terms "subject," "individual," and "patient" are used interchangeably and refer to a mammal from which a biological sample is taken, unless otherwise specified. In some embodiments, the subject is an individual taking at least one antiepileptic drug. In some embodiments, a typical subject is an epileptic patient.
As used herein, the terms "sample" or "biological sample" or "specimen" or "clinical specimen" or "biological matrix" are used interchangeably and refer to biological material isolated from a subject. In the present invention, "biological matrix" includes blood (e.g., whole blood, plasma, and serum) and saliva and fractions thereof separated by pretreatment. In some preferred embodiments, the biological matrix is plasma or saliva. The pretreatment method comprises filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents and the like.
As used herein, the term "analyte" or "target analyte" refers to a molecule or ion that can be found in a biological matrix and requires detection or quantification. In the present invention, "analyte" or "analyte of interest" specifically includes one or more of levetiracetam, carbamazepine, lamotrigine, gabapentin, zonisamide, pamidone, clonazepam, lacosamide, valproic acid, phenytoin, pirampanel, carbamazepine-10,11-epoxide, and licarbazepine.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is obvious that the drawings in the following description are some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive exercise.
FIG. 1a is a diagram showing a summary of information on reagent consumables used in an embodiment of the present invention;
FIG. 1b is a summary chart of information on standards and internal standards used in the examples of the present invention;
FIG. 2 is a summary plot of mass spectral parameters of the analyte and internal standard of the present invention;
FIG. 3a is a chromatogram of an analyte in a plasma quality control sample in positive ion mode;
FIG. 3b is a chromatogram of an analyte in a plasma quality control sample in negative ion mode;
FIG. 4a is a chromatogram of an internal standard in a plasma quality control sample in positive ion mode;
FIG. 4b is a chromatogram of an internal standard in a plasma quality control sample in negative ion mode;
FIG. 5a is a chromatogram of a blank plasma sample in positive ion mode;
FIG. 5b is a chromatogram of a blank plasma sample in negative ion mode;
FIG. 6 is a summary plot of the results of various analyte standard curves of the present invention;
FIG. 7a is a summary plot of the results of intra/inter-batch precision and accuracy of the analytes LEV, LTG, LC, CBZ and CBZO of the present invention;
FIG. 7b is a summary plot of the results of the intra/inter-lot precision and accuracy of the analytes LAC, CLN, PMD and GBP of the present invention;
FIG. 7c is a summary plot of the results of the intra/inter-batch precision and accuracy of the analytes PER, PHT, VPA and ZNS of the present invention;
FIG. 8 is a graph showing the results of actual plasma samples tested using the method of the present invention;
FIG. 9 is a graph showing the results of the test on the actual saliva sample using the method of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Herein "and/or" includes any and all combinations of one or more of the associated listed items.
By "plurality" herein is meant two or more, i.e. it includes two, three, four, five, etc.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a … …" does not exclude the presence of another identical element in a process, method, article, or apparatus that comprises the element.
As used in this specification, the term "about" typically means +/-5% of the stated value, more typically +/-4% of the stated value, more typically +/-3% of the stated value, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, and even more typically +/-0.5% of the stated value.
In this specification, certain embodiments may be disclosed in a range of formats. It should be understood that this description of "within a certain range" is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, the description of range 1-6 should be viewed as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, e.g., 1,2,3,4,5 and 6. The above rules apply regardless of the breadth of the range.
The first embodiment is as follows: preparation of stock solution and working solution
Specific information of the experimental materials involved in the present invention is shown in fig. 1a and 1 b.
1.1. Methanol with the volume fraction of 50 percent is taken as a working solution diluent, and the preparation method comprises the following steps: 50 mL methanol and 50 mL ultrapure water are respectively measured and mixed in a 200 mL solvent bottle, and stored at room temperature after 5 minutes of ultrasonic processing, and the solution is marked as solvent D.
1.2. Preparation of analyte and internal standard stocks:
1.2.1. preparing the analytes into stock solutions with the following concentrations (preparing two parts, namely an analyte standard curve stock solution and an analyte control stock solution respectively): levetiracetam 10 mg/mL, carbamazepine 10 mg/mL, lamotrigine 10 mg/mL, gabapentin 10 mg/mL, zonisamide 10 mg/mL, pamidone 10 mg/mL, clonazepam 0.1 mg/mL, lacosamide 10 mg/mL, sodium valproate (as valproic acid) 20 mg/mL, phenytoin sodium (as phenytoin) 10 mg/mL, licarbazepine 10 mg/mL, pyrinepalene 0.2 mg/mL, and carbamazepine-10,11-epoxide 2 zxft 4924/4924 mL. The analyte stocks were stored in a-40 ℃ refrigerator. Sodium valproate and sodium phenytoin are commercially available as common commercial products, and are sodium salts of valproic acid and phenytoin, respectively, which produce actual pharmacological effects. During the detection process, after sodium valproate and sodium phenytoin lose sodium ions, signals can be generated in the mass spectrum. The ions actually detected by the invention are valproic acid and phenytoin, therefore, when the sodium salt standard substance is weighed, the weight is converted according to the molecular weight of the valproic acid and the phenytoin, namely, the concentrations of the valproic acid and the phenytoin are corresponding to the concentration of the valproic acid and the phenytoin instead of the concentration of the sodium valproic acid and the phenytoin in of the '20 mg/mL' and the '10 mg/mL' in the sodium valproic acid stock solution and the sodium phenytoin stock solution.
1.2.2. Internal standards were formulated as stocks at the following concentrations: levetiracetam-d 6 mu g/mL, carbamazepine-d 10 mu g/mL, lamotrigine- 13 C, 15 N 4 100. mu.g/mL, gabapentin-d 6 mu.g/mL, zonisamide-d 4 100 mu.g/mL, pamidone-d 5 mu.g/mL, clonazepam-d 4 mu.g/mL, lacosamide-d 3 100 mu.g/mL, valproic acid-d 6 mg/mL, phenytoin-d 10 mu.g/mL, pyrinepalene-d 5 mu.g/mL, and licarbazepine-d 4 100 mu.g/mL. The internal standard stock solutions were stored in a-40 ℃ refrigerator.
1.3. Preparation of an analyte working solution:
1.3.1. the standard curve stock solution of the analyte prepared in 1.2.1: 100. mu.L levetiracetam, 40. Mu.L carbamazepine, 40. Mu.L lamotrigine, 80. Mu.L gabapentin, 100. Mu.L zonisamide, 40. Mu.L prometone, 200. Mu.L clonazepam, 80. Mu.L lacosamide, 250. Mu.L valproic acid, 80. Mu.L phenytoin, 200. Mu.L pirampanel, 100. Mu.L carbamazepine-10,11-epoxide and 100. Mu.L licarbazepine are mixed with 590. Mu.L solvent D to give a mixed standard curve working solution at the highest concentration point of 2 mL. Diluting the mixed standard curve working solution at the highest concentration point by a solvent D to seven standard curve working solutions (simply called "series of standard curve working solutions") with different concentrations, wherein the concentration points (including the highest concentration point) of the series of standard curve working solutions are respectively as follows:
the levetiracetam, licarbazepine and zonisamide concentrations are the same, and the standard curve working solution concentrations are respectively as follows: 0.5, 1,2, 10, 50, 125, 250, 500 μ g/mL; the concentrations of phenytoin, lacosamide and gabapentin are the same, and the concentrations of the standard curve working solution are respectively as follows: 0.4, 0.8, 1.6, 8, 40, 100, 200, 400 μ g/mL; the concentrations of lamotrigine, carbamazepine and primidone are the same, and the concentrations of standard curve working solutions are respectively as follows: 0.2, 0.4, 0.8, 4, 20, 50, 100, 200 μ g/mL; the standard curve working solution concentrations for carbamazepine-10,11-epoxide were: 0.1, 0.2, 0.4, 2, 10, 25, 50, 100 μ g/mL; the concentrations of the standard curve working solution of clonazepam are respectively as follows: 0.01, 0.02, 0.04, 0.2, 1, 2.5, 5, 10 μ g/mL; the concentrations of the working solution of the standard curve of the Perampanel are respectively as follows: 0.02, 0.04, 0.08, 0.4, 2, 5, 10, 20 μ g/mL; the concentrations of the working solutions of the standard curve of valproic acid were: 2.5, 5, 10, 50, 250, 625, 1250, 2500 mug/mL. The standard curve working solutions were stored in a refrigerator at-40 ℃.
1.3.2. The analytical material control stock solution prepared in 1.2.1: 75. mu.L levetiracetam, 30. Mu.L carbamazepine, 60. Mu.L lamotrigine, 60. Mu.L gabapentin, 75. Mu.L zonisamide, 30. Mu.L prometone, 150. Mu.L clonazepam, 60. Mu.L lacosamide, 187.5. Mu.L valproic acid, 60. Mu.L phenytoin, 150. Mu.L pirampanel, 75. Mu.L carbamazepine-10,11-epoxide and 75. Mu.L licarbazepine are mixed with 942.5. Mu.L solvent D to give 2 mL mixed high concentration quality control (HQC) working solution. Diluting the HQC working solution by a solvent D step by step into a medium-concentration quality control (MQC) working solution, a low-concentration quality control (LQC) working solution and a quantitative Lower Limit (LLOQ) working solution, wherein the concentration points of the quality control working solution are respectively as follows:
the concentrations of levetiracetam, licarbazepine and zonisamide are the same, and the concentrations of HQC, MQC, LQC and LLOQ working solutions are respectively as follows: 375. 75, 1.5 and 0.5 mu g/mL; the concentrations of phenytoin, lacosamide and gabapentin are the same, and the concentrations of HQC, MQC, LQC and LLOQ working solutions are respectively as follows: 300. 60, 12 and 0.4 mu g/mL; the concentrations of lamotrigine, carbamazepine and primidone are the same, and the concentrations of HQC, MQC, LQC and LLOQ working solutions are as follows: 150. 30, 6 and 0.2 mu g/mL; the concentrations of HQC, MQC, LQC, and LLOQ working solutions for carbamazepine-10,11-epoxide were: 75. 15, 3, 0.1 mu g/mL; the concentrations of HQC, MQC, LQC and LLOQ working solutions of clonazepam are respectively as follows: 7.5, 1.5, 0.3, 0.01 μ g/mL; the concentrations of HQC, MQC, LQC and LLOQ working solutions of Perampanel are respectively as follows: 15. 3, 0.6 and 0.02 mu g/mL; the concentrations of HQC, MQC, LQC and LLOQ working solutions of valproic acid are respectively as follows: 1875. 375, 7.5, 2.5. Mu.g/mL. The quality control working solution is stored in a refrigerator at the temperature of minus 40 ℃.
1.4. Preparing an internal standard working solution: taking the internal standard substance stock solutions prepared in 1.2.2 respectively: 125. mu.L levetiracetam- d 6, 50 mu.L carbamazepine- d 10, 50 mu.L lamotrigine- 13 C, 15 N 4 100 mu L of gabapentin-d 6, 125 mu L of zonisamide- d 4, 50 mu L of prometrone- d 5, 25 mu L of clonazepam- d 4, 100 mu L of lacosamide- d 3, 100 mu L of valproic acid- d 6, 100 mu L of phenytoin- d 10, 20 mu L of perampanel-d 5 and 125 mu L of licarbazepine-d 4, and adding a protein precipitant acetonitrile to 80 8978 zft 8978 to obtain an internal standard working solution (namely the protein precipitant containing the internal standard) which is stored in a refrigerator at 4 ℃.
1.5. The preparation method adopts a mobile phase A as a diluent before sample introduction, and comprises the following steps: 7.708 g ammonium acetate was weighed and dissolved in 20 mL ultrapure water to prepare a 5M ammonium acetate solution. 0.2 mL of 5M ammonium acetate solution was transferred to a solvent bottle, 200 mL ultrapure water and 0.2 mL formic acid were added, mixed well by sonication, and stored at room temperature, labeled as solvent A.
Example two: pretreatment of human plasma (or saliva) samples
2.1. Preparation of blank plasma (or saliva) samples:
2.1.1. 50 μ L of human blank plasma (or saliva) and 200 μ L of acetonitrile were added sequentially to a 1.5 mL polypropylene centrifuge tube and vortex mixed for 2 minutes to give a mixed blank sample.
2.1.2. The mixed blank sample was centrifuged at 13000 rpm for 10 minutes at room temperature and the supernatant collected. Transferring 50 mu L of supernatant into a new centrifuge tube, adding 200 mu L of solvent A for dilution, mixing by vortex for 1 minute, and transferring into a sample injection bottle for analysis and detection.
2.2. Preparation of null plasma (or saliva) samples:
essentially the same as the preparation of the blank plasma (or saliva) sample, except that: 50 μ L of human blank plasma (or saliva) and 200 μ L of the internal standard-containing protein precipitant prepared in example one were added to a 1.5 mL polypropylene centrifuge tube (the same procedure as 2.1 for the rest).
2.3. Preparation of standard curve sample:
basically the same method as the preparation of the above samples is used, except that: 10 μ L of the working solution of the series of standard curves prepared in example I was transferred and added into a 1.5 mL polypropylene centrifuge tube containing 90 μ L of blank plasma (or saliva), and after vortex mixing, samples of the series of standard curves were obtained. To 50 μ L of the series of standard curve samples, 200 μ L of protein precipitant containing an internal standard was added (the remaining steps were as in 2.1.). The concentration points of the series of standard curve samples are respectively:
the concentrations of levetiracetam, licarbazepine and zonisamide are the same, and the concentrations of the standard curve samples are respectively as follows: 0.05, 0.1, 0.2, 1, 5, 12.5, 25, 50 μ g/mL; the concentrations of phenytoin, lacosamide and gabapentin are the same, and the concentrations of the standard curve samples are respectively as follows: 0.04, 0.08, 0.16, 0.8, 4, 10, 20, 40 μ g/mL; the concentrations of lamotrigine, carbamazepine and primidone are the same, and the concentrations of the standard curve samples are respectively: 0.02, 0.04, 0.08, 0.4, 2, 5, 10, 20 μ g/mL; the concentrations of the standard curve samples of carbamazepine-10,11-epoxide were: 0.01, 0.02, 0.04, 0.2, 1, 2.5, 5, 10 μ g/mL; the concentrations of the clonazepam standard curve samples were: 0.001, 0.002, 0.004, 0.02, 0.1, 0.25, 0.5, 1 mug/mL; the concentrations of the standard curve samples of Perampanel are respectively as follows: 0.002, 0.004, 0.008, 0.04, 0.2, 0.5, 1,2 mug/mL; the concentrations of the standard curve samples of valproic acid were: 0.25, 0.5, 1, 5, 25, 62.5, 125, 250. Mu.g/mL.
2.4. Preparing a quality control sample:
basically the same method as the preparation of the above samples is used, except that: 10 μ L of the working solution prepared in example I was transferred to a 1.5 mL polypropylene centrifuge tube containing 90 μ L of blank plasma (or saliva), and vortex mixing was performed to obtain HQC, MQC, LQC, and LLOQ samples. To 50. Mu.L of the quality control sample, 200. Mu.L of a protein precipitant containing an internal standard was added (the same procedure as in 2.1 ℃ C.). The concentration points of the quality control working solution are respectively as follows:
the levetiracetam, licarbazepine and zonisamide concentrations were the same, and the HQC, MQC, LQC and LLOQ concentrations were: 37.5, 7.5, 0.15, 0.05 μ g/mL; the concentrations of phenytoin, lacosamide and gabapentin were the same, and the concentrations of HQC, MQC, LQC and LLOQ samples were: 30. 6, 1.2 and 0.04 mu g/mL; the concentrations of lamotrigine, carbamazepine and primidone were the same, and the concentrations of HQC, MQC, LQC and LLOQ samples were: 15. 3, 0.6 and 0.02 mu g/mL; the concentrations of HQC, MQC, LQC, and LLOQ samples of carbamazepine-10,11-epoxide were: 7.5, 1.5, 0.3, 0.01 μ g/mL; the concentrations of HQC, MQC, LQC and LLOQ samples of clonazepam were: 0.75, 0.15, 0.03, 0.001 mug/mL; the concentrations of HQC, MQC, LQC, and LLOQ samples for perampanel were: 1.5, 0.3, 0.06, 0.002 mug/mL; the concentrations of HQC, MQC, LQC and LLOQ samples of valproic acid were: 187.5, 37.5, 0.75, 0.25. Mu.g/mL.
2.5. The preparation of a patient plasma (or saliva) sample is essentially the same as that described above, except that: 50 μ L of unknown patient plasma (or saliva) was transferred to a 1.5 mL polypropylene centrifuge tube and 200 μ L of protein precipitant containing an internal standard was added (the remaining steps are as in 2.1.).
Both blank plasma (or saliva) samples from healthy individuals and patient plasma (or saliva) samples were centrifuged within 30 minutes after sampling and stored frozen at-40 ℃ in a freezer.
Example three: UPLC-MS/MS analysis and detection
3.1. The main apparatus is as follows: an ultra-high performance liquid tandem mass spectrometry system (UPLC-MS/MS) consists of ultra-high performance liquid chromatography of Waters company and 5500 type triple quadrupole mass spectrometry of SCIEX company, and is provided with an electrospray ionization source (ESI) and an Analyst data acquisition and processing system; XPE26 electronic analytical balance (METTLER corporation); VX-II multi-tube vortex oscillator (Tiangen Biochemical technology Co., ltd.); TGL-19 high speed refrigerated centrifuge (Sichuan Instrument Co., ltd.).
3.2. Experimental methods
3.2.1. Chromatographic conditions are as follows: the chromatographic column adopts an ACQUITY UPLC BEH C18 chromatographic column of Waters company, and the specification is 1.7 μm and 2.1 × 50 mm; the mobile phase A is an aqueous solution containing 5mM ammonium acetate and 0.1% formic acid, the mobile phase B is acetonitrile, the column temperature is 40 ℃, and the flow rate is 0.3-0.5 mL/min. The elution procedure for gradient elution was: 0 min (10% B,0.3 mL/min) → 0.50 min (10% B,0.3 mL/min) → 1.5 min (45% B,0.3 mL/min) → 2.5 min (55% B,0.3 mL/min) → 3.00 min (80% B,0.3 mL/min) → 3.5 min (80% B,0.3 mL/min) → 3.6 min (95% B,0.5 mL/min) → 4.5 min (95% B,0.5 mL/min) → 4.6 min (10% B,0.3 mL/min) → 5.5 min (stopping sampling). Preferably, the injection volume is 7.5. Mu.L.
3.2.2. Mass spectrum conditions: adopting an electrospray positive and negative ion switching mode to carry out multi-reaction monitoring scanning on a target analyte and an internal standard substance, wherein the capillary voltages are respectively as follows: (positive) 5500V, (negative) -4500V; the ion source temperatures were: 500 ℃ plus and 600 ℃ minus; each channel dwell time is 15 msec. The mass spectral parameters for each target analyte and internal standard are shown in figure 2.
3.3. Methodology validation
3.3.1. And (3) selectivity: blank plasma (or saliva) samples and quality control samples prepared in example two were taken and injected separately. In the chromatogram of the blank plasma (or saliva) sample, no interference peak appears at the retention time of each target analyte and the internal standard substance, which indicates that the method has good selectivity. Fig. 3 is a chromatogram of an analyte in a plasma quality control sample in positive and negative ion mode (where fig. 3a is positive ion mode, fig. 3b is negative ion mode, and cps represents counts per second). Fig. 4 is a chromatogram of an internal standard in a plasma quality control sample in positive and negative ion mode (where fig. 4a is positive ion mode, fig. 4b is negative ion mode, and cps represents counts per second). Fig. 5 is a chromatogram of a blank plasma sample in positive and negative ion mode (where fig. 5a is positive ion mode, fig. 5b is negative ion mode, and cps represents counts per second).
3.3.2. Calibration curve: sample the standard curve prepared in example two, and quantify by isotope internal standard method with weight factor of 1/x 2 . The calibration curve established in the Analyst software was linear well with correlation coefficients greater than 0.99, and the quantitative linear range and standard curve for each analyte are shown in fig. 6.
3.3.3. Accuracy and precision: and (3) taking the blank sample, the zero sample, the series of standard curve samples and the quality control samples with different concentrations prepared in the second embodiment to form analysis batches, and continuously analyzing the analysis batches. 1 analysis batch comprised: 1 blank sample +1 zero sample +2 series standard curve samples (1 series standard curve sample comprises 8 samples with different concentrations) +5 quality control samples with different concentrations (1 quality control sample comprises 4 quality control samples with concentrations (namely HQC, MQC, LQC and LLOQ)), and the total number of the samples is 38. The in/between-batch accuracy and precision of each target analyte is shown in fig. 7a, 7b and 7 c.
Example four: practical application
The method provided by the invention is applied to 276 plasma or saliva samples of different epileptic patients, and the result shows that the method provided by the invention can quickly and accurately detect the specific medicine-taking type of the epileptic patient (partial experiment results are shown in fig. 8 and fig. 9, concentration units are all mu g/mL, and ND indicates that no detection is carried out). For example, as shown in fig. 8, comparing the measured plasma concentration of the antiepileptic drug with the therapeutic concentration range can help doctors to evaluate the in vivo drug content and the reasonability of the dosage of the antiepileptic drug, so as to avoid the risk of therapy deficiency or poisoning of the epileptic (especially children, pregnant women, and patients with liver and kidney damage). It is noted that the "LEV" preceding the values corresponding to P1, P2 and P16 patients in fig. 8 indicates that the dosage regimen for the patient is not levetiracetam, but that levetiracetam is detected in the actual clinical specimen, indicating that levetiracetam may be present in the patient. After repeated tests, the detection of the levetiracetam is not a measurement deviation, namely the detection method provided by the invention is real and reliable, the detection of the levetiracetam is not a false positive result, and the patient is prompted to possibly take medicine by mistake or hide the situation of taking medicine, so that more detailed follow-up is required and a medicine taking scheme is normalized in time. Furthermore, "#" at "LEV" of the P22 patient in fig. 8 indicates that the patient's dosing regimen contained levetiracetam, but that levetiracetam was not detected. After retesting, it is also confirmed that the undetected levetiracetam is not a measurement deviation, i.e., the undetected levetiracetam is not a "false negative" result, and it is suggested that the patient may miss taking the drug and needs to be given intensive medication instructions to improve treatment compliance. The data of the four patients show that the condition that the epilepsy patient does not take medicine according to the advice generally exists, and the method provided by the invention can accurately and reliably help the clinician to monitor the medicine taking condition of the patient.
The detection method provided by the invention is not only suitable for detecting the plasma sample, but also suitable for detecting the saliva sample. Compared with blood sampling, saliva sampling is simple, convenient, quick and noninvasive, continuous therapeutic drug monitoring within a certain time is facilitated, and then more close therapeutic drug monitoring can be realized for some special patients or patients with treatment risks (such as the patients who are easy to take by mistake, leak or hide medicines, children, pregnant women, patients with liver and kidney damage and the like) mentioned above. In conclusion, the method provided by the invention can be used for detecting the change of the concentration of the medicament in the saliva of the patient so as to master the daily medicament taking condition of the patient, discover potential medicament taking risks and adjust the treatment scheme in time (figure 9).
To summarize:
the method provided by the invention has the advantages of rapid and reliable analysis, conformity with the international biological analysis verification requirements, and no interference of endogenous substances in plasma and saliva samples on the determination of each analyte. The method provided by the invention can realize close therapeutic drug monitoring on subjects with various dosing schedules, in particular to special patient groups (such as children, pregnant women, patients with liver and kidney damage and the like with complicated changes of drug concentration in vivo) which are easily influenced by physiological characteristics and drug interaction. The method provided by the invention is particularly suitable for judging whether the situations of drug concealing, drug missing and the like exist in a subject or not and evaluating the rationality of the drug administration dose and the drug administration scheme.
While the present invention has been described with reference to the embodiments shown in the drawings, the present invention is not limited to the embodiments, which are illustrative and not restrictive, and it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (9)
1. A method for rapid detection of the concentration of an antiepileptic drug in a clinical sample for non-diagnostic purposes, the antiepileptic drug comprising levetiracetam, carbamazepine, lamotrigine, gabapentin, zonisamide, pamiprone, clonazepam, lacosamide, valproic acid, phenytoin, piranphane, carbamazepine-10,11-epoxide, and licarbazepine, the method comprising:
s1, establishing a calibration curve by adopting a stable isotope internal standard quantitative method, taking the concentration ratio of the standard substance of the antiepileptic drug to the internal standard substance of the antiepileptic drug as an X axis, and taking the peak area ratio of the standard substance of the antiepileptic drug to the internal standard substance of the antiepileptic drug as a Y axis;
s2, detecting the processed clinical sample by ultra high performance liquid chromatography tandem mass spectrometry, wherein the clinical sample comprises plasma or saliva, and the chromatographic conditions comprise: performing gradient elution by using an aqueous solution containing 5mM ammonium acetate and 0.1% formic acid as a mobile phase A and acetonitrile as a mobile phase B, wherein the gradient elution procedure comprises the following steps: the flow velocity of the mobile phase is 0.3 mL/min when the flow velocity is 0-0.5 min, and the volume ratio of the mobile phase A to the mobile phase B is 90; 1.50 At min, the flow rate of the mobile phase is 0.3 mL/min, and the volume ratio of the mobile phase A to the mobile phase B is 55; 2.50 At min, the flow rate of the mobile phase is 0.3 mL/min, and the volume ratio of the mobile phase A to the mobile phase B is 45; 3.00-3.50 min, the flow rate of the mobile phase is 0.3 mL/min, and the volume ratio of the mobile phase A to the mobile phase B is 20; at 3.60-4.50 min, the flow rate of the mobile phase is 0.5 mL/min, and the volume ratio of the mobile phase A to the mobile phase B is 5;4.60 At min, the flow rate of the mobile phase is 0.3 mL/min, and the volume ratio of the mobile phase A to the mobile phase B is 90; 5.5 At min, the flow rate of the mobile phase is 0.3 mL/min, and the sample injection is stopped; the processed clinical sample is the clinical sample after being diluted by 5 times by a diluent; the diluent is the mobile phase A; the conditions of the mass spectrum include: a detection mode of positive and negative ion switching by electrospray is adopted, wherein the levetiracetam, the carbamazepine, the lamotrigine, the gabapentin, the primidone, the clonazepam, the lacosamide, the phenytoin, the piracetam, the carbamazepine-10,11-epoxide and the licarbazepine adopt a positive ion mode, and the corresponding collision voltages are 45V, 60V, 25V, 32V, 22V, 25V, 34V, 38V, 35V, 30V and 28V respectively; the zonisamide and the valproic acid adopt a negative ion mode, and the corresponding collision voltages are-12V and-11V respectively;
and S3, calculating and obtaining the concentration of the antiepileptic drug in the clinical sample by using the calibration curve.
2. The method of claim 1, wherein the internal standard for an antiepileptic drug comprises levetiracetam-d 6, carbamazepine-d 10, lamotrigine- 13 C, 15 N 4 Gabapentin-d 6, zonisamide-d 4, primidone-d 5,Clonazepam-d 4, lacosamide-d 3, valproic acid-d 6, phenytoin-d 10, perampanel-d 5, and licarbazepine-d 4.
3. The method of claim 1, wherein the method of pre-treating comprises mixing the clinical sample with a protein precipitant containing the internal standard substance, and centrifuging to obtain a supernatant, wherein the supernatant is the pre-treated clinical sample.
4. The method of claim 3, wherein the protein precipitating agent comprises acetonitrile.
5. The method of claim 2, wherein the conditions of mass spectrometry further comprise: said levetiracetam-d 6, said carbamazepine-d 10, said lamotrigine- 13 C, 15 N 4 The gabapentin-d 6, the paminone-d 5, the clonazepam-d 4, the lacosamide-d 3, the phenytoin-d 10, the perampanel-d 5 and the licarbazepine-d 4 adopt a positive ion mode, and the corresponding collision voltages are 42V, 58V, 36V, 25V, 16V, 25V, 36V, 30V, 35V and 30V, respectively; the zonisamide-d 4 and the valproic acid-d 6 adopt a negative ion mode, and the corresponding collision voltages are-20V and-11V respectively.
6. The method of claim 3, wherein the volume ratio of the clinical sample to the protein precipitant containing the internal standard is 1:4.
7. The method of claim 1, wherein the mass spectrometry conditions further comprise a mass spectrometry scan mode using multiple reaction monitoring, capillary voltage 5500V ESI +/4500V ESI-, and ion source temperature 500 ℃ ESI +/600 ℃ ESI-.
8. The method of claim 1, wherein the chromatographic conditions further comprise the use of ACQUITY TM Ultra-high performance liquid chromatograph and ACQUAn ITY UPLC BEH C18 column having a column temperature of 40 ℃.
9. The method of claim 1, wherein the detecting comprises the step of washing the injection needle with a multi-element needle wash solution comprising a methanol-acetonitrile-isopropanol-water-formic acid mixed solution 25: 80:1 v/v.
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