CN111579680A - Detection kit for antiepileptic drug in serum and application thereof - Google Patents
Detection kit for antiepileptic drug in serum and application thereof Download PDFInfo
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Abstract
The invention discloses a detection kit for an antiepileptic drug in serum and application thereof, belonging to the technical field of drug analysis. The kit provided by the invention can detect 10 common antiepileptic drugs at one time by adopting an LC-MS/MS method, has the advantages of simple pretreatment process, low cost, high sensitivity and strong specificity, can complete the separation and detection of the antiepileptic drugs within 6min, basically meets the requirements on accuracy and precision, can be used for quantitative analysis of the antiepileptic drugs in clinic, and provides a reliable detection method for monitoring the treatment concentration of the antiepileptic drugs in clinic.
Description
Technical Field
The invention belongs to the technical field of drug analysis, and particularly relates to a detection kit for an antiepileptic drug in serum and application thereof.
Background
Epilepsy is a chronic disease characterized by recurrent, paroxysmal, stereotypical central nervous system dysfunction of cerebral neurons with sudden abnormal discharge, resulting in transient cerebral dysfunction. Epilepsy has complex and various etiologies, including genetic factors, brain diseases, systemic diseases and the like. Epilepsy is the second most persistent ailment of the neurological diseases, which is second to cerebrovascular diseases, about 900 thousands of epilepsy patients exist in China, 500-600 thousands of epilepsy patients are active epilepsy patients, about 40 thousands of epilepsy patients are newly increased every year, and epilepsy has become the second most common disease of the neurology department, which is second to headache. When a epileptic patient has a seizure, the motor and sensory functions, autonomic nervous states, consciousness level and the like show different degrees of disorders, the epileptic persistent seizure promotes cerebral neurons to have severe hypoxia and induces degeneration and necrosis of the cerebral neurons, the patients in later period can have cognitive dysfunction such as learning and memory decline, and other mental disorders such as depression, anxiety, schizophrenia and the like are complicated in other few patients. Therefore, timely and effective control of epileptic seizure is helpful for preventing and treating epileptic development and occurrence of complications thereof.
Clinically common antiepileptic drugs include Levetiracetam (LTT), 10-Hydroxycarbazepine (HCBZ), Carbamazepine (CBZ), Oxcarbazepine (OCP), Phenytoin (PHT), Lamotrigine (LMT), valproic acid (VPA), Phenobarbital (PBB), Topiramate (TPM), Clonazepam (CNP), and the like. Because epilepsy needs to be maintained and treated for a long time, the effective blood concentration range of the antiepileptic drug is narrow, the metabolic process in vivo has larger individual difference, the drug poisoning event is easy to occur, and the pharmacokinetic characteristic has a correlation with the antiepileptic effect and the adverse drug reaction, so that the monitoring of the drug concentration in blood thereof realizes the individualized administration, reduces the adverse reaction and avoids the important reference of the malignant cycle of the administration, and has great significance in clinical administration.
At present, the method for determining the blood concentration of the Antiepileptic drug at home and abroad mainly adopts a high performance Liquid Chromatography and an immunization method, and the article 'Simple and Accurate Quantitative Analysis of Ten Antiepileptic Drugs in human Plasma by Liquid Chromatography/Tandem Mass Spectrometry' reports a method for simultaneously detecting Ten Antiepileptic Drugs, wherein Ten substances only adopt two isotope internal standards, the matrix interference is larger, the sample collection time is longer, and each needle needs more than 10 minutes; the article "Development, validation and localization of an online-SPE-LC-HRMS/MS for the quantitative analysis of a phenobarbital, phenobion, carbamazepine, and active metabolite carbamazepine10, 11-epoxide" describes the processing of samples by online solid phase extraction, high requirements for configuration, long sample collection times, etc. Patent CN 101093214A discloses a method for determining the blood concentration of antiepileptic drugs, and patent CN 101093214A discloses a method for simultaneously determining the blood concentration of multiple antiepileptic drugs, wherein the former adopts high performance liquid chromatography to detect four antiepileptic drugs, the latter adopts high performance liquid chromatography to detect six antiepileptic drugs, the high performance liquid chromatography has poor selectivity, the types of the detected drugs are limited, the flow rate is high, the cost of the required solvent is high, the analysis time is long, the former 9min detects 4 antiepileptic drugs, and the latter 20min detects 6 antiepileptic drugs, but the invention can detect 10 antiepileptic drugs only in 6min, and is more suitable for monitoring the clinical blood concentration.
Disclosure of Invention
The invention aims to provide a detection kit for an antiepileptic drug in serum based on the prior art.
The invention also aims to provide application of the kit in detecting the antiepileptic drugs in the serum by using the ultra-performance liquid chromatography tandem mass spectrometry technology.
In order to achieve the purpose, the invention adopts the following technical scheme:
a test kit for an antiepileptic drug in serum, the antiepileptic drug comprising: levetiracetam, 10-hydroxycarbazepine, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, valproic acid, phenobarbital, topiramate and clonazepam;
the kit comprises: mobile phase, calibrator solution, mixed internal standard solution, protein precipitant and quality control material;
the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is formic acid-ammonium formate aqueous solution, and the mobile phase B is acetonitrile;
the calibrator solution comprises a series of mixed solutions of levetiracetam, 10-hydroxycarbazepine, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, valproic acid, phenobarbital, topiramate and clonazepam, which are prepared from blank serum and have known concentrations;
the mixed internal standard solution is a mixed solution of levetiracetam-d 3, 10-hydroxycarbazepine-d 4, carbamazepine-d 2,15N, oxcarbazepine-d 4, phenytoin-d 10, lamotrigine-13C 3, valproic acid-d 6, phenobarbital-d 5, topiramate-d 12 and clonazepam-d 4, which have known concentration and are prepared by a methanol aqueous solution;
the quality control product comprises a mixed solution of levetiracetam, 10-hydroxycarbazepine, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, valproic acid, phenobarbital, topiramate and clonazepam which have known concentration and are prepared from blank serum.
Further, the mobile phase A is 0.01-0.2% formic acid-0.1 mM-10 mM ammonium formate aqueous solution, and preferably 0.1% formic acid-2 mM ammonium formate aqueous solution.
Further, the calibrator solution comprises mixed solutions with 7 concentration ratios.
Furthermore, the concentrations of levetiracetam-d 3, 10-hydroxycarbazepine-d 4, carbamazepine-d 2,15N, oxcarbazepine-d 4, phenytoin-d 10, lamotrigine-13C 3, valproic acid-d 6, phenobarbital-d 5, topiramate-d 12 and clonazepam-d 4 in the mixed internal standard solution are LTT-d 000 320000 ng/mL, HCBZ-d450 45000ng/mL, CBZ-d2,15N10000ng/mL, OCP-d420000ng/mL, PHT-d 1020000 ng/mL, VPA-d6100000ng/mL, TPM-d1220000ng/mL, CNP-d4500ng/mL, LMT-13C 00 35000 ng/mL and PBB-d51000ng/mL, respectively.
Further, the protein precipitator is a mixed solution of methanol and acetonitrile, and the volume ratio of the methanol to the acetonitrile is 1: 1-5; preferably, the volume ratio of methanol to acetonitrile in the protein precipitant is 1: 3.
Further, the quality control product comprises mixed liquid with low, medium and high concentration ratios.
The kit is applied to the detection of the antiepileptic drug in serum by using an ultra-performance liquid chromatography tandem mass spectrometry technology.
Has the advantages that:
when the kit provided by the invention is used for detecting the antiepileptic drug in serum, the pretreatment process is simple, the cost is low, the sensitivity is high, the specificity is strong, the separation and detection of the antiepileptic drug can be completed within 6min, the accuracy and the precision basically meet the requirements, the kit can be used for quantitative analysis of the antiepileptic drug in clinic, and a reliable detection method is provided for monitoring the treatment concentration of the antiepileptic drug in clinic.
Drawings
Fig. 1 is an extracted ion flow spectrum of the anti-epileptic drug standard in example 1.
FIG. 2 is an ion flow chart of the antiepileptic drug in serum of example 1.
Detailed Description
The invention provides a detection kit for antiepileptic drugs in serum, wherein the antiepileptic drugs comprise: levetiracetam (LTT), 10-Hydroxycarbazepine (HCBZ), Carbamazepine (CBZ), Oxcarbazepine (OCP), Phenytoin (PHT), Lamotrigine (LMT), valproic acid (VPA), Phenobarbital (PBB), Topiramate (TPM) and Clonazepam (CNP).
The corresponding isotope internal standard substances selected aiming at the antiepileptic medicine are respectively as follows: levetiracetam-d 3(LTT-d3), 10-hydroxycarbazepine-d 4(HCBZ-d4), carbamazepine-d 2,15N (CBZ-d2,15N), oxcarbazepine-d 4(OCP-d4), phenytoin-d 10(PHT-d10), lamotrigine-13C 3(LMT-13C3), valproic acid-d 6(VPA-d6), phenobarbital-d 5(PBB-d5), topiramate-d 12(TPM-d12) and clonazepam-d 4(CNP-d 4).
The kit comprises the following reagents:
(1) mobile phase:
mobile phase A: 0.01 to 0.2 percent of 0.1 to 10mM of 0.1mM of formic acid ammonium formate aqueous solution; mobile phase B: acetonitrile;
(2) calibration solution:
preparing a mixed standard solution into a calibrator solution with seven different concentration points by using a blank serum matrix, wherein the seven concentration points of the calibrator solution are as follows:
LTT/PHT/OCP/TPM：100ng/mL、250ng/mL、500ng/mL、2500ng/mL、5000ng/mL、25000ng/mL、50000ng/mL
CBZ：50ng/mL、125ng/mL、250ng/mL、1250ng/mL、2500ng/mL、12500ng/mL、25000ng/mL
HCBZ/LMT：20ng/mL、50ng/mL、100ng/mL、500ng/mL、1000ng/mL、5000ng/mL、10000ng/mL
CNP：0.5ng/mL、1.25ng/mL、2.5ng/mL、12.5ng/mL、25ng/mL、125ng/mL、250ng/mL
VPA：500ng/mL、1250ng/mL、2500ng/mL、12500ng/mL、25000ng/mL、125000ng/mL、250000ng/mL
PBB：200ng/mL、500ng/mL、1000ng/mL、5000ng/mL、10000ng/mL、50000ng/mL、100000ng/mL；
(3) mixing internal standard solutions:
methanol aqueous solution containing LTT-d 320000 ng/mL, HCBZ-d45000ng/mL, CBZ-d2,15N10000ng/mL, OCP-d420000ng/mL, PHT-d 1020000 ng/mL, VPA-d6100000ng/mL, TPM-d1220000ng/mL, CNP-d4500ng/mL, LMT-13C 35000 ng/mL, PBB-d51000 ng/mL;
(4) protein precipitant:
a mixed solution of methanol and acetonitrile;
(5) quality control product:
blank serum matrix containing antiepileptic drug comprises low, medium and high concentrations of QC (L), QC (M) and QC (H), wherein,
QC (L) QC (M) 10-fold dilution with blank serum matrix;
QC (M) is the above mixed standard solution diluted to 500 times with blank serum substrate;
QC (H) is the above mixed standard solution diluted 50-fold with blank serum matrix.
In a preferable embodiment, the mobile phase A is 0.01-0.2% formic acid 0.1 mM-10 mM ammonium formate aqueous solution, preferably 0.1% formic acid 2mM ammonium formate aqueous solution.
In one scheme, the volume ratio of methanol to acetonitrile in the protein precipitator is 1: 1-5; preferably, the volume ratio of methanol to acetonitrile in the protein precipitant is 1: 3.
The mixed standard solution mentioned in the present invention is prepared as follows: the antiepileptic drugs LTT 20mg/mL, CBZ10 mg/mL, PHT 20mg/mL, LMT 10mg/mL, CNP 1mg/mL, HCBZ 5mg/mL, OCP 5mg/mL, VPA50mg/mL, PBB 10mg/mL and TPM20mg/mL are respectively prepared into standard solutions containing LTT 1000000ng/mL, CBZ500000ng/mL, PHT 1000000ng/mL, LMT200000ng/mL, CNP 5000ng/mL, HCBZ 200000ng/mL, OCP 1000000ng/mL, VPA5000000ng/mL, PBB 2000000/mL and TPM 1000000ng/mL by using methanol.
The mixed internal standard solution mentioned in the invention is prepared according to the following method: an isotope mixed solution containing LTT-d 31 mg/mL, HCBZ-d41mg/mL, CBZ-d2,15N 1mg/mL, OCP-d42 mg/mL, PHT-d 101 mg/mL, VPA-d 65 mg/mL, TPM-d121mg/mL, CNP-d 40.1mg/mL, LMT-13C 31 mg/mL, PBB-d51 mg/mL mother liquor and methanol water solution is prepared into an isotope mixed solution containing LTT-d 320000 ng/mL, HCBZ-d45000ng/mL, CBZ-d2,15N10000ng/mL, OCP-d420000ng/mL, PHT-d 1020000 ng/mL, VPA-d6100000ng/mL, TPM-d1220000ng/mL, CNP-d4500ng/mL, LMT-13C 35000 ng/mL and PBB-d 350 51000 ng/51000/mL.
When preparing a mixed standard solution and a mixed internal standard solution, the adopted methanol aqueous solution is 50-95% methanol aqueous solution; preferably 70 to 90 percent of methanol aqueous solution; more preferably 80% aqueous methanol.
In the preparation of the mixed standard solution, the blank serum matrix is blank serum without antiepileptic drugs.
The concentration of the aqueous methanol solution referred to in the present invention generally means a volume concentration.
The serum mentioned in the invention is human or animal serum.
In a preferred scheme, the kit for detecting the antiepileptic drug in the serum by the ultra-high performance liquid chromatography tandem mass spectrometry technology comprises the following reagents:
(1) mobile phase:
mobile phase A: 0.1mM to 10mM ammonium formate aqueous solution containing 0.01 percent to 0.2 percent of formic acid; mobile phase B: acetonitrile;
(2) calibration solution:
the mother liquor of antiepileptic drug is prepared into standard solutions containing LTT 1000000ng/mL, CBZ500000ng/mL, PHT 1mg/mL, HCBZ 5mg/mL, OCP 5mg/mL, VPA50mg/mL, PBB 10mg/mL and TPM20mg/mL respectively with methanol, wherein the standard solutions contain LTT 1000000ng/mL, CBZ500000ng/mL, PHT 1000000ng/mL, LMT200000ng/mL, CNP 5000ng/mL, HCBZ 200000ng/mL, OCP 1000000ng/mL, VPA5000000ng/mL, PBB 2000000ng/mL and TPM 1000000 ng/mL. Preparing the mixed standard solution into calibration substance solutions of seven different concentration points by using blank serum without the antiepileptic drug;
(3) mixing internal standard solutions:
an isotope mixed solution containing LTT-d 31 mg/mL, HCBZ-d41mg/mL, CBZ-d2,15N 1mg/mL, OCP-d42 mg/mL, PHT-d 101 mg/mL, VPA-d 65 mg/mL, TPM-d121mg/mL, CNP-d 40.1mg/mL, LMT-13C 31 mg/mL, PBB-d51 mg/mL mother liquor and methanol water solution is prepared into an isotope mixed solution containing LTT-d 320000 ng/mL, HCBZ-d45000ng/mL, CBZ-d2,15N10000ng/mL, OCP-d420000ng/mL, PHT-d 1020000 ng/mL, VPA-d6100000ng/mL, TPM-d1220000ng/mL, CNP-d4500ng/mL, LMT-13C 35000 ng/mL and PBB-d 350 51000 ng/51000/mL.
(4) Protein precipitant:
the volume ratio of the methanol to the acetonitrile is 1: 1-5.
(5) Quality control product:
the mixed standard solution is prepared into QC (L), QC (M) and QC (H) with three different concentrations by blank serum without the antiepileptic drug.
In a more preferred embodiment, the kit for detecting the antiepileptic drug in the serum by the ultra-high performance liquid chromatography tandem mass spectrometry technology comprises the following reagents:
(1) mobile phase:
mobile phase A: 0.1% formic acid 0.1mM to 10mM ammonium formate aqueous solution; mobile phase B: acetonitrile;
(2) calibration solution:
respectively preparing mother liquor of antiepileptic drug with the concentrations of LTT 20mg/mL, CBZ10 mg/mL, PHT 20mg/mL, LMT 10mg/mL, CNP 1mg/mL, HCBZ 5mg/mL, OCP 5mg/mL, VPA50mg/mL, PBB 10mg/mL and TPM20mg/mL by using 80% methanol to prepare standard solutions containing LTT 1000000ng/mL, CBZ500000ng/mL, PHT 1000000ng/mL, LMT200000ng/mL, CNP 5000ng/mL, HCBZ 200000ng/mL, OCP 1000000ng/mL, VPA5000000ng/mL, PBB 2000000ng/mL and TPM 1000000ng/mL, and preparing the mixed standard solutions into calibrator solutions of seven different concentration points by using serum without antiepileptic drug;
(3) mixing internal standard solutions:
preparing isotope mixed solution containing LTT-d 31 mg/mL, HCBZ-d41mg/mL, CBZ-d2,15N 1mg/mL, OCP-d42 mg/mL, PHT-d 101 mg/mL, VPA-d 65 mg/mL, TPM-d121mg/mL, CNP-d 40.1mg/mL, LMT-13C 31 mg/mL, PBB-d51 mg/mL mother liquor with 80% methanol water solution to obtain isotope mixed solution containing LTT-d 320000 ng/mL, HCBZ-d45000ng/mL, CBZ-d2,15N10000ng/mL, OCP-d420000ng/mL, PHT-d 1020000 ng/mL, VPA-d6100000ng/mL, TPM-d1220000ng/mL, CNP-d4500ng/mL, LMT-13C 35000 ng/mL, PBB 35000-d 51000 ng/51000/mL;
(4) protein precipitant:
the volume ratio of methanol to acetonitrile is 1: 3;
(5) quality control product:
QC (L) QC (M) 10-fold dilution with blank serum matrix;
QC (M) is the above mixed standard solution diluted to 500 times with blank serum substrate;
QC (H) is the above mixed standard solution diluted 50-fold with blank serum matrix.
Preparing the mixed standard solution into QC (L), QC (M) and QC (H) with three different concentrations by using blank serum without the antiepileptic drug, wherein the corresponding concentrations of the antiepileptic drug quality control products of the QC (L), the QC (M) and the QC (H) are shown in a table 1;
TABLE 1 antiepileptic drug substance control corresponding concentration (unit: ng/mL)
QC (L) includes: LTT/PHT/OCP/TPM200 ng/mL, CBZ100 ng/mL, LMT/HCBZ40 ng/mL, CNP 1ng/mL, VPA1000 ng/mL, PBB400 ng/mL
QC (M) comprises: LTT/PHT/OCP/TPM2000 ng/mL, CBZ1000 ng/mL, LMT/HCBZ400 ng/mL, CNP10 ng/mL, VPA10000 ng/mL, PBB4000 ng/mL
QC (H) includes: LTT/PHT/OCP/TPM 20000ng/mL, CBZ 10000ng/mL, LMT/HCBZ4000ng/mL, CNP 100ng/mL, VPA 100000ng/mL, PBB 40000 ng/mL.
In a more preferred embodiment, the mixed internal standard solution is prepared as follows:
accurately transferring a certain volume of the isotope internal standard mother liquor of the antiepileptic drug, adding 884 microliter of 80% methanol aqueous solution, and uniformly mixing to obtain 1mL isotope internal standard solution. The frozen food is recommended to be stored in a refrigerator at the temperature of 80 ℃ below zero and is taken out for use. See table 2 below for concentrations.
TABLE 2 preparation of mixed internal standard solutions
In a more preferred embodiment, the calibrator solution is prepared as follows:
accurately transferring a certain volume of antiepileptic drug standard mother liquor, adding 785 μ L of 80% methanol aqueous solution, and mixing well to obtain 1mL of mixed standard solution with the concentration shown in Table 3 below.
TABLE 3 Mixed Standard solution
Preparing standard yeast by gradient dilution method, taking out standard solution from refrigerator at-20 deg.C, vortex for 10s, preparing maximum concentration point of standard yeast with standard solution within 2min, and storing at-80 deg.C. The specific procedure is as follows in Table 4 (unit: ng/mL):
TABLE 4 Standard koji preparation
The application of the kit in detecting the antiepileptic drug in the serum by using the ultra-performance liquid chromatography tandem mass spectrometry technology is also within the protection scope of the invention.
The specific detection method comprises the following steps:
a method for detecting antiepileptic drugs in serum by an ultra-performance liquid chromatography tandem mass spectrometry technology,
the antiepileptic drugs are respectively: levetiracetam (LTT), 10-Hydroxycarbazepine (HCBZ), Carbamazepine (CBZ), Oxcarbazepine (OCP), Phenytoin (PHT), Lamotrigine (LMT), valproic acid (VPA), Phenobarbital (PBB), Topiramate (TPM), Clonazepam (CNP)
The isotope internal standard substances corresponding to the antiepileptic medicine are respectively as follows: levetiracetam-d 3(LTT-d3), 10-hydroxycarbazepine-d 4(HCBZ-d4), carbamazepine-d 2,15N (CBZ-d2,15N), oxcarbazepine-d 4(OCP-d4), phenytoin-d 10(PHT-d10), lamotrigine-13C 3(LMT-13C3), valproic acid-d 6(VPA-d6), phenobarbital-d 5(PBB-d5), topiramate-d 12(TPM-d12), clonazepam-d 4(CNP-d4)
Detecting the antiepileptic drug in the preprocessed serum by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, firstly separating a target substance to be detected from interfering components in a serum matrix by utilizing the ultra-high performance liquid chromatography, then establishing a calibration curve by utilizing a mass spectrum isotope internal standard quantitative method and taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the content of the antiepileptic drug in the serum, wherein the specific chromatographic conditions are as follows:
(1) ultra-high performance liquid chromatography conditions:
mobile phase A: 0.01 to 0.2 percent of 0.1 to 10mM of 0.1mM of formic acid ammonium formate aqueous solution; mobile phase B: acetonitrile;
the type of the chromatographic column: ACQUITY UPLC BEH C18 (2.1X 50mm, 1.7 μm);
the method adopts a gradient elution mode with the mobile phase A and the mobile phase B as a mixed mobile phase, the initial ratio of the mobile phase A to the mobile phase B is 92:8, and the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 92:8 to 35:65 at a constant speed within 0-3.0 minutes; the volume ratio of the mobile phase A to the mobile phase B is gradually changed from 35:65 to 2:98 at a constant speed within 3.0-3.5 minutes; changing the volume ratio of the mobile phase A to the mobile phase B from 2:98 to 92:8 within 3.5-6.0 minutes, wherein the flow rate is 0.3mL/min, the collection time of each sample is 6.0min, the column temperature is 45 ℃, and the sample injection volume is 1 mu L;
(2) mass spectrum conditions:
in an electrospray ionization (ESI) mode, adopting multi-reaction monitoring (MRM) to carry out positive and negative ion switching scanning; spray voltage ESI + (3.0kV), ESI- (2.5 kV); the source temperature is 120 ℃; the temperature of atomizing gas is 400 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper holes is 150L/h; each target and its corresponding isotope internal standard were monitored simultaneously.
In order to improve the chromatographic separation selectivity, it may be considered to adjust the polarity of the mobile phase. The invention adds formic acid and ammonium formate in the mobile phase A, can effectively improve the ionization efficiency of certain target compounds, has higher sensitivity for detecting the antiepileptic drug in serum compared with the LC-MS/MS method adopted in the prior art under the coordination of other conditions, has simple pretreatment process, low cost, high sensitivity and strong specificity, and completes the separation and detection of the antiepileptic drug within 5 min. In a preferred embodiment, the mobile phase A is 0.01-0.2% formic acid 0.1-10 mM ammonium formate aqueous solution without affecting the effect of the invention. In a more preferred embodiment, mobile phase A is 0.1% formic acid in 2mM ammonium formate in water.
In chromatography, the choice of the chromatographic column is important and the requirements for the chromatographic column: high column efficiency, good selectivity, high analysis speed and the like. The invention adopts 0.01 to 0.2 percent of 0.1 to 10mM of 0.1mM of formic acid ammonium aqueous solution and acetonitrile as mobile phases, and the types of chromatographic columns are as follows: the detection kit has the advantages that the detection of the sample is not interfered by endogenous substances under the coordination of other conditions, the detection kit is high in sensitivity, strong in specificity, low in cost and simple in pretreatment process, the separation and detection can be completed within 6.0min, and the precision and the accuracy meet the requirements, wherein the detection kit is ACQUITY UPLC BEH C18(2.1 multiplied by 50mm, 1.7 mu m).
When the internal standard method is adopted, the selection of the internal standard substance is very important work. The ideal internal standard should be capable of being added to the sample in an accurate, known amount, and have substantially the same or as consistent as possible physicochemical properties, chromatographic behavior, and response characteristics as the sample being analyzed; under chromatographic conditions, the internal standard must be sufficiently separated from the components of the sample. The invention respectively adopts levetiracetam-d 3(LTT-d3), 10-hydroxycarbazepine-d 4(HCBZ-d4), carbamazepine-d 2,15N (CBZ-d2,15N), oxcarbazepine-d 4(OCP-d4), phenytoin-d 10(PHT-d10), lamotrigine-13C 3(LMT-13C3), valproic acid-d 6(VPA-d6), phenobarbital-d 5(PBB-d5), topiramate-d 12(TPM-d12) and clonazepam-d 4(CNP-d4) as internal standards, the deuterated internal standard and the substance to be tested have the same retention time, chemical properties and anti-matrix effect, and the reproducibility and accuracy in the determination of the epileptic drugs are better.
In one embodiment, the flow rate is 0.1-0.4 mL/min, preferably 0.3 mL/min.
Further, the column temperature is 35-50 ℃, preferably 45 ℃.
Furthermore, the injection volume is 1-5 μ L, preferably 1 μ L.
In a preferred scheme, when the ultra-high performance liquid chromatography tandem mass spectrometry technology is adopted to detect the antiepileptic drug in serum, the specific chromatographic conditions are as follows:
(1) high performance liquid chromatography conditions:
mobile phase A: 0.1% formic acid 2mM ammonium formate-water solution;
mobile phase B: acetonitrile;
the type of the chromatographic column: ACQUITY UPLC BEH C18 (2.1X 50mm, 1.7 μm);
the gradient elution mode is adopted, see table 5; the flow rate is 0.3mL/min, the column temperature is 45 ℃, and the sample injection volume is 1 mu L;
TABLE 5 mobile phase gradient elution parameters
| Time (min) | Flow rate (mL/min) | ％A | ％B | Curve |
| 0.0 | 0.3 | 92 | 8 | - |
| 3.0 | 0.3 | 35 | 65 | 6 |
| 3.5 | 0.3 | 2 | 98 | 6 |
| 6.0 | 0.3 | 92 | 8 | 1 |
(2) Mass spectrum conditions:
in an electrospray ionization (ESI) mode, adopting multi-reaction monitoring (MRM) to carry out positive and negative ion switching scanning; spray voltage ESI + (3.0kV), ESI- (2.5 kV); the source temperature is 120 ℃; the temperature of atomizing gas is 400 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper holes is 150L/h; meanwhile, the antiepileptic drugs and the corresponding isotope internal standards thereof are monitored, and the mass spectrum acquisition parameters of each target substance to be detected are shown in table 6.
TABLE 6 substance Spectrum parameters of antiepileptic drugs
The serum mentioned in the invention is human or animal serum.
In one protocol, pre-treated serum was prepared as follows: adding a protein precipitator containing an internal standard into the serum, and then oscillating and centrifuging to obtain a supernatant; the protein precipitant containing the internal standard is prepared by mixing a mixed internal standard solution and the protein precipitant. The protein precipitator is a mixed solution of methanol and acetonitrile; preferably, the volume ratio of the methanol to the acetonitrile in the mixed solution of the methanol and the acetonitrile is 1: 1-5.
In a preferred embodiment, the pre-treated serum is prepared as follows: putting 50 μ L of serum into a 1.5mL centrifuge tube, adding 200 μ L of protein precipitant containing internal standard, and oscillating at high speed (maximum oscillation speed) for 5 min; centrifuging at 12000-15000 r/min at 1-5 ℃ for 4-10 min, and taking 60 mu L of supernatant for sample injection. The protein precipitant containing the internal standard is prepared by mixing a mixed internal standard solution and a protein precipitant, wherein the volume ratio of the mixed internal standard solution to the protein precipitant is 0.1-0.3: 19.7 to 19.9.
In a more preferred embodiment, the pre-treated serum is prepared as follows: putting 50 μ L of serum into a 1.5mL centrifuge tube, adding 200 μ L of protein precipitant containing internal standard (volume ratio of methanol to acetonitrile is 1:3), and oscillating at high speed (maximum oscillation speed) for 5 min; centrifuging at 14000r/min at 4 ℃ for 5 min; transfer 60. mu.L of supernatant from the EP tube to a plastic lined tube in a 1. mu.L sample volume. The protein precipitant containing the internal standard is prepared by mixing a mixed internal standard solution and the protein precipitant, wherein the volumes of the mixed internal standard solution and the protein precipitant are 0.2: 19.8, the same as below.
In one embodiment, the protein precipitant containing the internal standard is prepared as follows:
an internal standard solution containing LTT-d 31 mg/mL, HCBZ-d41mg/mL, CBZ-d2,15N 1mg/mL, OCP-d42 mg/mL, PHT-d 101 mg/mL, VPA-d 65 mg/mL, TPM-d121mg/mL, CNP-d 40.1mg/mL, LMT-13C 31 mg/mL, PBB-d51 mg/mL, and 80% methanol aqueous solution is prepared into a mixed solution containing LTT-d 320000 ng/mL, HCBZ-d45000ng/mL, CBZ-d2,15N10000ng/mL, OCP-d420000ng/mL, PHT-d 00 1020000 ng/mL, VPA-d6100000ng/mL, TPM-d1220000ng/mL, CNP-d4500ng/mL, LMT-13C 35000 ng/mL, PBB 35000-d 51000 ng/mL. And adding 200 mu L of the mixed internal standard solution into 19.8mL of protein precipitator (the volume ratio of methanol to acetonitrile is 1:3) to obtain the protein precipitator containing the internal standard.
In one embodiment, the standard solution is prepared as follows:
the mother liquor of antiepileptic drugs is prepared into standard solutions containing LTT 1000000ng/mL, CBZ500000ng/mL, PHT 50mg/mL, PBB 10mg/mL and TPM20mg/mL by 80% methanol respectively, wherein the concentrations of the mother liquor are respectively LTT 20mg/mL, CBZ10 mg/mL, PHT 20mg/mL, HCBZ 5mg/mL, OCP 5mg/mL, VPA50mg/mL, PBB 10mg/mL and TPM20mg/mL, and the standard solutions are prepared into standard solutions containing LTT 1000000ng/mL, CBZ500000ng/mL, PHT 1000000ng/mL, LMT200000ng/mL, CNP 5000ng/mL, HCBZ 200000ng/mL, OCP 1000000ng/mL, VPA5000000ng/mL, PBB 2000000ng/mL and TPM 1000000 ng/mL.
Preparing the mixed standard solution into calibration solution with seven different concentration points by using a blank serum substrate, wherein the seven concentration points of the calibration solution are as follows:
LTT/PHT/OCP/TPM：100ng/mL、250ng/mL、500ng/mL、2500ng/mL、5000ng/mL、25000ng/mL、50000ng/mL
CBZ：50ng/mL、125ng/mL、250ng/mL、1250ng/mL、2500ng/mL、12500ng/mL、25000ng/mL
HCBZ/LMT：20ng/mL、50ng/mL、100ng/mL、500ng/mL、1000ng/mL、5000ng/mL、10000ng/mL
CNP：0.5ng/mL、1.25ng/mL、2.5ng/mL、12.5ng/mL、25ng/mL、125ng/mL、250ng/mLVPA：500ng/mL、1250ng/mL、2500ng/mL、12500ng/mL、25000ng/mL、125000ng/mL、250000ng/mL
PBB：200ng/mL、500ng/mL、1000ng/mL、5000ng/mL、10000ng/mL、50000ng/mL、100000ng/mL。
taking 50 mu L of each concentration point sample, putting the sample into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to acetonitrile is 1:3), and oscillating at high speed (maximum oscillation speed) for 5 min; centrifuging at 14000r/min at 4 ℃ for 5 min; transfer 60. mu.L of supernatant from the EP tube to a plastic lined tube in a 1. mu.L sample volume.
In one scheme, the quality control product is prepared according to the following method: preparing the mixed standard solution into three different concentrations of QC (L), QC (M) and QC (H) by using blank serum without the antiepileptic drug:
QC (L) includes: LTT/PHT/OCP/TPM200 ng/mL, CBZ100 ng/mL, LMT/HCBZ40 ng/mL, CNP 1ng/mL, VPA1000 ng/mL, PBB400 ng/mL
QC (M) comprises: LTT/PHT/OCP/TPM2000 ng/mL, CBZ1000 ng/mL, LMT/HCBZ400 ng/mL, CNP10 ng/mL, VPA10000 ng/mL, PBB4000 ng/mL
QC (H) includes: LTT/PHT/OCP/TPM 20000ng/mL, CBZ 10000ng/mL, LMT/HCBZ4000ng/mL, CNP 100ng/mL, VPA 100000ng/mL, PBB 40000 ng/mL.
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
Example 1
First, experimental material and instrument
1. Material
The samples for the kit study experiments were from serum samples collected from the 11 month outpatient service in 2019 of Shanghai Zhongshan Hospital.
(1) The instrument comprises the following steps: xevo TQ-S triple quadrupole mass spectrometer (Waters Corporation); UPLC I-Class ultra high performance liquid chromatography system (with autosampler, Waters Corporation); SCILOGEX D2012 high speed bench top centrifuge (usa); ultra pure water meter (ELGA LabWater, uk); multi-tube Vortex mixer (Vortex genie2, usa); an adjustable pipettor (Eppendorf 0.5-10 muL, 10-100 muL, 100-1000 muL); glassware, graduated cylinders, and the like.
(2) Reagent consumables: MS grade methanol (Fisher, usa); MS grade acetonitrile (Fisher, usa); HPLC grade acetonitrile (Honeywell, usa); MS grade formic acid (Fisher, usa); HPLC grade methanol (Honeywell, usa); a chromatographic column: ACQUITY UPLC BEH C18 (2.1X 50mm, 1.7 μm).
(3) And (3) standard substance: the standards and their corresponding internal standards are shown in table 7 below.
TABLE 7 Standard and internal standards
| Serial number | Name of Chinese | Manufacturer of the product | |
| 1 | Levetiracetam | TRC | |
| 2 | Levetiracetam-d 3 | TRC | |
| 3 | 10-hydroxycarbamazepine | TRC | |
| 4 | 10-hydroxycarbamazepine-d 4 | TRC | |
| 5 | Carbamazepine | Dr.Ehrenstorfer | |
| 6 | Carbamazepine-d 2,15N | TRC | |
| 7 | Oxcarbazepine | TRC | |
| 8 | Oxcarbazepine-d 4 | TRC | |
| 9 | | Dr.Ehrenstorfer | |
| 10 | Phenytoin- |
TRC | |
| 11 | Lamotrigine | Bailingwei-medicine | |
| 12 | lamotrigine-13C 3 | TRC | |
| 13 | Valproic acid | TRC | |
| 14 | Valproic acid-d 6 | TRC | |
| 15 | Phenobarbital | Cerilliant | |
| 16 | Phenobarbital-d 5 | TRC | |
| 17 | Topiramate | TRC | |
| 18 | Topiramate-d 12 | TRC | |
| 19 | Clonazepam | Cerilliant | |
| 20 | Clonazepam-d 4 | Sigma |
(4) Quality control product: blank serum matrix containing antiepileptic drugs has low, medium and high concentrations, which are QC (L), QC (M) and QC (H), respectively, as shown in Table 1.
The upper and lower peripheries of the kit are coated, the shockproof and heat preservation are carried out, mobile phases A and B are placed at the upper left part, 11 ampoule bottles are respectively placed at the lower left part, and the standard solution, the quality control product and the mixed internal standard solution are respectively contained; to the right, 25mL of protein precipitant was placed.
Second, liquid condition
(1) Chromatographic conditions are as follows: mobile phase A: 0.1% formic acid 2mM ammonium formate-water solution; mobile phase B: and (3) acetonitrile. The type of the chromatographic column: ACQUITY UPLC BEH C18 (2.1X 50mm, 1.7 μm), using gradient elution, as detailed in Table 5. The flow rate was 0.3mL/min, the column temperature was 45 ℃ and the injection volume was 1. mu.L.
(2) Mass spectrum conditions: in an electrospray ionization (ESI) mode, adopting multi-reaction monitoring (MRM) to carry out positive and negative ion switching scanning; spray voltage ESI + (3.0kV), ESI- (2.5 kV); the desolvation temperature is 120 ℃; the temperature of atomizing gas is 400 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper holes is 150L/h; each target was monitored simultaneously with the isotope internal standard. The mass spectrometric acquisition parameters for each target analyte are shown in table 6.
Third, the experimental process
(1) Preparing a standard substance:
the mother liquor of antiepileptic drugs is prepared into standard solutions containing LTT 1000000ng/mL, CBZ500000ng/mL, PHT 50mg/mL, PBB 10mg/mL and TPM20mg/mL by 80% methanol respectively, wherein the concentrations of the mother liquor are respectively LTT 20mg/mL, CBZ10 mg/mL, PHT 20mg/mL, HCBZ 5mg/mL, OCP 5mg/mL, VPA50mg/mL, PBB 10mg/mL and TPM20mg/mL, and the standard solutions are prepared into standard solutions containing LTT 1000000ng/mL, CBZ500000ng/mL, PHT 1000000ng/mL, LMT200000ng/mL, CNP 5000ng/mL, HCBZ 200000ng/mL, OCP 1000000ng/mL, VPA5000000ng/mL, PBB 2000000ng/mL and TPM 1000000 ng/mL. (see Table 3 for details).
The mixed standard solution is prepared into a calibrator solution (see table 4) with seven different concentration points by using a blank serum matrix, wherein the concentration of each calibration point is as listed in table 4, and the seven concentration points of the calibrator solution are as follows:
LTT/PHT/OCP/TPM：100ng/mL、250ng/mL、500ng/mL、2500ng/mL、5000ng/mL、25000ng/mL、50000ng/mL
CBZ：50ng/mL、125ng/mL、250ng/mL、1250ng/mL、2500ng/mL、12500ng/mL、25000ng/mL
HCBZ/LMT：20ng/mL、50ng/mL、100ng/mL、500ng/mL、1000ng/mL、5000ng/mL、10000ng/mL
CNP：0.5ng/mL、1.25ng/mL、2.5ng/mL、12.5ng/mL、25ng/mL、125ng/mL、250ng/mLVPA：500ng/mL、1250ng/mL、2500ng/mL、12500ng/mL、25000ng/mL、125000ng/mL、250000ng/mL
PBB：200ng/mL、500ng/mL、1000ng/mL、5000ng/mL、10000ng/mL、50000ng/mL、100000ng/mL。
(2) preparation of mixed internal standard solution
An isotope solution mixture comprising LTT-d 31 mg/mL, HCBZ-d41mg/mL, CBZ-d2,15N 1mg/mL, OCP-d42 mg/mL, PHT-d 101 mg/mL, VPA-d 65 mg/mL, TPM-d121mg/mL, CNP-d 40.1mg/mL, LMT-13C 31 mg/mL, PBB-d51 mg/mL, and 80% methanol aqueous solution is prepared into an isotope solution mixture comprising LTT-d 320000 ng/mL, HCBZ-d45000ng/mL, CBZ-d2,15N10000ng/mL, OCP-d420000ng/mL, PHT-d 1020000 ng/mL, VPA-d6100000ng/mL, TPM-d1220000ng/mL, CNP-d4500ng/mL, LMT-d 6313C ng/mL, PBB 3508/00/mL and PBB-d 5108/mL. (see Table 2 for details).
(3) Preparing a quality control product:
the mixed standard solution is prepared into QC (L), QC (M) and QC (H) with three different concentrations by using blank serum without the antiepileptic drug, and the details are shown in table 1.
QC (L) includes: LTT/PHT/OCP/TPM200 ng/mL, CBZ100 ng/mL, LMT/HCBZ40 ng/mL, CNP 1ng/mL, VPA1000 ng/mL, PBB400 ng/mL
QC (M) comprises: LTT/PHT/OCP/TPM2000 ng/mL, CBZ1000 ng/mL, LMT/HCBZ400 ng/mL, CNP10 ng/mL, VPA10000 ng/mL, PBB4000 ng/mL
QC (H) includes: LTT/PHT/OCP/TPM 20000ng/mL, CBZ 10000ng/mL, LMT/HCBZ4000ng/mL, CNP 100ng/mL, VPA 100000ng/mL, PBB 40000 ng/mL.
(4) Sample processing
1) Pretreatment of a standard product: taking 50 mu L of each concentration point sample, putting the sample into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of methanol to acetonitrile is 1:3), and oscillating at high speed (maximum oscillation speed) for 5 min; centrifuging at 14000r/min at 4 ℃ for 5 min; transfer 60. mu.L of supernatant from the EP tube to a plastic lined tube in a 1. mu.L sample volume.
2) Pretreatment of a serum sample: putting 50 μ L of serum into a 1.5mL centrifuge tube, adding 200 μ L of protein precipitant containing internal standard (volume ratio of methanol to acetonitrile is 1:3), and oscillating at high speed (maximum oscillation speed) for 5 min; centrifuging at 14000r/min at 4 ℃ for 5 min; transfer 60. mu.L of supernatant from the EP tube to a plastic lined tube in a 1. mu.L sample volume.
3) Pretreatment of quality control products: the quality control solutions QC (L), QC (M), QC (H) are respectively taken and 50 μ L of each quality control solution QC (L), QC (M), QC (H) are respectively put into a 1.5mL centrifuge tube, and then the quality control solutions QC (L), QC (M), QC (H) are consistent with the pretreatment of the serum sample, and the details are not.
The components of the assay kit are shown in Table 8.
TABLE 8 preparation of antiepileptic drug assay kit Components (100 persons)
Fourth, method verification
1. Extracting an ion current chromatogram: the peak shapes of the standard product of the antiepileptic drug and the serum sample are symmetrical, and no interference of miscellaneous peaks exists, which indicates that the standard product of the antiepileptic drug can be well detected under the condition, and fig. 1 is an extracted ion flow spectrogram of the standard product of the antiepileptic drug; FIG. 2 is an extracted ion flow chromatogram of an antiepileptic drug in a serum sample.
2. Calibration curve: and establishing a calibration curve by adopting an isotope internal standard quantitative method and utilizing TargetLynx software to calculate the concentration of the substance to be detected in the serum by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis. The linear fitting equation of the antiepileptic drugs in the respective concentration ranges has good linearity, the correlation coefficient is more than 0.99, and the quantitative requirements are met, which is shown in table 9.
TABLE 9 linear regression equation and linear correlation coefficient for antiepileptic drugs
| Serial number | Compound (I) | Retention time (min) | Linear range (ng/mL) | Linear equation of equations | Coefficient of correlation (r) |
| 1 | LTT | 1.5 | 100-50000 | Y＝0.000757509X+0.00994791 | 0.999 |
| 2 | HCBZ | 2.29 | 20-10000 | Y＝0.000142015X-0.000604281 | 0.990 |
| 3 | CBZ | 2.88 | 50-25000 | Y＝4.99326e-005X+0.000147965 | 0.998 |
| 4 | OCP | 2.6 | 100-50000 | Y＝0.000135441X+3.31354e-005 | 0.998 |
| 5 | PHT | 2.87 | 100-50000 | Y＝0.000364933X+0.0119435 | 0.998 |
| 6 | LMT | 1.77 | 20-10000 | Y＝0.000632911X+0.00193997 | 0.999 |
| 7 | VPA | 3.47 | 500-250000 | Y＝0.000135112X+0.00110046 | 0.996 |
| 8 | PBB | 2.49 | 200-100000 | Y＝0.000905464X-0.039066 | 0.999 |
| 9 | TPM | 2.72 | 100-50000 | Y＝5.11742e-005X-0.000496442 | 0.992 |
| 10 | CNP | 3.09 | 0.5-250 | Y＝2.57054X+0.16262 | 0.999 |
3. Accuracy survey: and evaluating the accuracy of the method by adopting a standard recovery rate test. A mixed blank serum sample is prepared, 3 concentrations of mixed standard substances of low, medium and high are respectively added, the same steps are repeated to determine 5 times, the result shows that the adding standard recovery rate of the antiepileptic drug is between 87.69% and 107.64%, the RSD of 5 repeated tests is in the range of 1.7% to 8.01%, and the statistical result is shown in Table 10.
TABLE 10 results of recovery of antiepileptic drugs with a standard
4. And (3) precision test: taking an interference-free blank serum sample, adding antiepileptic drug standards with different concentrations to obtain serum samples with low, medium and high concentrations, repeatedly processing 6 batches in one day for three days continuously, quantitatively determining the concentration of the antiepileptic drug by an isotope internal standard method, wherein the batch precision is 1.37-13.12%, processing 3 batches in three days, and calculating the batch precision to be 2.74-12.73%, and the result is shown in Table 11.
TABLE 11 results of inter-batch precision measurements
The invention establishes a method for simultaneously measuring the antiepileptic drugs in human serum by ID-UPLC-MS/MS. The serum dosage is less (only 50 mu L), the pretreatment is simple, and the analysis of various substances by one injection only needs 6min, and the method is simple and quick.
The isotope internal standard method is adopted for quantification, so that the matrix interference can be greatly eliminated, the result is not influenced by conditions such as a pretreatment process, instrument response fluctuation and the like, and accurate quantification can be achieved. The result of the method for evaluating the accuracy of the standard recovery test shows that the standard recovery of the antiepileptic drug is 87.69-107.64%, the RSD of 5 repeated tests is 1.7-8.01%, and the accuracy is good.
The reproducibility result of the method shows that the precision of the antiepileptic drug in batches is 1.37-13.12%, the precision between the batches is 2.74-12.73% by 3-batch processing within three days, and the reproducibility of the method is good.
In a word, the method has the advantages of high sensitivity, strong specificity, accuracy and simple pretreatment process, completes the separation and detection of the compound within 6min, meets the requirements on accuracy and precision, can be used for quantitative analysis of clinical serum antiepileptic drugs, and provides a reliable detection method for monitoring related drug concentrations.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: modifications of the technical solutions described in the foregoing embodiments are still possible, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A detection kit for an antiepileptic drug in serum is characterized in that: the antiepileptic medicine comprises: levetiracetam, 10-hydroxycarbazepine, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, valproic acid, phenobarbital, topiramate and clonazepam;
the kit comprises: mobile phase, calibrator solution, mixed internal standard solution, protein precipitant and quality control material;
the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is formic acid-ammonium formate aqueous solution, and the mobile phase B is acetonitrile;
the calibrator solution comprises a series of mixed solutions of levetiracetam, 10-hydroxycarbazepine, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, valproic acid, phenobarbital, topiramate and clonazepam, which are prepared from blank serum and have known concentrations;
the mixed internal standard solution is a mixed solution of levetiracetam-d 3, 10-hydroxycarbazepine-d 4, carbamazepine-d 2,15N, oxcarbazepine-d 4, phenytoin-d 10, lamotrigine-13C 3, valproic acid-d 6, phenobarbital-d 5, topiramate-d 12 and clonazepam-d 4, which have known concentration and are prepared by a methanol aqueous solution;
the quality control product comprises a mixed solution of levetiracetam, 10-hydroxycarbazepine, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, valproic acid, phenobarbital, topiramate and clonazepam which have known concentration and are prepared by blank serum;
the protein precipitator is a mixed solution of methanol and acetonitrile.
2. The kit of claim 1, wherein: the mobile phase A is 0.01% -0.2% formic acid-0.1 mM-10 mM ammonium formate aqueous solution.
3. The kit of claim 2, wherein: the mobile phase A was 0.1% formic acid-2 mM aqueous ammonium formate solution.
4. The kit of claim 1, wherein: the calibrator solution comprises the following 7 mixed solutions:
mixed solution 1: the concentrations of levetiracetam, 10-hydroxycarbazepine, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, valproic acid, phenobarbital, topiramate and clonazepam were: levetiracetam 100ng/mL, 10-hydroxycarbazepine 20ng/mL, carbamazepine 50ng/mL, oxcarbazepine 100ng/mL, phenytoin 100ng/mL, lamotrigine 20ng/mL, valproic acid 500ng/mL, phenobarbital 200ng/mL, topiramate 100ng/mL and clonazepam 0.5 ng/mL;
and (2) mixed solution: the concentrations of levetiracetam, 10-hydroxycarbazepine, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, valproic acid, phenobarbital, topiramate and clonazepam were: levetiracetam 250 ng/mL, 10-hydroxycarbazepine 50ng/mL, carbamazepine 125 ng/mL, oxcarbazepine 250 ng/mL, phenytoin 250 ng/mL, lamotrigine 50ng/mL, valproic acid 1250 ng/mL, phenobarbital 500ng/mL, topiramate 250 ng/mL and clonazepam 1.25 ng/mL;
and (3) mixed solution: the concentrations of levetiracetam, 10-hydroxycarbazepine, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, valproic acid, phenobarbital, topiramate and clonazepam were: levetiracetam 500ng/mL, 10-hydroxycarbazepine 100ng/mL, carbamazepine 250 ng/mL, oxcarbazepine 500ng/mL, phenytoin 500ng/mL, lamotrigine 100ng/mL, valproic acid 2500 ng/mL, phenobarbital 1000ng/mL, topiramate 500ng/mL and clonazepam 2.5 ng/mL;
and (4) mixed solution: the concentrations of levetiracetam, 10-hydroxycarbazepine, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, valproic acid, phenobarbital, topiramate and clonazepam were: levetiracetam 2500 ng/mL, 10-hydroxycarbazepine 500ng/mL, carbamazepine 1250 ng/mL, oxcarbazepine 2500 ng/mL, phenytoin 2500 ng/mL, lamotrigine 500ng/mL, valproic acid 12500 ng/mL, phenobarbital 5000ng/mL, topiramate 2500 ng/mL, and clonazepam 12.5 ng/mL;
and (5) mixed solution: the concentrations of levetiracetam, 10-hydroxycarbazepine, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, valproic acid, phenobarbital, topiramate and clonazepam were: levetiracetam 5000ng/mL, 10-hydroxycarbazepine 1000ng/mL, carbamazepine 2500 ng/mL, oxcarbazepine 5000ng/mL, phenytoin 5000ng/mL, lamotrigine 1000ng/mL, valproic acid 25000 ng/mL, phenobarbital 10000ng/mL, topiramate 5000ng/mL and clonazepam 25 ng/mL;
and (6) mixed solution: the concentrations of levetiracetam, 10-hydroxycarbazepine, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, valproic acid, phenobarbital, topiramate and clonazepam were: levetiracetam 25000 ng/mL, 10-hydroxycarbazepine 5000ng/mL, carbamazepine 12500 ng/mL, oxcarbazepine 25000 ng/mL, phenytoin 25000 ng/mL, lamotrigine 5000ng/mL, valproic acid 125000 ng/mL, phenobarbital 50000 ng/mL, topiramate 25000 ng/mL and clonazepam 125 ng/mL;
and (3) mixed solution 7: the concentrations of levetiracetam, 10-hydroxycarbazepine, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, valproic acid, phenobarbital, topiramate and clonazepam were: levetiracetam 50000 ng/mL, 10-hydroxycarbazepine 10000ng/mL, carbamazepine 25000 ng/mL, oxcarbazepine 50000 ng/mL, phenytoin 50000 ng/mL, lamotrigine 10000ng/mL, valproic acid 250000 ng/mL, phenobarbital 100000ng/mL, topiramate 50000 ng/mL and clonazepam 250 ng/mL.
5. The kit of claim 1, wherein: the concentrations of levetiracetam-d 3, 10-hydroxycarbazepine-d 4, carbamazepine-d 2,15N, oxcarbazepine-d 4, phenytoin-d 10, lamotrigine-13C 3, valproic acid-d 6, phenobarbital-d 5, topiramate-d 12 and clonazepam-d 4 in the mixed internal standard solution are respectively levetiracetam-d 320000 ng/mL, 10-hydroxycarbazepine-d 45000ng/mL, carbamazepine-d 2,15N10000ng/mL, oxcarbazepine-d 420000ng/mL, phenytoin-d 1020000 ng/mL, valproic acid-d 6100000ng/mL, topiramate-d 1220000ng/mL, clonazepam-d 4500ng/mL, lamotrigine-13C 35000 ng/mL, phenobarbital-d 51000 ng/mL.
6. The kit of claim 1, wherein: the volume ratio of methanol to acetonitrile in the protein precipitator is 1: 1-5.
7. The kit of claim 6, wherein: the volume ratio of methanol to acetonitrile in the protein precipitant is 1: 3.
8. The kit of claim 1, wherein: the quality control product comprises the following mixed liquid with 3 concentration ratios:
low concentration quality control solution: the concentrations of levetiracetam, 10-hydroxycarbazepine, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, valproic acid, phenobarbital, topiramate and clonazepam were: levetiracetam 200ng/mL, 10-hydroxycarbazepine 40ng/mL, carbamazepine 100ng/mL, oxcarbazepine 200ng/mL, phenytoin 200ng/mL, lamotrigine 40ng/mL, valproic acid 1000ng/mL, phenobarbital 400ng/mL, topiramate 200ng/mL and clonazepam 1 ng/mL;
medium concentration quality control solution: the concentrations of levetiracetam, 10-hydroxycarbazepine, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, valproic acid, phenobarbital, topiramate and clonazepam were: levetiracetam 2000ng/mL, 10-hydroxycarbazepine 400ng/mL, carbamazepine 1000ng/mL, oxcarbazepine 2000ng/mL, phenytoin 2000ng/mL, lamotrigine 400ng/mL, valproic acid 10000ng/mL, phenobarbital 4000ng/mL, topiramate 2000ng/mL and clonazepam 10 ng/mL;
high concentration quality control solution: the concentrations of levetiracetam, 10-hydroxycarbazepine, carbamazepine, oxcarbazepine, phenytoin, lamotrigine, valproic acid, phenobarbital, topiramate and clonazepam were: levetiracetam 20000ng/mL, 10-hydroxycarbazepine 4000ng/mL, carbamazepine 10000ng/mL, oxcarbazepine 20000ng/mL, phenytoin 20000ng/mL, lamotrigine 4000ng/mL, valproic acid 100000ng/mL, phenobarbital 40000ng/mL, topiramate 20000ng/mL and clonazepam 100 ng/mL.
9. The use of the kit of claim 1 for detecting antiepileptic drugs in serum using ultra performance liquid chromatography tandem mass spectrometry.
10. Use according to claim 9, characterized in that: the mixed internal standard solution is mixed with the protein precipitator to prepare the protein precipitator containing the internal standard, and then the protein precipitator is used for detection, wherein the volume ratio of the mixed internal standard solution to the protein precipitator is 0.1-0.3: 19.7 to 19.9.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112946101A (en) * | 2021-01-26 | 2021-06-11 | 山东英盛生物技术有限公司 | Method for simultaneously determining content of multiple antiepileptic drugs in blood and application thereof |
| CN112946100A (en) * | 2021-01-26 | 2021-06-11 | 山东英盛生物技术有限公司 | Method and kit for determining carbamazepine and 10-hydroxycarbazepine and application thereof |
| CN113092615A (en) * | 2021-04-01 | 2021-07-09 | 四川大学华西医院 | Method for detecting antiepileptic drug and metabolite thereof in hair by liquid chromatography-mass spectrometry |
| CN113192649A (en) * | 2021-06-01 | 2021-07-30 | 山东英盛生物技术有限公司 | System for guiding individualized and accurate medication of epileptic diseases |
| CN113884592A (en) * | 2021-09-27 | 2022-01-04 | 昭衍（苏州）新药研究中心有限公司 | Method for detecting concentration of carbamazepine drug in dry blood spots |
| CN115639306A (en) * | 2022-12-26 | 2023-01-24 | 四川大学华西医院 | Method for rapidly detecting concentration of antiepileptic drug in clinical sample |
| CN117825592A (en) * | 2024-03-05 | 2024-04-05 | 北京水木济衡生物技术有限公司 | Quality control product, kit and method for detecting concentration of antiepileptic drugs in human blood matrix |
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| CN112946101A (en) * | 2021-01-26 | 2021-06-11 | 山东英盛生物技术有限公司 | Method for simultaneously determining content of multiple antiepileptic drugs in blood and application thereof |
| CN112946100A (en) * | 2021-01-26 | 2021-06-11 | 山东英盛生物技术有限公司 | Method and kit for determining carbamazepine and 10-hydroxycarbazepine and application thereof |
| CN112946101B (en) * | 2021-01-26 | 2022-05-13 | 山东英盛生物技术有限公司 | Method for simultaneously determining content of multiple antiepileptic drugs in blood and application thereof |
| CN113092615A (en) * | 2021-04-01 | 2021-07-09 | 四川大学华西医院 | Method for detecting antiepileptic drug and metabolite thereof in hair by liquid chromatography-mass spectrometry |
| CN113192649A (en) * | 2021-06-01 | 2021-07-30 | 山东英盛生物技术有限公司 | System for guiding individualized and accurate medication of epileptic diseases |
| CN113192649B (en) * | 2021-06-01 | 2023-09-22 | 山东英盛生物技术有限公司 | System for guiding individual accurate medication of epileptic diseases |
| CN113884592A (en) * | 2021-09-27 | 2022-01-04 | 昭衍（苏州）新药研究中心有限公司 | Method for detecting concentration of carbamazepine drug in dry blood spots |
| CN115639306A (en) * | 2022-12-26 | 2023-01-24 | 四川大学华西医院 | Method for rapidly detecting concentration of antiepileptic drug in clinical sample |
| CN115639306B (en) * | 2022-12-26 | 2023-03-28 | 四川大学华西医院 | Method for rapidly detecting concentration of antiepileptic drug in clinical sample |
| CN117825592A (en) * | 2024-03-05 | 2024-04-05 | 北京水木济衡生物技术有限公司 | Quality control product, kit and method for detecting concentration of antiepileptic drugs in human blood matrix |
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