CN112946100A - Method and kit for determining carbamazepine and 10-hydroxycarbazepine and application thereof - Google Patents
Method and kit for determining carbamazepine and 10-hydroxycarbazepine and application thereof Download PDFInfo
- Publication number
- CN112946100A CN112946100A CN202110105348.7A CN202110105348A CN112946100A CN 112946100 A CN112946100 A CN 112946100A CN 202110105348 A CN202110105348 A CN 202110105348A CN 112946100 A CN112946100 A CN 112946100A
- Authority
- CN
- China
- Prior art keywords
- carbamazepine
- hydroxycarbazepine
- mobile phase
- acetonitrile
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention provides a method for determining carbamazepine and 10-hydroxycarbazepine, a kit and application thereof, and belongs to the technical field of medicine detection. The method adopts a precipitated protein method combined with HPLC-MS/MS to determine the content of antiepileptic drugs of carbamazepine and 10-hydroxycarbazepine in human plasma, fully verifies the determination method of the antiepileptic drugs of carbamazepine and 10-hydroxycarbazepine in human plasma from the aspects of specificity, linearity, sensitivity, accuracy, precision, matrix effect, recovery rate, stability and the like, is finally applied to human pharmacokinetic research of oral administration, and can calculate pharmacokinetic parameters through a non-atrioventricular model, thereby having good value of practical application.
Description
Technical Field
The invention belongs to the technical field of medicine detection, and particularly relates to a method for determining carbamazepine and 10-hydroxycarbazepine, a kit and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Epilepsy is a functional disorder caused by abnormal discharge of cerebral neuron cells, is induced by various reasons, has the characteristics of short, sudden and repeated attack, and brings heavy physical and psychological pain and medical burden to patients and families. Generally, patients with established epilepsy need to take antiepileptic drugs on a regular basis to reduce the cognitive function invasion caused by recurrent seizures. Carbamazepine and oxcarbazepine are used as clinical common antiepileptic drugs, and the combined treatment of the drugs is commonly used for intractable epilepsy. The anticonvulsant mechanism of carbamazepine is not clear and it may be able to increase the inactivation efficacy of sodium channels, restrict postsynaptic neurons and block presynaptic Na+Channels, thereby limiting the action potentials of pre-and post-synaptic neurons. Oxcarbazepine is one of the carbamazepine derivatives, and a plurality of researches prove that oxcarbazepine is an antiepileptic drug with definite curative effect and high safety, and a metabolic active product 10-hydroxycarbazepine is usually taken as the focus of clinical drug concentration detection. When oxcarbazepine and carbamazepine are used together, the simultaneous detection of carbamazepine and 10-hydroxycarbazepine with very similar properties is a new problem for the detection of antiepileptic drugs.
Papers on the quantification of antiepileptic drugs in biological fluids using high performance liquid chromatography-ultraviolet chromatography (HPLC-UV), Gas Chromatography (GC) and high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) have been published. Liu Xuan et al established a content determination method for simultaneously detecting 4 anti-epileptic drugs in 2020 by HPLC-UV method, but the analysis time is too long for 40 minutes to realize high throughput. The generation of quiet et al reported that HPLC-MS/MS was used for the first time to measure 12 antiepileptic drugs in serum, and the method detects carbamazepine and oxcarbazepine and does not detect 10-hydroxycarbazepine because the difference between the properties of carbamazepine and 10-hydroxycarbazepine is small and the separation on a common C18 chromatographic column is difficult. The reports of Jiangyuan et al develop a precipitated protein method combined with an ultra-high performance liquid chromatography method, the method uses 10 mu L of sample volume, the lowest limit of quantitation (LLOQ) of carbamazepine and 10-hydroxycarbazepine is only 1 mu g/mL and 2.5 mu g/mL, the high sample injection amount cannot obtain ideal limit of quantitation, the detection of clinical drug concentration is difficult, the total analysis time is as long as 24 minutes, the chromatographic peak is trailing, and the baseline is higher. Therefore, in the pharmacokinetic research, a method for simultaneously measuring the content of the active metabolite 10-hydroxycarbazepine of carbamazepine and oxcarbazepine with high sensitivity and high resolution is provided to be in urgent need to be solved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for measuring carbamazepine and 10-hydroxycarbazepine, a kit and application thereof. The method adopts a precipitated protein method combined with HPLC-MS/MS to determine the content of antiepileptic drugs of carbamazepine and 10-hydroxycarbazepine in human plasma, fully verifies the determination method of the antiepileptic drugs of carbamazepine and 10-hydroxycarbazepine in human plasma from the aspects of specificity, linearity, sensitivity, accuracy, precision, matrix effect, recovery rate, stability and the like, is finally applied to human pharmacokinetic research of oral administration, and can calculate pharmacokinetic parameters through a non-atrioventricular model, thereby having good value of practical application.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided a method for the determination of carbamazepine and 10-hydroxycarbazepine, said method comprising: preparing a standard curve by using a standard substance for quantification, simultaneously performing quality control by using a quality control substance, and detecting a sample to be detected based on HPLC-MS/MS;
specifically, quality control is performed by adopting quality control products at four levels of quantitative lower limit, low level, medium level and high level or by adopting quality control products at three levels of low level, medium level and high level.
Wherein the quantitative lower limit, the low concentration, the medium concentration and the high concentration of the quality control products of the carbamazepine and the 10-hydroxycarbazepine are respectively 0.1, 0.25, 2.5 and 25 ng/mL.
The preparation method of the sample to be detected comprises the following steps: mixing the test sample with the internal standard working solution, centrifuging and taking the supernatant to obtain the product.
The test sample is a subject blood sample, including whole blood, plasma or serum, and more preferably plasma.
The internal standard working solution is a carbamazepine (carbamazepine-d 10) and 10-hydroxycarbazepine (10-hydroxycarbazepine-d 3) solution with isotope internal standards, and the preparation method specifically comprises the following steps: dissolving the raw materials of the isotope internal standard carbamazepine and the 10-hydroxycarbazepine with acetonitrile to prepare an internal standard stock solution, and then diluting the internal standard stock solution by using a precipitated protein solvent.
The precipitated protein solvent is a mixed solution of acetonitrile and methanol, and the volume ratio of the acetonitrile to the methanol is 2-4: 1-3, preferably 3: 2. At this dosage-to-volume ratio, the precipitation efficiency is highest.
The specific method for detecting the sample to be detected by HPLC-MS/MS comprises the following steps:
the liquid chromatography conditions include:
gradient elution was used, mobile phase a: water (2mM ammonium acetate), mobile phase B phase: methanol;
the chromatographic column is a C18 chromatographic column; the flow rate of the mobile phase is 0.3-0.5 ml/min (preferably 0.4 ml/min); the column temperature is 25-40 ℃ (preferably 35 ℃); the sample injection amount is 0.5-2 muL (preferably 1 muL);
in particular, the chromatographic column is a Kinetex Polor C18 chromatographic column, and researches show that the chromatographic column can separate carbamazepine and 10-hydroxycarbazepine in a short time, has symmetrical peak shapes and is reliable in method. Under the optimized high performance liquid chromatography conditions, the retention time of the carbamazepine and the 10-hydroxycarbazepine is 2.13 and 2.75 minutes; the total run time was 4.5 minutes. Thereby significantly shortening the detection time.
The gradient elution mode is specifically as follows: 0-1.5min, and 5-5% of mobile phase B; 1.5-1.9min, and 5-70% of mobile phase B; 1.9-2.0min, and 70-95% of mobile phase B; 2.0-3.5min, 95-95% of mobile phase B; 3.5-3.6min, and 95-5% of mobile phase B; 3.6-4.5min, and 5-5% of mobile phase B.
The mass spectrometry conditions include:
an ion source: electrospray (ESI); the scanning mode is as follows: multiple Reaction Monitoring (MRM); an ionization mode: a positive ion; ion source voltage: 5000V; ion source temperature: 650 ℃; air curtain air: 15 psi; atomizing: 45 psi; auxiliary gas: 55 psi.
In a second aspect of the invention, there is provided a kit for detecting carbamazepine and 10-hydroxycarbazepine, said kit comprising, a standard of carbamazepine and 10-hydroxycarbazepine;
standard of carbamazepine-d 10 and 10-hydroxycarbazepine-d 3;
the diluent is acetonitrile or acetonitrile/methanol mixed solution;
the volume ratio of the acetonitrile/methanol mixed solution is 2-4: 1-3, and preferably 3: 2.
Further, the kit also comprises blank plasma.
In a third aspect of the invention there is provided the use of a method and/or test kit as described above for the determination of carbamazepine and 10-hydroxycarbazepine in pharmacokinetic studies.
Specifically, the application includes: the pharmacokinetics of orally administered drugs in humans were studied based on the above-described test methods.
Compared with the prior art, one or more technical schemes have the following beneficial technical effects:
the technical scheme provides a method for determining the content of antiepileptic drugs of carbamazepine and 10-hydroxycarbazepine in human plasma by combining a precipitated protein method with HPLC-MS/MS, fully verifies the determination of the carbamazepine and 10-hydroxycarbazepine in the human plasma from the aspects of specificity, linearity, sensitivity, accuracy, precision, matrix effect, recovery rate, stability and the like, and is finally applied to human pharmacokinetic research of oral administration. The technical scheme provides an efficient protein precipitation extraction method for carbamazepine and 10-hydroxycarbazepine for the first time, and the protein precipitation extraction method shows obvious superiority in human body pharmacokinetic research.
The liquid chromatography-mass spectrometry quantitative determination method has the advantages of accuracy, reliability, high sensitivity, lower specificity detection limit and quantitative limit and the like, and has good practical application value for analyzing the content of the drug components in the blood plasma.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 shows the structural formulae and cleavage patterns of carbamazepine, 10-hydroxycarbazepine, carbamazepine-D10 and 10-hydroxycarbazepine-D3 in example 1 of the present invention (A: carbamazepine, B: 10-hydroxycarbazepine, C: carbamazepine-D10, D: 10-hydroxycarbazepine-D3);
figure 2 is a plot of mean plasma concentration versus time following oral administration of carbamazepine, oxcarbazepine;
FIG. 3 is a chromatogram and mass spectrum of carbamazepine and 10-hydroxycarbazepine established in example 1 of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
As mentioned above, when oxcarbazepine is used in combination with carbamazepine, the simultaneous detection of carbamazepine and 10-hydroxycarbazepine, which have very similar properties, is a new problem in the detection of antiepileptic drugs.
In view of the above, the invention provides a method for determining the content of antiepileptic drugs, namely carbamazepine and 10-hydroxycarbazepine, in human plasma by combining a precipitated protein method with HPLC-MS/MS, optimizes a sample pretreatment method, instrument detection conditions and the like, fully verifies the determination of the carbamazepine and the 10-hydroxycarbazepine in the human plasma from the aspects of specificity, linearity, sensitivity, accuracy, precision, matrix effect, recovery rate, stability and the like, and is finally applied to human pharmacokinetic research of oral administration.
In one embodiment of the present invention, the quantitative determination method comprises the steps of:
(1) accurately weighed standard reference substances, namely carbamazepine and 10-hydroxycarbazepine, are dissolved by acetonitrile respectively to obtain stock solutions of carbamazepine and 10-hydroxycarbazepine, wherein the final concentration of the carbamazepine and the 10-hydroxycarbazepine is 1000 mu g/mL. The accurate volume of 0.10mL carbamazepine and 10-hydroxycarbazepine standard solution is respectively transferred into a 10mL volumetric flask, and the volume is determined by acetonitrile, thus obtaining the working solution of 10 mu g/mL carbamazepine and 10-hydroxycarbazepine. Dilution with acetonitrile yielded working solutions of 2, 4, 10, 20, 100, 200, 400 and 600 ng/mL. Meanwhile, 10.00mg of carbamazepine-d 10 and 10-hydroxycarbazepine-d 3 bulk drug with accurate weights were transferred to a 10mL volumetric flask and dissolved with a certain volume of acetonitrile to prepare an internal standard stock solution of 1000. mu.g/mL. The working solution concentration of carbamazepine-d 10 and 10-hydroxycarbazepine-d 3 is 50ng/mL, and the dilution solvent is Acetonitrile (ACN) and Methanol (MEOH) is 3:2 (V/V). All drug stocks were stored in light-shielded containers at 4 ℃ for at least 60 days with no change.
Calibration standards were prepared in drug concentration points in the plasma of 0.1, 0.2, 0.25, 0.5, 1.0, 2.5, 5.0, 10, 20, 25, 30ng/mL carbamazepine and 10-hydroxycarbazepine. For accuracy and precision studies, QCs was prepared as 6 replicates at 4 concentration levels, including lower quantitative limit (LLOQ), low (L: defined as three times LLOQ), medium (M: defined as medium range), and high (H: defined as high range), according to FDA guidelines for selecting quality control points (QCs). For the other experiments (during analysis of samples from volunteers), only samples at 3 concentration levels (LQC, MQC and HQC) were used. For carbamazepine and 10-hydroxycarbazepine, LLOQ, LQC, MQC and HQC were prepared at 0.1, 0.25, 2.5 and 25ng/mL, respectively.
mu.L of human blank plasma was placed in a 2.0mL centrifuge tube and a precise volume of 5. mu.L of carbamazepine and 10-hydroxycarbazepine 2-600ng/mL working solution was added to obtain a plasma concentration of 0.1-30ng/mL of carbamazepine and 10-hydroxycarbazepine. Then 150. mu.L of precipitated protein solvent (ACN: MEOH ═ 3:2) containing 50ng/mL carbamazepine-d 10 and 10-hydroxycarbazepine-d 3 was added and the analytes and internal isotope standards were extracted with shaking for 10min and the upper organic and lower aqueous phases were separated by centrifugation at 14000rpm for 15min at 4 ℃. Dissolving 100 mu L of supernatant into 100 mu L of water phase, and mixing for 2 minutes by vortex to obtain a reference substance solution;
(2) and (3) determination: carrying out HPLC-MS/MS analysis on the reference substance solution obtained in the step (1), and carrying out gradient elution to obtain a mobile phase A: water (2mM ammonium acetate), mobile phase B phase: methanol.
In yet another embodiment of the present invention, in step (1), 45. mu.L of human blank plasma is placed in a 2.0mL centrifuge tube and a precise volume of 5. mu.L of the working solution of carbamazepine and 10-hydroxycarbazepine 2-600ng/mL is added to obtain a plasma concentration of carbamazepine and 10-hydroxycarbazepine of 0.1-30 ng/mL. Then, 100-200. mu.L of precipitated protein solvent (ACN: MEOH. RTM.3: 2, V/V) containing 50ng/mL of carbamazepine-d 10 and 10-hydroxycarbazepine-d 3 was added and the analyte and the isotope internal standard were extracted with shaking for 10min and the upper organic phase and the lower aqueous phase were separated by centrifugation at 13000-15000rpm for 10-20min at 4 ℃. 100 μ L of the supernatant was dissolved in 100 μ L of the aqueous phase and vortexed for 2 minutes to provide a control solution.
The present invention has made several attempts on mobile phase systems in order to obtain satisfactory chromatographic behavior and to maximize the ionization response of the analyte and the isotopic internal standard. In view of stability of the pH range of the mobile phase and elimination of splitting of chromatographic peaks, 2mM ammonium acetate was added to the mobile phase. Elution experiments were performed on the analytes and the isotopic internal standard with different ratios of methanol-water and acetonitrile-water, and methanol was found to have lower background noise and better resolution than acetonitrile. The Aijier Feinuomei Kinetex Polor C18 chromatographic column has better retention effect on carbamazepine and 10-hydroxycarbazepine, and the response of 2mM ammonium acetate on the carbamazepine and 10-hydroxycarbazepine is obviously enhanced. Since 10-hydroxycarbazepine has an additional hydroxyl group at position 10 of the structure of carbamazepine, the 10-hydroxycarbazepine molecule will be cleaved in the ion source portion, resulting in the quantitative parent molecular weight of carbamazepine. The Kinetex Polor C18 chromatographic column can separate the carbamazepine and the 10-hydroxycarbazepine in a short time (the separation degree is more than 1.5), and the method is reliable due to the symmetrical peak shape. Under optimized high performance liquid chromatography conditions, carbamazepine and 10-hydroxycarbazepine were detected with retention times of 2.13 and 2.75 minutes. The total run time was 4.5 minutes.
The invention optimizes the positive ion mode by utilizing HPLC-MS/MS analysis and MS parameters, and improves the response of MRM measurement to an ESI source. The fragmentation of the MRM is converted to m/z 237.1 to 194.1 for carbamazepine (FIG. 1), m/z 247.1 to 204.1 for carbamazepine-d 10 (FIG. 1), m/z 255.1 to 237.1 for 10-hydroxycarbazepine (FIG. 1) and m/z 258.1 to 240.1 for 10-hydroxycarbazepine-d 3 (FIG. 1), the residence time of each conversion being 300 ms. The collision energy per compound was 22 ev. The separation potential was maintained at 40V. The source parameters for all analytes were 15psi curtain gas, medium collision gas (CAD), 650 deg.C, 5000V ion ejection voltage, and 60psi ion source gas.
In the optimization aspect of sample preparation, compared with an ethyl acetate liquid-liquid extraction method, the protein precipitation method has the advantages of high precision, high recovery rate, simplicity in operation and the like. The sample preparation was performed by protein precipitation. The quantitation limits for carbamazepine and 10-hydroxycarbazepine are useful for the quantitative analysis of pharmacokinetics in human plasma samples.
Initially, the solvents for precipitation of the protein were acetonitrile and methanol, but this resulted in a partial loss of carbamazepine and 10-hydroxycarbazepine content, probably due to the inability of acetonitrile and methanol to effectively desorb the analyte from the protein. Factors that influence the charge state distribution include solvent pH and drug solubility. When acetonitrile is used: as the precipitating protein solvent, methanol 3:2 was found to precipitate most efficiently, so acetonitrile was chosen: methanol 3:2 as the precipitated protein solvent.
In still another embodiment of the present invention, the column in the step (2) is a Kinetex Polor C18 column (2.1 mm. times.100 mm, 2.7 μm, Ijel Feynomel, Inc.); the flow rate of the mobile phase is 0.4 ml/min; the column temperature was 35 ℃; the sample size was 1. mu.L.
The present invention examined the effect of four flow rates (0.3mL/min,0.4mL/min and 0.5mL/min) on the assay results. The results show that: when the flow rate is 0.4mL/min, the separation effect is optimal, the retention time of each chromatographic peak is proper, the separation degree is good, the base line is stable, and the peak shapes are symmetrical, so that the flow rate is selected to be 0.4 mL/min.
Meanwhile, the invention tests the influence of four different column temperatures (such as 25 ℃, 30 ℃, 35 ℃ and 40 ℃) on the mass spectrum chromatographic detection result. The result shows that when the column temperature is 35 ℃, the retention time of chromatographic peaks is appropriate, the base line is stable, the resolution of each chromatographic peak is good, and the peak shapes are symmetrical, so the column temperature is selected to be 35 ℃.
Mass spectrometry parameters for carbamazepine, 10-hydroxycarbazepine, carbamazepine-d 10 and 10-hydroxycarbazepine-d 3 are shown in Table 1.
Table 1 mass spectral parameters of analytes and isotopic internal standards
The mass spectrum conditions are optimized simultaneously, the mass spectrum conditions of the analyte and the isotope internal standard are optimized by adopting a multi-reaction ion detection mode (MRM) of the API5500 type triple quadrupole mass spectrometer, the high-response peak of each pair of ions is ensured, the detection results are shown in table 1, and specific parent ions and specific daughter ions are found from the analyte and the isotope internal standard and are used for quantitative analysis.
In the selection of the analysis time, the invention records 10min chromatogram when selecting the elution time of the chromatogram. The result shows that no obvious chromatographic peak appears after 4.5min, and in order to take care of the difference of the batch samples, the characteristic peaks of all the batch samples can be detected, so 4.5min is selected as the analysis time.
In another embodiment of the present invention, the gradient elution mode in step (3) is: 0-1.5min, and 5-5% of mobile phase B; 1.5-1.9min, and 5-70% of mobile phase B; 1.9-2.0min, and 70-95% of mobile phase B; 2.0-3.5min, 95-95% of mobile phase B; 3.5-3.6min, and 95-5% of mobile phase B; 3.6-4.5min, and 5-5% of mobile phase B;
in another embodiment of the present invention, the medium mass spectrum conditions are: an ion source: electrospray (ESI); the scanning mode is as follows: multiple Reaction Monitoring (MRM); an ionization mode: a positive ion; ion source voltage: 5000V; ion source temperature: 650 ℃; air curtain air: 15 psi; atomizing: 45 psi; auxiliary gas: 55 psi;
in yet another embodiment of the invention, carbamazepine-d 10 and 10-hydroxycarbazepine-d 3 are used as isotopic internal standard compounds.
According to the invention, an HPLC-MS/MS liquid chromatography-mass spectrometry analysis method is adopted, a selective ion detection (SIM) mode is tried to determine in the experimental process, the response of each component is low and the baseline is high, the matrix influence is large, and quantitative analysis cannot be realized, however, when a multi-reaction detection (MRM) method is used for scanning parent ions and daughter ions of characteristic fragments, the response intensity of an ion peak is obviously higher than that of the selective ion detection (SIM) mode, and the baseline is low, so that the quantitative analysis can be realized. Therefore, the experiment selects a multi-reaction detection (MRM) scanning mode for quantifying the carbamazepine and the 10-hydroxycarbazepine, and the defects of long time consumption, difficult separation, high detection limit and the like exist in the conventional liquid phase method for detection, so that the method is not beneficial to the test.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
A method for determining the content of antiepileptic drugs of carbamazepine and 10-hydroxycarbazepine in human plasma by combining a protein precipitation method with HPLC-MS/MS comprises the following steps:
the first step is as follows:
preparation of a reference solution: accurately weighed standard reference substances, namely carbamazepine and 10-hydroxycarbazepine, are dissolved by acetonitrile respectively to obtain stock solutions of carbamazepine and 10-hydroxycarbazepine, wherein the final concentration of the carbamazepine and the 10-hydroxycarbazepine is 1000 mu g/mL. The accurate volume of 0.10mL carbamazepine and 10-hydroxycarbazepine standard solution is respectively transferred into a 10mL volumetric flask, and the volume is determined by acetonitrile, thus obtaining the working solution of 10 mu g/mL carbamazepine and 10-hydroxycarbazepine. Dilution with acetonitrile yielded working solutions of 2, 4, 10, 20, 100, 200, 400 and 600 ng/mL. Meanwhile, 10.00mg of carbamazepine-d 10 and 10-hydroxycarbazepine-d 3 bulk drug with accurate weights were transferred to a 10mL volumetric flask and dissolved with a certain volume of acetonitrile to prepare an internal standard stock solution of 1000. mu.g/mL. The working solution concentration of carbamazepine-d 10 and 10-hydroxycarbazepine-d 3 is 50ng/mL, and the dilution solvent is Acetonitrile (ACN) and Methanol (MEOH) which are 3: 2. All drug stocks were stored in light-shielded containers at 4 ℃ for at least 60 days with no change.
Calibration standards were prepared in drug concentration points in the plasma of 0.1, 0.2, 0.25, 0.5, 1.0, 2.5, 5.0, 10, 20, 25, 30ng/mL carbamazepine and 10-hydroxycarbazepine. For accuracy and precision studies, QCs was prepared as 6 replicates at 4 concentration levels, including lower quantitative limit (LLOQ), low (L: defined as three times LLOQ), medium (M: defined as medium range), and high (H: defined as high range), according to FDA guidelines for selecting quality control points (QCs). For the other experiments (during analysis of samples from volunteers), only samples at 3 concentration levels (LQC, MQC and HQC) were used. For carbamazepine and 10-hydroxycarbazepine, LLOQ, LQC, MQC and HQC were prepared at 0.1, 0.25, 2.5 and 25ng/mL, respectively.
mu.L of human blank plasma was placed in a 2.0mL centrifuge tube and a precise volume of 5. mu.L of carbamazepine and 10-hydroxycarbazepine 2-600ng/mL working solution was added to obtain a plasma concentration of 0.1-30ng/mL of carbamazepine and 10-hydroxycarbazepine. Then 150. mu.L of precipitated protein solvent (ACN: MEOH ═ 3:2, V/V) containing 50ng/mL carbamazepine-d 10 and 10-hydroxycarbazepine-d 3 was added and the analytes and internal isotope were extracted with shaking for 10min and the upper organic phase and lower aqueous phase were separated by centrifugation at 14000rpm for 15min at 4 ℃. Dissolving 100 mu L of supernatant into 100 mu L of water phase, and mixing for 2 minutes by vortex to obtain a reference substance solution;
the second step is that:
and (3) determination: carrying out HPLC-MS/MS analysis on the reference substance solution obtained in the step (1), and carrying out gradient elution to obtain a mobile phase A: water (2mM ammonium acetate), mobile phase B phase: methanol.
In this example, the column was a Kinetex Polor C18 column (2.1 mm. times.100 mm, 2.7 μm, Ejel Feinum); the flow rate of the mobile phase is 0.4 ml/min; the column temperature was 35 ℃; the sample size was 1. mu.L. The mass spectrum parameters of each effective component are shown in table 1. The gradient elution mode is as follows: 0-1.5min, and 5-5% of mobile phase B; 1.5-1.9min, and 5-70% of mobile phase B; 1.9-2.0min, and 70-95% of mobile phase B; 2.0-3.5min, 95-95% of mobile phase B; 3.5-3.6min, and 95-5% of mobile phase B; 3.6-4.5min, and 5-5% of mobile phase B. FIG. 3 is a chromatogram of a carbamazepine and 10-hydroxycarbazepine chromatogram established in accordance with the present invention.
The mass spectrum conditions are as follows: an ion source: electrospray (ESI); the scanning mode is as follows: multiple Reaction Monitoring (MRM); an ionization mode: a positive ion; ion source voltage: 5000V; ion source temperature: 650 ℃; air curtain air: 15 psi; atomizing: 45 psi; auxiliary gas: 55 psi;
the third step:
the method for investigating the feasibility of the established high performance liquid chromatography-tandem mass spectrometry method comprises specificity, quantitative limit, precision, accuracy, stability, matrix effect and extraction recovery rate.
The specificity is as follows: specific and endogenous interferences were assessed by comparing chromatograms of six persons blank plasma samples, clinical plasma samples obtained from one of the subjects at 0.5 hours after the last dose, plasma samples with carbamazepine and 10-hydroxycarbazepine added at a dose of 30ng/mL and plasma samples with carbamazepine and 10-hydroxycarbazepine added at a dose of 0.1 ng/mL. Specificity and exogenous interference were assessed by comparing chromatograms of LLOQ and isotopic internal standard (50ng/mL) of 50% acetonitrile and tri-distilled water (0.1ng/mL) with addition of carbamazepine and 10-hydroxycarbazepine. All blank plasma samples were prepared and analyzed to ensure that there were no interfering peaks. Under the established chromatographic conditions, there was no endogenous interference in plasma, indicating that the selectivity of the method was acceptable;
and (4) quantitative limit: by 1/X2A weighted linear least squares regression model, using peak area ratios of carbamazepine/carbamazepine-d 10, 10-hydroxycarbazepine/10-hydroxycarbazepine-d 3 to plasma concentrations to construct calibration curves. LLOQ indicates the lowest concentration of analyte in the linear range, and can be used with acceptable essenceThe degree and accuracy were determined.
Precision: six replicate samples of LLOQ and QC at four concentrations (0.1, 0.25, 2.5, and 25ng/mL) were analyzed on the same day to assess precision and accuracy within the day. Daytime precision and accuracy were assessed by analyzing LLOQ and QC samples for three consecutive days. The precision and accuracy of the method are expressed in Relative Standard Deviation (RSD) and Relative Error (RE), respectively. Neither RSD nor RE must exceed 15%. However, at LLOQ, RE and RSD < + > 20% are acceptable. The precision and accuracy results for carbamazepine and 10-hydroxycarbazepine in the LLOQ and QC samples are shown in Table 2. The precision (RSD) of each sample level of carbamazepine and 10-hydroxycarbazepine was less than 9.99%. The accuracy of the sample levels of carbamazepine and 10-hydroxycarbazepine was between 1.48% and 8.31%, with measurements within acceptable ranges.
TABLE 2 precision and accuracy of the method for determining the content of carbamazepine and 10-hydroxycarbazepine in human plasma
Matrix effect and extraction recovery: the extraction recovery was performed by comparing the absolute peak area of the analyte to IS ratio extracted from three levels of QC samples prepared in 6 different batches of plasma with the absolute peak area of blank plasma, highly hemolytic plasma and high fat plasma extracted followed by fortification of LQC, MQC, HQC with the same concentration of pure analyte solution. Matrix effects were assessed by comparing the absolute peak area of analyte in the six samples at LQC, MQC, HQC levels in the blank plasma extract fortified with the same concentration level of analyte in the blank water extract fortified with the IS ratio. In human plasma blanks, the average matrix effect for homogenization of the standard carbamazepine and 10-hydroxycarbazepine is 101.2-106.0%, while the average matrix effect for the high hemolysis is 99.9-101.4%. The matrix effect of carbamazepine and 10-hydroxycarbazepine is 95.5-101.1% in high-lipid plasma. As shown in table 3, all relative standard deviation values were between 0.75% and 9.17%, indicating that the effect of plasma matrix was negligible for the analysis. The average extraction recovery rate of the homogenized standard substance of carbamazepine and 10-hydroxycarbazepine is 96.7-103.8%, and the extraction recovery rate results of carbamazepine and 10-hydroxycarbazepine under different concentrations are accurate and good in reproducibility.
Table 3 extraction recovery and matrix effect of the method for determining the content of carbamazepine and 10-hydroxycarbazepine in human plasma (n ═ 6).
And (3) stability test: three QC samples at different concentrations were analyzed for stability under different conditions: (1) three consecutive freeze-thaw cycles (from-20 ℃ to 23 ℃); (2) the first 3h of preparation at room temperature (23 ℃); (3) 20 hours after the preparation at the refrigerator temperature (4 ℃) and 6 hours after the preparation at the room temperature (23 ℃); (4) the automatic sample injector is prepared for 24 hours at 10 ℃; (5) the refrigerator temperature (-20 ℃) was 3, 8, 31 days before preparation. The stability of the solution was evaluated by comparing the average concentration of the stored QC samples and the freshly prepared samples. The samples were considered stable with a deviation from the nominal concentration of ± 15.0%. All stability test samples were analyzed in 6 replicates and deviations were determined from freshly prepared samples. After three freeze-thaw cycles and at room temperature for at least 3h, there was no significant difference (< 15%) in the CV% (7.35%) responses of carbamazepine and 10-hydroxycarbazepine, indicating that carbamazepine and 10-hydroxycarbazepine are stable under these conditions. The CV values (11.92%) of the plasma samples were stable over at least three freeze/thaw cycles. The treated samples were stable in the autosampler for 24 hours and in the room temperature tray for 3 hours with CV% values of at least 6.88% and 7.35%, respectively. Plasma samples were stable at-20 ℃ for at least 4 weeks with no significant loss (< 8.43%). The results are shown in tables 4 and 5.
Table 4 sample stability of carbamazepine and 10-hydroxycarbazepine (n ═ 6, expressed as Mean ± R.E%)
Table 5 carbamazepine and 10-hydroxycarbazepine solution stability (n ═ 6)
Example 2
A method for determining the content of antiepileptic drugs of carbamazepine and 10-hydroxycarbazepine in human plasma by combining a protein precipitation method with HPLC-MS/MS and application thereof comprise the following steps:
the first step is as follows:
pharmacokinetic studies were performed on 12 healthy male subjects. The ethical committee approved the protocol and the volunteers had informed written consent. Fasting was 12 hours before dosing and 3 hours after dosing. Following oral administration of carbamazepine and oxcarbazepine, 2mL blood samples were drawn from the jugular vein before and at 0. Then blood was taken at 0.083, 0.167, 0.333, 0.5, 0.75, 1, 1.25, 1.75, 2, 2.5, 3, 4, 5, 6, 8, 12 and 24 hours, respectively. During the experiment, water can be freely drunk. Plasma was then prepared by centrifugation at 14000 Xg for 10 minutes and immediately frozen at-80 ℃.
mu.L of human blank plasma was placed in a 2.0mL centrifuge tube, and the analyte and isotope internal standard were extracted by adding 150. mu.L of precipitated protein solvent (ACN: MEOH 3:2, V/V) containing 50ng/mL isotope internal standard with shaking for 10min, and the upper organic phase and lower aqueous phase were separated by centrifugation at 14000rpm for 15min at 4 ℃. Dissolving 100 mu L of supernatant into 100 mu L of water phase, and mixing for 2 minutes by vortex to obtain a test solution;
the second step is that:
and (3) determination: carrying out HPLC-MS/MS analysis on the test solution obtained in the step (1), and adopting gradient elution to obtain a mobile phase A: water (2mM ammonium acetate), mobile phase B phase: methanol.
Pharmacokinetic analysis adopted DAS2 non-compartmental model software program (China Committee for mathematical and pharmacological sciences, Shanghai, China) to calculate AUC and Cmax、Tmax、T1/2Vz/F and CLz/F. Data are expressed as Mean ± SD.
The mean plasma concentration-time curves after oral administration of carbamazepine and oxcarbazepine are shown in figure 2. The pharmacokinetic parameters are shown in table 6.
Table 6 non-compartmental pharmacokinetic parameters after oral administration of carbamazepine and oxcarbazepine (mean ± standard deviation, n ═ 12)
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A method for assaying carbamazepine and 10-hydroxycarbazepine comprising: and preparing a standard curve by using a standard substance for quantification, performing quality control by using a quality control substance, and detecting a sample to be detected based on HPLC-MS/MS.
2. The method of claim 1, wherein the quality control is performed using four levels of quality control materials of lower limit, low, medium and high of quantitation or three levels of quality control materials of low, medium and high of quantitation;
preferably, the lower limit, the low concentration, the medium concentration and the high concentration of the quality control substances of the carbamazepine and the 10-hydroxycarbazepine are respectively 0.1, 0.25, 2.5 and 25 ng/mL.
3. The method of claim 1, wherein the sample to be tested is formulated by: mixing a test sample with an internal standard working solution, centrifuging and taking a supernatant to obtain the test sample;
the test sample is a blood sample of a subject, including whole blood, plasma or serum, and is further preferably plasma;
the preparation method of the isotope internal standard carbamazepine (carbamazepine-d 10) and 10-hydroxycarbazepine (10-hydroxycarbazepine-d 3) solution comprises the following steps: dissolving the raw materials of the isotope internal standard carbamazepine and the 10-hydroxycarbazepine with acetonitrile to prepare an internal standard stock solution, and then diluting the internal standard stock solution by using a precipitated protein solvent.
4. The method according to claim 3, wherein the precipitated protein solvent is a mixed solution of acetonitrile and methanol, and the volume ratio of the acetonitrile to the methanol is 2-4: 1-3, preferably 3: 2.
5. The method of claim 1, wherein the HPLC-MS/MS is used for detecting the sample to be detected by the following specific method:
the liquid chromatography conditions include:
gradient elution was used, mobile phase a: water (2mM ammonium acetate), mobile phase B phase: methanol.
6. The method of claim 5, wherein the chromatography column is a C18 chromatography column; the flow rate of the mobile phase is 0.3-0.5 ml/min (preferably 0.4 ml/min); the column temperature is 25-40 ℃ (preferably 35 ℃); the amount of sample is 0.5-2 μ L (preferably 1 μ L).
7. The method according to claim 5, wherein the gradient elution mode is in particular: 0-1.5min, and 5-5% of mobile phase B; 1.5-1.9min, and 5-70% of mobile phase B; 1.9-2.0min, mobile phase B70-95%; 2.0-3.5min, 95-95% of mobile phase B; 3.5-3.6min, and 95-5% of mobile phase B; 3.6-4.5min, and 5-5% of mobile phase B.
8. The method of claim 5, wherein the mass spectrometry conditions comprise:
an ion source: electrospray (ESI); the scanning mode is as follows: multiple Reaction Monitoring (MRM); an ionization mode: a positive ion; ion source voltage: 5000V; ion source temperature: 650 ℃; air curtain air: 15 psi; atomizing: 45 psi; auxiliary gas: 55 psi.
9. A kit for detecting carbamazepine and 10-hydroxycarbazepine, comprising a standard of carbamazepine and 10-hydroxycarbazepine;
standard of carbamazepine-d 10 and 10-hydroxycarbazepine-d 3;
the diluent is acetonitrile or acetonitrile/methanol mixed solution;
preferably, the volume ratio of the acetonitrile/methanol mixed solution is 2-4: 1-3, and preferably 3: 2.
Preferably, the kit further comprises blank plasma.
10. Use of the method for the determination of carbamazepine and 10-hydroxycarbazepine according to any one of claims 1 to 8 and/or the kit according to claim 9 in pharmacokinetic studies;
preferably, the application comprises: the pharmacokinetics of orally administered human subjects were studied based on the test method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110105348.7A CN112946100A (en) | 2021-01-26 | 2021-01-26 | Method and kit for determining carbamazepine and 10-hydroxycarbazepine and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110105348.7A CN112946100A (en) | 2021-01-26 | 2021-01-26 | Method and kit for determining carbamazepine and 10-hydroxycarbazepine and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112946100A true CN112946100A (en) | 2021-06-11 |
Family
ID=76237148
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110105348.7A Pending CN112946100A (en) | 2021-01-26 | 2021-01-26 | Method and kit for determining carbamazepine and 10-hydroxycarbazepine and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112946100A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105136957A (en) * | 2015-09-11 | 2015-12-09 | 无锡市人民医院 | Detection method for simultaneously measuring OXC in human plasma and metabolite MHD and MHD-G |
| CN109655568A (en) * | 2019-01-22 | 2019-04-19 | 杭州度安医学检验实验室有限公司 | Efficient LC-MS measures the method and kit of 35 kinds of psychotropic agents simultaneously |
| CN111579680A (en) * | 2020-05-29 | 2020-08-25 | 南京品生医疗科技有限公司 | Detection kit for antiepileptic drug in serum and application thereof |
| CN111812216A (en) * | 2020-05-29 | 2020-10-23 | 南京品生医学检验实验室有限公司 | Method for detecting concentration of antiepileptic drug in serum |
-
2021
- 2021-01-26 CN CN202110105348.7A patent/CN112946100A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105136957A (en) * | 2015-09-11 | 2015-12-09 | 无锡市人民医院 | Detection method for simultaneously measuring OXC in human plasma and metabolite MHD and MHD-G |
| CN109655568A (en) * | 2019-01-22 | 2019-04-19 | 杭州度安医学检验实验室有限公司 | Efficient LC-MS measures the method and kit of 35 kinds of psychotropic agents simultaneously |
| CN111579680A (en) * | 2020-05-29 | 2020-08-25 | 南京品生医疗科技有限公司 | Detection kit for antiepileptic drug in serum and application thereof |
| CN111812216A (en) * | 2020-05-29 | 2020-10-23 | 南京品生医学检验实验室有限公司 | Method for detecting concentration of antiepileptic drug in serum |
Non-Patent Citations (2)
| Title |
|---|
| KASHIF UL HAQ，ET AL: "DEVELOPMENT AND VALIDATION OF LC-MS/MS METHOD FOR THE SIMULTANEOUSQUANTITATIVE ANALYSIS OF OXCARBAZEPINE AND ITS METABOLITE 10-HYDROXYCARBAZEPINE IN K2EDTA PLASMA", 《INTERNATIONAL JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES》 * |
| LEI YIN1,ET AL: "A Parallel‑Column LC–MS/MS Method for High‑Throughput Analysis of Eight Antiepileptic Drugs in Clinical Therapeutic Drug Monitoring", 《CHROMATOGRAPHIA》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ho et al. | Electrospray ionisation mass spectrometry: principles and clinical applications | |
| Dolder et al. | Development and validation of a rapid turboflow LC-MS/MS method for the quantification of LSD and 2-oxo-3-hydroxy LSD in serum and urine samples of emergency toxicological cases | |
| CN112946101B (en) | Method for simultaneously determining content of multiple antiepileptic drugs in blood and application thereof | |
| CN108918722B (en) | Method for detecting NMDA receptor antagonist JCC-02 blood concentration based on HPLC-MS/MS technology | |
| CN111766311A (en) | Method for detecting anti-tuberculosis drugs in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology | |
| Wang et al. | Simultaneous determination of topiramate, carbamazepine, oxcarbazepine and its major metabolite in human plasma by SFC-ESI-MS/MS with polarity switching: application to therapeutic drug monitoring | |
| CN113588804A (en) | Kit for detecting concentration of 5-hydroxytryptamine and melatonin in serum | |
| CN113917049A (en) | Biological analysis method for clinical research of chlorpromazine and metabolite concentration in plasma sample | |
| CN110031568B (en) | Method for determining concentration of Sacubitril, desethylSacubitril and valsartan in human plasma | |
| CN111912921A (en) | Method for detecting 3 lipids in plasma by ultra-high performance liquid chromatography tandem mass spectrometry technology | |
| CN115326960A (en) | Analysis method for simultaneously detecting concentrations of 8 antiepileptic drugs and 1 active metabolite in human plasma | |
| CN111458417B (en) | Method and kit for combined detection of multiple antibiotics in sample to be detected | |
| Sun et al. | Simultaneous quantitative analysis of phosphocreatine, creatine and creatinine in plasma of children by HPLC–MS/MS method: Application to a pharmacokinetic study in children with viral myocarditis | |
| Yang et al. | Determination of palonosetron in human plasma by ultra performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study | |
| Sun et al. | Simultaneous determination of ivabradine, metoprolol and their metabolites in rat plasma by ultra-performance liquid chromatography tandem mass spectrometry and its application in a pharmacokinetic study | |
| CN112180018A (en) | Liquid chromatography-mass spectrometry method for detecting 13 ceramides in plasma | |
| CN113325100B (en) | Method for simultaneously determining contents of various fatty acids in blood and application thereof | |
| CN112946100A (en) | Method and kit for determining carbamazepine and 10-hydroxycarbazepine and application thereof | |
| Ji et al. | Development and validation of a liquid chromatography‐tandem mass spectrometry method with triple‐stage fragmentation to determine levetiracetam in epileptic patient serum and its application in therapeutic drug monitoring | |
| CN114624361A (en) | Method for simultaneously measuring concentration of allopurinol and oxyallopurinol in human plasma | |
| CN113030343B (en) | Liquid chromatography tandem mass spectrometry detection method for pyrroloquinoline quinone in blood plasma | |
| Shi et al. | A LC-MS 3 strategy to determine lamotrigine by QQ-trap tandem mass spectrometry coupled with triple stage fragmentation to enhance sensitivity and selectivity | |
| CN113820424A (en) | HPLC-MS/MS method for simultaneously determining concentration of 14 antidepressants in human plasma | |
| CN112903849B (en) | Method and kit for detecting eszopiclone content in blood and application of kit | |
| Lee et al. | Development of liquid chromatography tandem mass spectrometry method for determination of spironolactone in human plasma: application to a bioequivalence study of Daewon Spiracton tablet®(spironolactone 50 mg) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |