CN115639306A - 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 detection of antiepileptic drugs, in particular to a method for rapidly detecting the concentration of antiepileptic drugs in a clinical sample. The antiepileptic drug comprises one or more of levetiracetam, carbamazepine, lamotrigine, gabapentin, zonisamide, paminone, clonazepam, lacosamide, valproic acid, phenytoin, piranepalene, 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 clinical samples, wherein the antiepileptic drugs comprise Levetiracetam (LEV), carbamazepine (CBZ), lamotrigine (LTG), gabapentin (GBP), zonisamide (ZONISAMIDE, ZNS), primidone (PMD), clonazepam (CLN), lacosamide (LAC), valproic acid (VPA), phenytoin (PHYTOIN, PHT), pyrimopalene (Perampanel, PER), carbamazepine metabolite, carbamazepine-10,11-epoxide (Carbamazepine-10, 11-oxazole, CBZ) and oxcarbazepine-10, licarbazepine (LC)).
Background
Epilepsy is a chronic neurological disease with brain dysfunction caused by highly synchronized abnormal discharges of brain 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 epileptic seizure also has negative effects on the psychology and character of the patient, and influences the 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 epileptics require long-term medication, their "compliance" is also an important factor affecting efficacy. 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, the key to anti-epileptic treatment is to make and adjust individual administration strategies according to the type and course of disease development of patients 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 the plasma concentration is higher than 20 μ g/mL, the patient with phenytoin will 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 who cannot be controlled by single medicine, combined medicine is 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 ultra-high performance liquid chromatography tandem mass spectrometry, which not only covers a few types of antiepileptic drugs, but also has a high lower limit of quantitation, and cannot meet the requirements for detecting antiepileptic drugs for various types of epileptic patients (e.g., common epileptic patients, epileptic patients using a complicated medication scheme, epileptic patients with poor medication compliance, children, the elderly, hepatic and renal insufficiency, pregnant women and other special groups). Chinese patent CN115326960A discloses an analysis method for simultaneously detecting concentrations of 8 anti-epileptic drugs and 1 active metabolite in human plasma, which not only covers a few types of anti-epileptic drugs, but also has a high upper limit of quantification, which is almost the upper limit of the therapeutic concentration range, so that it is impossible to accurately detect the concentrations of the anti-epileptic drugs taken in excess or in vivo of patients affected by the interaction of the drugs. In addition, according to the technical scheme of the chinese patent CN115326960A, when a plurality of analytes share the same internal standard substance, 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 patients with various types of epilepsy.
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 the active metabolite 10, 11-dihydro-10-hydroxy-carbamazepine (also called licarbazepine) in vivo to play a pharmacological role after being taken, the invention uses the licarbazepine as a substitute detection substance of oxcarbazepine. The active metabolite, namely the carbamazepine-10,11-epoxide, is generated in vivo by the carbamazepine, and the carbamazepine exert the pharmacological action of resisting epilepsy together, but the content of the carbamazepine-10,11-epoxide in vivo is lower than that of the parent carbamazepine, so that 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, CBZ and CBZO respectively), 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 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 initial mobile phase of the gradient elution is that the volume ratio of the mobile phase A to the mobile phase B is 90; the processed clinical sample is the clinical sample after being diluted by 5 times by diluent, and the diluent is the mobile phase A; the conditions of the mass spectrum include: employing a detection mode that switches between electrospray positive and negative ions, wherein said levetiracetam, said carbamazepine, said lamotrigine, said gabapentin, said prometone, said clonazepam, said lacosamide, said phenytoin, said piracetam, said carbamazepine-10,11-epoxide, and said licarbazepine employ a positive ion mode with corresponding collision voltages of 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 improved the response of valproic acid in negative ion mode, which helped 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.
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 was an aqueous solution containing 5mM ammonium acetate and 0.1% formic acid (v/v), mobile phase A. The pre-treated clinical sample is diluted with the diluent to comprise about 16% of the organic phase prior to injection, similar to the initial mobile phase (which comprises about 10% of the 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: 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.
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, stopping sampling when the flow rate of the mobile phase is 0.3 mL/min.
In some embodiments, the conditions of the mass spectrum further include a mass spectrometry scan mode using multiple reaction monitoring, with a capillary voltage of 5500V (ESI +)/4500V (ESI-), and an ion source temperature 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 are subjected to 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 the cycle of drug therapy 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. It is precisely because epileptics need to take medicines for a long time and even dynamically adjust the medication regimen that epileptics may suffer from poor compliance, such as concealing actual or missed medication (including unauthorized taking of other types of antiepileptic drugs, increasing the dosage of medication taken, unauthorized withdrawal of medication). This not only presents an obstacle for the physician to develop and adjust individualized dosing strategies, but also may make it more dangerous that seizure is difficult to control effectively, and in particular, may create huge potential risks such as drug interactions, overdose, etc. in pregnant women, children, and special populations with other underlying diseases, which present a life risk 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 which is one of the antiepileptic drugs is a small molecular compound (the molecular weight is only 144), and the valproic acid is difficult to crack in a mass spectrum, and the sensitivity is lower than that of other antiepileptic drugs, so that a sample is not diluted before sample injection and detection in the prior art, and the sensitivity for detecting the valproic acid is improved. 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, thereby not only optimizing the response of a chromatographic peak type and a mass spectrum, but also reducing biological matrix components entering a liquid system, and reducing the influence of a 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 needing to take multiple antiepileptic drugs and patients needing 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 damaged liver and kidney, etc.) who need to closely monitor the therapeutic drugs, the plasma samples of the patients are often collected for multiple times, which further reduces the acceptance and the matching degree of children, old people, and epileptics with blood drawing difficulties, etc., and is not beneficial to closely monitor the therapeutic drugs for the epileptics. A series of experiments prove that the method provided by the invention not only can accurately detect the concentration of the antiepileptic drug in a plasma sample, but also can accurately detect the concentration of the antiepileptic drug in a saliva sample which is more easily obtained, 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 favorable for 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, the 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, "biomatrix" 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 testing 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 phrases "comprising a component of' 8230; \8230;" does not exclude the presence of another like 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 taken 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 related to 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 of methanol and 50 mL of ultrapure water were measured and mixed in a 200 mL solvent bottle, sonicated for 5 minutes and stored at room temperature, labeled as solvent D.
1.2. Preparation of analyte and internal standard stock solutions:
1.2.1. preparing the analytes into stock solutions with the following concentrations (two parts are prepared, 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, piraampanel 0.2 mg/mL, and carbamazepine-10,11-epoxide 2 mg/mL. The analyte stocks were stored in a-40 ℃ refrigerator. Sodium valproate and phenytoin are commercially available as common 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 method are valproic acid and phenytoin, so 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 concentrations of the valproic acid and the phenytoin but not the concentrations of the sodium valproic acid and the phenytoin in the sodium valproic acid stock solution and the sodium phenytoin stock solution of the phenytoin which the concentrations of the valproic acid and the phenytoin are corresponding to the concentrations of the valproic acid and the phenytoin in.
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 all 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 of solvent D to give 2 mL of mixed standard curve working solution at the highest concentration point. Gradually diluting the mixed standard curve working solution at the highest concentration point into seven standard curve working solutions with different concentrations (referred to as "series of standard curve working solutions" for short) by using a solvent D, wherein the concentration points (including the highest concentration point) of the series of standard curve working solutions are respectively as follows:
the concentrations of levetiracetam, licarbazem and zonisamide are the same, and the concentrations of the standard curve working solution 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 the standard curve working solution 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 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 of 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 respectively 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 fluids of valproic acid were: 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 sprayButyl-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, adding a protein precipitant acetonitrile into the mixture to 80 mL to obtain an internal standard working solution (namely the protein precipitant containing the internal standard), and storing the internal standard working solution 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 of ammonium acetate was weighed and dissolved in 20 mL of 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 of ultrapure water and 0.2 mL of formic acid were added, mixed well by sonication, and stored at room temperature, labeled solvent A.
The second embodiment: pretreatment of human plasma (or saliva) samples
2.1. Preparation of blank plasma (or saliva) samples:
2.1.1. mu.L of human blank plasma (or saliva) and 200. Mu.L of acetonitrile were sequentially added 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: mu.L of human blank plasma (or saliva) and 200. Mu.L of the protein precipitant containing the internal standard formulated in example one were added to a 1.5 mL polypropylene centrifuge tube (the same procedure as 2.1.).
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 one was removed, added to a 1.5 mL polypropylene centrifuge tube containing 90 μ L of blank plasma (or saliva), and vortexed to obtain a series of standard curve samples. 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, licarbazem 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 μ g/mL; the concentrations of the samples of the calibration curve 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 into a 1.5 mL polypropylene centrifuge tube containing 90 μ L of blank plasma (or saliva), and vortexed to obtain HQC, MQC, LQC, and LLOQ samples. To 50. Mu.L of quality control sample, 200. Mu.L of protein precipitant containing an internal standard was added (the same procedure as in 2.1.). 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 samples were at the following concentrations: 37.5, 7.5, 0.15 and 0.05 mu 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 mu g/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 taken into a 1.5 mL polypropylene centrifuge tube and 200 μ L of protein precipitant containing an internal standard was added (the same procedure as for 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 analytical 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 analysis 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 has a specification of 1.7 μm and 2.1 × 50 mm; mobile phase a was an aqueous solution containing 5mM ammonium acetate and 0.1% formic acid, mobile phase B was acetonitrile, the column temperature was 40 ℃, and the flow rate was 0.3-0.5 mL/min. The elution procedure for the 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,0.5 mL/min) → 4.5 min (95% B,0.5 mL/min) → 4.6 min (10% B,0.3 mL/min) → 5.5 min (10% B,0.3 mL/min) → 5 min) (stopping injection). 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: 5500V, (positive) 4500V, (negative); 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 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 curves established in the Analyst software were well linear 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 P22 patient in fig. 8 indicates that levetiracetam was included in the patient's dosing regimen, but 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 further more close therapeutic drug monitoring can be realized for some special patients or patients with treatment risks (for example, the patients who are easy to miss, miss or hide medicines, children, pregnant women, patients with liver and kidney damage and other special groups 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 a treatment scheme in time (fig. 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 schemes, 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 subjects have the conditions of concealed medicines, missed medicines and the like and evaluating the rationality of the administration dosage and the medication scheme.
While the present invention has been described with reference to the particular illustrative embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but is intended to cover various modifications, equivalent arrangements, and equivalents thereof, which may be made by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A method for rapidly detecting the concentration of an antiepileptic drug comprising one or more of levetiracetam, carbamazepine, lamotrigine, gabapentin, zonisamide, pamidone, clonazepam, lacosamide, valproic acid, phenytoin, piraampane, carbamazepine-10,11-epoxide, and licarbazepine in a clinical sample, 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 initial mobile phase of the gradient elution is that the volume ratio of the mobile phase A to the mobile phase B is 90; 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: 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.
2. The method of claim 1 wherein the internal standard for antiepileptic drugs comprises levetiracetam-d 6, carbamazepine-d 10, lamotrigine- 13 C, 15 N 4 Gabapentin-d 6,One or more of zonisamide-d 4, prometrone-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: the levetiracetam-d 6, the carbamazepine-d 10, the lamotrigine- 13 C, 15 N 4 The gabapentin-d 6, the prometrone-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 1, wherein the gradient elution procedure 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, stopping sampling, wherein the flow rate of the mobile phase is 0.3 mL/min.
7. The method of claim 3, wherein the volume ratio of the clinical sample to the protein precipitant comprising the internal standard is 1.
8. The method of claim 1, wherein the mass spectrometry conditions 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-).
9. The method of claim 1, wherein 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 ℃.
10. The method according to claim 1, wherein the before and after the detection comprises a step of washing the injection needle with a multi-component needle wash comprising a methanol-acetonitrile-isopropanol-water-formic acid mixed solution (25.
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| 代静 等: "超高效液相色谱-串联质谱法同时测定血清中12种抗癫痫药物" * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115248263A (en) * | 2021-04-28 | 2022-10-28 | 四川大学华西医院 | HPLC-MS/MS method for quantitatively detecting antiepileptic drug in saliva |
| CN115248263B (en) * | 2021-04-28 | 2023-12-15 | 四川大学华西医院 | HPLC-MS/MS method for quantitatively detecting antiepileptic drugs in saliva |
| CN117092046A (en) * | 2023-08-03 | 2023-11-21 | 首都医科大学附属北京安定医院 | Method for detecting whether oral cavity of mental patient is hidden with medicine |
| CN117092046B (en) * | 2023-08-03 | 2024-03-08 | 首都医科大学附属北京安定医院 | Method for detecting whether oral cavity of mental patient is hidden with medicine |
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| CN115639306B (en) | 2023-03-28 |
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