CN111781290A - Kit and detection method for accurately determining blood concentration of multiple antiepileptic drugs in human serum - Google Patents
Kit and detection method for accurately determining blood concentration of multiple antiepileptic drugs in human serum Download PDFInfo
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Abstract
The invention discloses a kit and a detection method for accurately determining the blood concentration of various antiepileptic drugs in human serum, wherein the kit mainly comprises: mother liquor of a calibrator: methanol or acetonitrile is used as a diluent to prepare a mother liquor of a calibrator with 6 concentration points, and the types of the mother liquor of the calibrator are as follows in sequence: lamotrigine, phenobarbital, oxcarbazepine, carbamazepine, phenytoin; also comprises an internal standard, a quality control product, a matrix correction fluid and an extraction liquid: the extract is tert-butyl methyl ether solution, redissolution and mobile phase. Complex purification steps are not needed, and the required analysis time is short; the speed of detection is improved.
Description
Technical Field
The invention relates to the technical field of medical inspection, in particular to a kit and a detection method for accurately determining the blood concentration of multiple antiepileptic drugs in human serum.
Background
The antiepileptic drugs such as Phenobarbital (PB), Phenytoin (PHT), Carbamazepine (CBZ), Oxcarbazepine (Oxcarbazepine, OXC) and Lamotrigine (LTG) belong to controlled-form drugs, and most epileptic patients need to take the drugs for a long time and have great individual difference in drug response. PB, PHT and CBZ are all first-generation antiepileptic drugs commonly used in clinic, OXC and LTG are second-generation antiepileptic drugs on the market in nearly ten years, and due to different pharmacological characteristics and clinical indications of the drugs, the drugs are commonly combined in clinic.
But the therapeutic concentration range is narrow, the individual difference of metabolism is large, the toxic concentration is close to the therapeutic concentration range, the self-induction effect is realized, the in-vivo treatment process is complex, and the individual difference of concentration and effect exists. The anti-epileptic effect and the toxic effect are closely related to the blood concentration, the adverse reaction is more after long-term administration, and the drug resistance is easy to generate. In addition, although the single drug is used for clinically treating the epilepsy, a certain number of patients belong to intractable epilepsy and need to be combined, or the combined application of the traditional antiepileptic drugs, such as the combined application of carbamazepine, phenobarbital, phenytoin sodium and valproic acid, or the combined application of the traditional antiepileptic drugs and new antiepileptic drugs, such as LTG and the currently commonly used antiepileptic drugs, such as PHT, CBZ and the like, have pharmacokinetic interaction, especially the traditional antiepileptic drugs are mostly liver drug enzyme inducers or inhibitors, and have influence on the drugs combined with the drugs. The pharmacokinetic behavior of the antiepileptic drug is more complex due to the interaction between the multi-drug combination drugs, so that the blood concentration difference between individual patients is large, and the monitoring and individual administration of the blood concentration are also needed during clinical use. PB has an induction effect on liver drug enzymes to influence the normal metabolism of other drugs, so that the blood concentration is reduced; CBZ has side effects in the range of blood concentration for treatment, and the blood concentration and clearance are greatly changed due to self-induced metabolism; PHT also has nonlinear kinetics, and individualized dosing is claimed clinically. When OXC is used in combination with PHT, PB and LTG, it can increase PHT blood concentration, reduce its metabolism, increase the toxicity of the latter, manifested as ataxia, nystagmus, hyperreflexia, etc., and also increase the metabolism of LTG by liver, reduce its blood concentration and weaken its antiepileptic action. Therefore, patients need to be treated regularly. Monitoring of blood drug levels and personalized administration are needed in order to improve the effectiveness and safety of clinical medication, reduce or avoid toxic side effects, and provide reliable objective basis for diagnosis and treatment of intoxication from overdose.
At present, many methods for measuring the concentration of serum antiepileptic drugs at home and abroad are available, and common monitoring methods include Fluorescence Polarization Immunoassay (FPIA), enzyme-enhanced immunoassay (EMIT), High Performance Liquid Chromatography (HPLC), liquid-mass spectrometry (LC-MS/MS) and the like. The immunization method is simple to operate, but the reagent needs to be imported, the price is high, the effective period is short, multiple medicines cannot be simultaneously measured, and the cost is relatively high for the primary hospitals with few sources of diseases, so that the development of the primary hospitals is not facilitated. The FPIA method utilizes mutual recognition and combination of antigen and antibody to generate a detection signal, and when some antigens have the same antigen surface mark as an object to be detected or the antibodies have a plurality of antigen binding sites, the FPIA method can cause the antibodies to generate cross reaction with maternal metabolites and endogenous substances with similar antigen structures, so that the determination result is higher than that of an HPLC method with stronger specificity. When the FPIA method is used for measuring the CBZ, the metabolism of the CBZ in a human body is 10, 11-epoxy Carbamazepine (CBZE), the pharmacological action and the toxic and side effect are similar to those of the CBZ, the measurement value of the FPIA method is the sum of the CBZE and the CBZ, and the interference of metabolites on the CBZ cannot be eliminated by the FPIA method. The HPLC method and the LC-MS/MS method have strong specificity and high sensitivity, metabolites of the methods do not interfere with the determination of analytes by adjusting proper chromatographic conditions, and can simultaneously determine a plurality of drugs and the metabolites thereof, the results are accurate, the linear range is relatively wide, the detection of clinical patients can be met, and compared with the HPLC, the LC-MS/MS method has higher instrument price and maintenance cost, higher requirements on personnel and environment, time and cost, and great distance for clinical use.
At present, liquid phase clinical examination methods are all methods established by laboratories, and have the problems of complex pretreatment operation, long analysis time, more required organic solvents, low sensitivity and the like.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method which does not need to carry out complicated purification steps and needs less analysis time; the detection speed is improved; the gradient elution mode is adopted, so that the efficiency of the chromatographic column can be better protected, and the chromatographic column can be matched with a liquid chromatograph for use and is suitable for the inspection of clinical samples; meanwhile, the product can simultaneously detect the concentration of 5 antiepileptic drugs such as phenobarbital, phenytoin, carbamazepine, oxcarbazepine and lamotrigine in serum, achieves the function of single-sample multi-index synchronous detection, and has the characteristics of high accuracy, short detection time, less reagent consumption, convenient operation, low cost and the like.
In order to solve the technical problems, the invention adopts the technical scheme that: a kit for accurately determining the blood concentration of a plurality of antiepileptic drugs in human serum mainly comprises:
(1) mother liquor of a calibrator: using methanol or acetonitrile as a diluent to prepare a mother liquor of a calibrator comprising 6 concentration points, wherein the 6 concentration points of the mother liquor of the calibrator are as follows in sequence:
lamotrigine: 10.00. mu.g/mL, 20.00. mu.g/mL, 50.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL;
phenobarbital: 20.00. mu.g/mL, 40.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL, 800.00. mu.g/mL;
oxcarbazepine: 15.00. mu.g/mL, 30.00. mu.g/mL, 75.00. mu.g/mL, 150.00. mu.g/mL, 300.00. mu.g/mL, 600.00. mu.g/mL;
carbamazepine: 15.00. mu.g/mL, 30.00. mu.g/mL, 75.00. mu.g/mL, 150.00. mu.g/mL, 300.00. mu.g/mL, 600.00. mu.g/mL;
phenytoin: 20.00. mu.g/mL, 40.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL, 800.00. mu.g/mL;
the mother liquor of the calibrator is a mixed solution of lamotrigine, phenobarbital, oxcarbazepine, carbamazepine and phenytoin; the concentration of each component in the total mixed solution is the concentration of each component in the six points; the concentration of the calibrator working solution is multiplied by 40/200;
(2) internal standard: the internal standard solution is ketoprofen methanol solution (prepared solution with ketoprofen as solute and methanol as solvent), and the concentration range of the ketoprofen in the solution is 10-1000 mug/mL;
(3) quality control product: comprises a quality control product 1 and a quality control product 2, wherein
The concentration range and the components of the quality control material 1 comprise: lamotrigine: 3.95-5.93 mu g/mL; phenobarbital: 8.76-11.80 mug/mL; oxcarbazepine: 5.59-9.32 mu g/mL; carbamazepine: 3.86-5.78 mu g/mL; phenytoin: 4.22-5.72 mu g/mL;
the concentration range and the components of the quality control material 2 comprise: lamotrigine: 11.40-17.00 mu g/mL; phenobarbital: 38.00-57.00 mu g/mL; oxcarbazepine: 15.80-23.70 mu g/mL; carbamazepine: 11.70-17.60 mu g/mL; phenytoin: 23.10-31.30 mu g/mL;
the quality control product 1 and the quality control product 2 are both composed of a mixture of lamotrigine, phenobarbital, oxcarbazepine, carbamazepine and phenytoin; the concentrations of the respective components are the respective concentrations in the mixture of the quality control material 1 or the quality control material 2;
(4) matrix correction fluid: the matrix correction fluid is blank serum freeze-dried powder;
(5) extracting liquid: the extract is tert-butyl methyl ether solution (namely 100 percent of tert-butyl methyl ether);
(6) compounding the solution: the complex solution is a mixture of methanol or acetonitrile and water, and the volume percentage content of the methanol or the acetonitrile in the mixture is 40-100%;
(7) mobile phase: the mobile phase A is aqueous solution, the mobile phase B is acetonitrile solution, and both the mobile phase A and the mobile phase B contain 0.05-0.2% of formic acid by volume percentage.
Preferably, the diluent (solvent) in the step (1) of the present invention is a methanol solution.
Preferably, the quality control substance 1 and the quality control substance 2 in the step (3) of the present invention are both composed of a mixture of lamotrigine, phenobarbital, oxcarbazepine, carbamazepine and phenytoin, and the form of the quality control substance 1 or the quality control substance 2 may be a freeze-dried powder, a serum matrix or a methanol solution matrix.
Preferably, the composite solution of step (6) of the present invention is a 50% methanol aqueous solution by volume percentage.
Preferably, in step (7) of the present invention, the content of formic acid in mobile phase a is 0.1% by volume, and the content of formic acid in mobile phase B is 0.05% by volume.
The kit of the invention also comprises an operation instruction.
The invention also provides a detection method of the kit for accurately measuring the concentration of the antiepileptic drug in blood in human serum, which comprises the following steps: adding methanol solution of internal standard ketoprofen into human serum, mixing, adding the extract, mixing and centrifuging, removing supernatant, drying with nitrogen, adding redissolution for redissolving, mixing and centrifuging, taking supernatant, detecting by using a liquid chromatograph-PDA detector, and quantifying by using an internal standard curve method.
The invention relates to a detection method of a kit for accurately measuring the blood concentration of an anti-epileptic drug in human serum, which comprises the following specific steps:
(1) test sample processing
(1.1) calibrator: accurately sucking 40 mu L of calibrator mother liquor into a centrifuge tube at room temperature, drying the calibrator mother liquor by nitrogen at 45 ℃, adding 200 mu L of matrix correction fluid, mixing in a vortex manner, adding 100 mu L of internal standard with known concentration, mixing in a vortex manner for 1min, adding 800 mu L of extract liquor, mixing in a vortex manner for 5min to completely mix the internal standard with the matrix correction fluid, centrifuging at 15000r/min for 2min, and drying the supernatant by nitrogen at 45 ℃; adding 150 μ L of redissolution for redissolution, mixing with vortex, centrifuging at 15000r/min for 5min, collecting supernatant 120 μ L, and adding into sample bottle for chromatographic analysis;
(1.2) quality control/serum sample: accurately sucking 200 μ L of quality control product/serum sample into a centrifuge tube at room temperature, adding 100 μ L of internal standard with known concentration, mixing for 1min by vortex, adding 800 μ L of extract, mixing for 5min by vortex, centrifuging at 15000r/min for 2min, collecting supernatant, and blow-drying with 45 deg.C nitrogen gas; adding 150 μ L of redissolution for redissolution, mixing with vortex, centrifuging at 15000r/min for 5min, collecting supernatant 120 μ L, and adding into sample bottle for chromatographic analysis;
(2) sample detection assay
(2.1) chromatographic conditions
Column chromatography: c18 column or equivalent;
the mobile phase: mobile phase a and mobile phase B;
detection wavelength: lamotrigine (265 nm); phenobarbital and phenytoin (230 nm); oxcarbazepine (240 nm);
carbamazepine (284 nm);
flow rate: 0.5 mL/min;
gradient elution: as shown in table 1 below;
TABLE 1
In the gradient elution process in the table above, the volume ratio of the mobile phase a to the mobile phase B in the mobile phase is continuously adjusted and changed with time, and the change in the proportion of the mobile phase B is used as an indication.
Column temperature: 40 ℃;
sample injector temperature: 8 ℃;
sample size: 8 mu L of the solution;
(2.2) injecting 8 mu L of each of the processed calibrator, the quality control material and the sample into a liquid chromatograph, and recording a chromatogram;
(2.3) calculation of detection result
(2.3.1) plotting and fitting of calibration curves: general liquid chromatography analysis software can automatically draw a calibration curve according to a calibration result, a concentration ratio of 6 calibrator working solutions to an internal standard is used as an abscissa (x), a ratio of actual measurement peak areas of the 6 calibrator working solutions to respective internal standard peak areas is used as an ordinate (y), linear regression is performed by using a least square method, a standard curve is automatically drawn, and a regression equation can be obtained: y is ax + b, wherein y is a vertical coordinate, x is a horizontal coordinate, a is a slope, b is an intercept, and r (correlation coefficient) value is calculated, and the r value is more than or equal to 0.9900;
(2.3.2) calculation of recovery: the measured concentration of each analyte in the quality control product can be obtained from a calibration curve according to the peak area ratio of each analyte in the quality control product to the corresponding internal standard, and the calculation formula of the recovery rate is as follows: the recovery rate (%) is that the determination concentration/indication concentration is multiplied by 100, and the recovery rate (%) is in the range of 85-115%;
(2.3.3) calculation of sample results: the measured concentration of each analyte in the sample can be obtained from the calibration curve based on the ratio of the peak areas of each analyte in the sample and the corresponding internal standard.
In the step (1), the vortex mixing speed is 2000rpm, and under the rotation speed, the mixed liquid is completely mixed, and the substance to be detected is completely extracted.
The volume of the extract liquid added in the step (1) is 800 mu L, so that the substance to be detected can be completely extracted from the sample; the volume of the reconstituted solution was 150. mu.L to ensure that the test substance was completely reconstituted from the sample.
The extraction liquid and the redissolution are added in the step (1) of the invention, mixed and centrifuged, namely, centrifuged at a low temperature and a high speed, namely, at a temperature of between 5 and 10 ℃, preferably at a temperature of between 8 ℃ and 15000 rpm. The centrifugation process is preferably performed at low temperature to ensure that the sample material is stable, coupled with high speed centrifugation to make the supernatant cleaner, and to protect the instrument and chromatography column.
The invention has the advantages and beneficial effects that:
the kit is adopted to process a serum sample, a very clean processing solution can be obtained without complicated purification and purification steps through a simple liquid-liquid extraction (one-step extraction) pretreatment method, high sensitivity is obtained through concentration, the required analysis time is short, the separation of 5 antiepileptic drugs and internal standards is completed within 9min, and the detection speed is improved; the gradient elution mode is adopted, so that the efficiency of the chromatographic column can be better protected, and the chromatographic column can be matched with a liquid chromatograph for use and is suitable for the inspection of clinical samples; meanwhile, the product can simultaneously detect the concentration of 5 antiepileptic drugs including phenobarbital, phenytoin, carbamazepine, oxcarbazepine and lamotrigine in serum, achieves the function of single-sample multi-index synchronous detection, and has the characteristics of high accuracy, short detection time, less reagent consumption, convenient operation, low cost and the like.
Drawings
FIG. 1 shows the chemical structural formulas of 5 antiepileptic drugs of phenobarbital, phenytoin, carbamazepine, oxcarbazepine and lamotrigine.
FIG. 2 is a chromatogram of 5 antiepileptic drugs of phenobarbital, phenytoin, carbamazepine, oxcarbazepine and lamotrigine and an internal standard solution.
FIG. 3 is a chromatogram of a blank serum sample of the present invention.
FIG. 4 is a chromatogram of an actual measurement serum sample of 5 antiepileptic drugs of phenobarbital, phenytoin, carbamazepine, oxcarbazepine and lamotrigine.
Fig. 5 is a standard curve for lamotrigine, linear range: (1.00-40.00) mu g/mL.
FIG. 6 is a standard curve of phenobarbital, linear range: (2.00-80.00) mu g/mL.
Figure 7 is a standard curve for oxcarbazepine, linear range: (1.50-60.00) mu g/mL.
Fig. 8 is a standard curve for carbamazepine, linear range: (1.50-60.00) mu g/mL.
Fig. 9 is a standard curve of phenytoin, linear range: (2.00-80.00) mu g/mL.
Detailed Description
The invention will now be further illustrated by the following non-limiting examples, and it will be apparent to those skilled in the art that modifications may be made without departing from the spirit of the invention, such modifications also falling within the scope of the invention.
The following experimental methods are all conventional methods unless otherwise specified, and the experimental materials used are readily available from commercial companies unless otherwise specified.
Example 1:
1. mother liquor of a calibrator: using methanol as a diluent to prepare a calibrator mother liquor comprising 6 concentration points, wherein the 6 concentration points of the calibrator mother liquor are as follows in sequence:
lamotrigine: 10.00. mu.g/mL, 20.00. mu.g/mL, 50.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL;
phenobarbital: 20.00. mu.g/mL, 40.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL, 800.00. mu.g/mL;
oxcarbazepine: 15.00. mu.g/mL, 30.00. mu.g/mL, 75.00. mu.g/mL, 150.00. mu.g/mL, 300.00. mu.g/mL, 600.00. mu.g/mL;
carbamazepine: 15.00. mu.g/mL, 30.00. mu.g/mL, 75.00. mu.g/mL, 150.00. mu.g/mL, 300.00. mu.g/mL, 600.00. mu.g/mL;
phenytoin: 20.00. mu.g/mL, 40.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL, 800.00. mu.g/mL;
the mother liquor of the calibrator is a mixed solution of lamotrigine, phenobarbital, oxcarbazepine, carbamazepine and phenytoin; the concentration of each component in the total mixed solution is the concentration of each component in the six points; the mother liquor of each concentration point contains lamotrigine, phenobarbital, oxcarbazepine, carbamazepine and phenytoin, and the concentration of the five substances in the mother liquor of each concentration point is generally formed by correspondingly proportioning the concentration from the low concentration to the high concentration in the concentration of the 6 concentration points, for example, the concentration of lamotrigine in the mother liquor of the calibrator of the first concentration point is 10.00 mu g/mL, the concentration of phenobarbital is 20.00 mu g/mL, the concentration of oxcarbazepine is 15.00 mu g/mL, the concentration of carbamazepine is 15.00 mu g/mL, and the concentration of the five substances in the mother liquor of each concentration point corresponds to the mode, and the following embodiment is also the same; the concentration of the calibrator working solution is multiplied by 40/200;
2. internal standard: ketoprofen solution in methanol, wherein the concentration of ketoprofen in the solution is 500 mu g/mL;
3. quality control product: comprises a quality control product 1 and a quality control product 2, wherein
The concentration range and the components of the quality control material 1 comprise: lamotrigine: 4.28. mu.g/mL; phenobarbital: 9.84. mu.g/mL; oxcarbazepine: 7.46 mu g/mL; carbamazepine: 4.82 μ g/mL; phenytoin: 5.03 mu g/mL;
the concentration range and the components of the quality control material 2 comprise: lamotrigine: 12.61. mu.g/mL; phenobarbital: 41.27 μ g/mL; oxcarbazepine: 19.80. mu.g/mL; carbamazepine: 12.51. mu.g/mL; phenytoin: 25.54. mu.g/mL;
the quality control product 1 and the quality control product 2 are both a mixture of lamotrigine, phenobarbital, oxcarbazepine, carbamazepine and phenytoin; the concentrations of the respective components are the respective concentrations in the mixture of the quality control material 1 or the quality control material 2 (the quality control material may be in the form of lyophilized powder, serum matrix or methanol solution matrix, and the quality control material in the form of lyophilized powder matrix is used herein, and the same is true in the following embodiment);
4. matrix correction fluid: blank serum lyophilized powder (1mL of lyophilized powder frozen from blank serum) is precisely measured and taken 1mL of purified water into a bottle when in use, and the mixture is mixed for 30min until the lyophilized powder is completely dissolved;
5. extracting liquid: t-butyl methyl ether solution (i.e., 100% t-butyl methyl ether);
6. compounding the solution: 50% methanol aqueous solution by volume;
7. mobile phase: mobile phase a (0.1% aqueous formic acid), mobile phase B (0.05% acetonitrile formic acid).
The detection is carried out by using the kit, and the steps are as follows:
(1) test sample processing
Accurately absorbing 40 mu L of calibrator mother liquor into a centrifuge tube at room temperature, drying by nitrogen at 45 ℃, adding 200 mu L of matrix correction fluid, mixing in a vortex manner, adding 100 mu L of internal standard with known concentration, mixing in a vortex manner for 1min, adding 800 mu L of extract liquor, mixing in a vortex manner for 5min to completely mix, centrifuging at 15000r/min for 2min, and drying supernatant by nitrogen at 45 ℃; adding 150 μ L of redissolution for redissolution, mixing with vortex, centrifuging at 15000r/min for 5min, collecting supernatant 120 μ L, and adding into sample bottle for chromatographic analysis;
quality control product/serum sample: accurately sucking 200 μ L of quality control product/serum sample into a centrifuge tube at room temperature, adding 100 μ L of internal standard with known concentration, mixing for 1min by vortex, adding 800 μ L of extract, mixing for 5min by vortex, centrifuging at 15000r/min for 2min, collecting supernatant, and blow-drying with 45 deg.C nitrogen gas; adding 150 μ L of redissolution for redissolution, mixing with vortex, centrifuging at 15000r/min for 5min, collecting supernatant 120 μ L, and adding into sample bottle for chromatographic analysis;
(2) sample detection assay
(2.1) chromatographic conditions
Column chromatography: c18 column or equivalent;
the mobile phase: mobile phase a and mobile phase B;
detection wavelength: lamotrigine (265 nm); phenobarbital and phenytoin (230 nm); oxcarbazepine (240 nm);
carbamazepine (284 nm);
flow rate: 0.5 mL/min;
gradient elution: as shown in table 2 below;
TABLE 2
In the gradient elution process in the table above, the volume ratio of the mobile phase a to the mobile phase B in the mobile phase is continuously adjusted and changed with time, and the change in the proportion of the mobile phase B is used as an indication.
Column temperature: 40 ℃;
sample injector temperature: 8 ℃;
sample size: 8 mu L of the solution;
example 2:
1. mother liquor of a calibrator: using methanol as a diluent to prepare a calibrator mother liquor comprising 6 concentration points, wherein the 6 concentration points of the calibrator mother liquor are as follows in sequence:
lamotrigine: 10.00. mu.g/mL, 20.00. mu.g/mL, 50.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL;
phenobarbital: 20.00. mu.g/mL, 40.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL, 800.00. mu.g/mL;
oxcarbazepine: 15.00. mu.g/mL, 30.00. mu.g/mL, 75.00. mu.g/mL, 150.00. mu.g/mL, 300.00. mu.g/mL, 600.00. mu.g/mL;
carbamazepine: 15.00. mu.g/mL, 30.00. mu.g/mL, 75.00. mu.g/mL, 150.00. mu.g/mL, 300.00. mu.g/mL, 600.00. mu.g/mL;
phenytoin: 20.00. mu.g/mL, 40.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL, 800.00. mu.g/mL;
the mother liquor of the calibrator is a mixed solution of lamotrigine, phenobarbital, oxcarbazepine, carbamazepine and phenytoin; the concentration of each component in the total mixed solution is the concentration of each component in the six points; the concentration of the calibrator working solution is multiplied by 40/200;
2. internal standard: ketoprofen in methanol at a concentration of 500 μ g/mL;
3. quality control product: comprises a quality control product 1 and a quality control product 2, wherein
The concentration range and the components of the quality control material 1 comprise: lamotrigine: 4.28. mu.g/mL; phenobarbital: 9.84. mu.g/mL; oxcarbazepine: 7.46 mu g/mL; carbamazepine: 4.82 μ g/mL; phenytoin: 5.03 mu g/mL;
the concentration range and the components of the quality control material 2 comprise: lamotrigine: 12.61. mu.g/mL; phenobarbital: 41.27 μ g/mL; oxcarbazepine: 19.80. mu.g/mL; carbamazepine: 12.51. mu.g/mL; phenytoin: 25.54. mu.g/mL;
the quality control product 1 and the quality control product 2 are both a mixture of lamotrigine, phenobarbital, oxcarbazepine, carbamazepine and phenytoin; the concentrations of the respective components are the respective concentrations in the mixture of the quality control material 1 or the quality control material 2;
4. matrix correction fluid: blank serum lyophilized powder (1mL of lyophilized powder frozen from blank serum) is precisely measured and taken 1mL of purified water into a bottle when in use, and the mixture is mixed for 30min until the lyophilized powder is completely dissolved;
5. extracting liquid: t-butyl methyl ether solution (i.e., 100% t-butyl methyl ether);
6. compounding the solution: 100% methanol solution;
7. mobile phase: mobile phase a (0.1% aqueous formic acid), mobile phase B (0.05% acetonitrile formic acid).
The detection was carried out using the above-mentioned kit in the same manner as in example 1.
Example 3:
1. mother liquor of a calibrator: using methanol as a diluent to prepare a calibrator mother liquor comprising 6 concentration points, wherein the 6 concentration points of the calibrator mother liquor are as follows in sequence:
lamotrigine: 10.00. mu.g/mL, 20.00. mu.g/mL, 50.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL;
phenobarbital: 20.00. mu.g/mL, 40.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL, 800.00. mu.g/mL;
oxcarbazepine: 15.00. mu.g/mL, 30.00. mu.g/mL, 75.00. mu.g/mL, 150.00. mu.g/mL, 300.00. mu.g/mL, 600.00. mu.g/mL;
carbamazepine: 15.00. mu.g/mL, 30.00. mu.g/mL, 75.00. mu.g/mL, 150.00. mu.g/mL, 300.00. mu.g/mL, 600.00. mu.g/mL;
phenytoin: 20.00. mu.g/mL, 40.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL, 800.00. mu.g/mL;
the mother liquor of the calibrator is a mixed solution of lamotrigine, phenobarbital, oxcarbazepine, carbamazepine and phenytoin; the concentration of each component in the total mixed solution is the concentration of each component in the six points; the concentration of the calibrator working solution is multiplied by 40/200;
2. internal standard: ketoprofen in methanol at a concentration of 500 μ g/mL;
3. quality control product: comprises a quality control product 1 and a quality control product 2, wherein
The concentration range and the components of the quality control material 1 comprise: lamotrigine: 4.28. mu.g/mL; phenobarbital: 9.84. mu.g/mL; oxcarbazepine: 7.46 mu g/mL; carbamazepine: 4.82 μ g/mL; phenytoin: 5.03 mu g/mL;
the concentration range and the components of the quality control material 2 comprise: lamotrigine: 12.61. mu.g/mL; phenobarbital: 41.27 μ g/mL; oxcarbazepine: 19.80. mu.g/mL; carbamazepine: 12.51. mu.g/mL; phenytoin: 25.54. mu.g/mL;
the quality control product 1 and the quality control product 2 are both a mixture of lamotrigine, phenobarbital, oxcarbazepine, carbamazepine and phenytoin; the concentrations of the respective components are the respective concentrations in the mixture of the quality control material 1 or the quality control material 2;
4. matrix correction fluid: blank serum lyophilized powder (1mL of lyophilized powder frozen from blank serum) is precisely measured and taken 1mL of purified water into a bottle when in use, and the mixture is mixed for 30min until the lyophilized powder is completely dissolved;
5. extracting liquid: t-butyl methyl ether solution (i.e., 100% t-butyl methyl ether);
6. compounding the solution: 50% acetonitrile in water by volume;
7. mobile phase: mobile phase a (0.05% aqueous formic acid), mobile phase B (0.05% acetonitrile formic acid).
The detection was carried out using the above-mentioned kit in the same manner as in example 1.
Example 4:
1. mother liquor of a calibrator: using methanol as a diluent to prepare a calibrator mother liquor comprising 6 concentration points, wherein the 6 concentration points of the calibrator mother liquor are as follows in sequence:
lamotrigine: 10.00. mu.g/mL, 20.00. mu.g/mL, 50.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL;
phenobarbital: 20.00. mu.g/mL, 40.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL, 800.00. mu.g/mL;
oxcarbazepine: 15.00. mu.g/mL, 30.00. mu.g/mL, 75.00. mu.g/mL, 150.00. mu.g/mL, 300.00. mu.g/mL, 600.00. mu.g/mL;
carbamazepine: 15.00. mu.g/mL, 30.00. mu.g/mL, 75.00. mu.g/mL, 150.00. mu.g/mL, 300.00. mu.g/mL, 600.00. mu.g/mL;
phenytoin: 20.00. mu.g/mL, 40.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL, 800.00. mu.g/mL;
the mother liquor of the calibrator is a mixed solution of lamotrigine, phenobarbital, oxcarbazepine, carbamazepine and phenytoin; the concentration of each component in the total mixed solution is the concentration of each component in the six points; the concentration of the calibrator working solution is multiplied by 40/200;
2. internal standard: ketoprofen in methanol at a concentration of 500 μ g/mL;
3. quality control product: comprises a quality control product 1 and a quality control product 2, wherein
The concentration range and the components of the quality control material 1 comprise: lamotrigine: 4.28. mu.g/mL; phenobarbital: 9.84. mu.g/mL; oxcarbazepine: 7.46 mu g/mL; carbamazepine: 4.82 μ g/mL; phenytoin: 5.03 mu g/mL;
the concentration range and the components of the quality control material 2 comprise: lamotrigine: 12.61. mu.g/mL; phenobarbital: 41.27 μ g/mL; oxcarbazepine: 19.80. mu.g/mL; carbamazepine: 12.51. mu.g/mL; phenytoin: 25.54. mu.g/mL;
the quality control product 1 and the quality control product 2 are both a mixture of lamotrigine, phenobarbital, oxcarbazepine, carbamazepine and phenytoin; the concentrations of the respective components are the respective concentrations in the mixture of the quality control material 1 or the quality control material 2;
4. matrix correction fluid: blank serum lyophilized powder (1mL of lyophilized powder frozen from blank serum) is precisely measured and taken 1mL of purified water into a bottle when in use, and the mixture is mixed for 30min until the lyophilized powder is completely dissolved;
5. extracting liquid: t-butyl methyl ether solution (i.e., 100% t-butyl methyl ether);
6. compounding the solution: 100% acetonitrile in water by volume;
7. mobile phase: mobile phase a (0.1% aqueous formic acid), mobile phase B (0.1% acetonitrile formic acid).
The detection was carried out using the above-mentioned kit in the same manner as in example 1.
Example 5:
1. mother liquor of a calibrator: using methanol as a diluent to prepare a calibrator mother liquor comprising 6 concentration points, wherein the 6 concentration points of the calibrator mother liquor are as follows in sequence:
lamotrigine: 10.00. mu.g/mL, 20.00. mu.g/mL, 50.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL;
phenobarbital: 20.00. mu.g/mL, 40.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL, 800.00. mu.g/mL;
oxcarbazepine: 15.00. mu.g/mL, 30.00. mu.g/mL, 75.00. mu.g/mL, 150.00. mu.g/mL, 300.00. mu.g/mL, 600.00. mu.g/mL;
carbamazepine: 15.00. mu.g/mL, 30.00. mu.g/mL, 75.00. mu.g/mL, 150.00. mu.g/mL, 300.00. mu.g/mL, 600.00. mu.g/mL;
phenytoin: 20.00. mu.g/mL, 40.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL, 800.00. mu.g/mL;
the mother liquor of the calibrator is a mixed solution of lamotrigine, phenobarbital, oxcarbazepine, carbamazepine and phenytoin; the concentration of each component in the total mixed solution is the concentration of each component in the six points; the concentration of the calibrator working solution is multiplied by 40/200;
2. internal standard: ketoprofen in methanol at a concentration of 500 μ g/mL;
3. quality control product: comprises a quality control product 1 and a quality control product 2, wherein
The concentration range and the components of the quality control material 1 comprise: lamotrigine: 4.28. mu.g/mL; phenobarbital: 9.84. mu.g/mL; oxcarbazepine: 7.46 mu g/mL; carbamazepine: 4.82 μ g/mL; phenytoin: 5.03 mu g/mL;
the concentration range and the components of the quality control material 2 comprise: lamotrigine: 12.61. mu.g/mL; phenobarbital: 41.27 μ g/mL; oxcarbazepine: 19.80. mu.g/mL; carbamazepine: 12.51. mu.g/mL; phenytoin: 25.54. mu.g/mL;
the quality control product 1 and the quality control product 2 are both a mixture of lamotrigine, phenobarbital, oxcarbazepine, carbamazepine and phenytoin; the concentrations of the respective components are the respective concentrations in the mixture of the quality control material 1 or the quality control material 2;
4. matrix correction fluid: blank serum lyophilized powder (1mL of lyophilized powder frozen from blank serum) is precisely measured and taken 1mL of purified water into a bottle when in use, and the mixture is mixed for 30min until the lyophilized powder is completely dissolved;
5. extracting liquid: t-butyl methyl ether solution (i.e., 100% t-butyl methyl ether);
6. compounding the solution: 50% methanol aqueous solution by volume;
7. mobile phase: mobile phase a (0.2% aqueous formic acid), mobile phase B (0.2% acetonitrile formic acid).
The detection was carried out using the above-mentioned kit in the same manner as in example 1.
The treated calibrator, quality control and sample obtained in examples 1 to 5 were each injected in an amount of 8. mu.L into a liquid chromatograph, a chromatogram was recorded, and the detection results were calculated according to the following procedure:
(1) drawing and fitting a calibration curve: general liquid chromatography analysis software can automatically draw a calibration curve according to a calibration result, a concentration ratio of 6 calibrator working solutions to an internal standard is used as an abscissa (x), a ratio of actual measurement peak areas of the 6 calibrator working solutions to respective internal standard peak areas is used as an ordinate (y), linear regression is performed by using a least square method, a standard curve is automatically drawn, and a regression equation can be obtained: y is ax + b, wherein y is a vertical coordinate, x is a horizontal coordinate, a is a slope, b is an intercept, and r (correlation coefficient) value is calculated, and the r value is more than or equal to 0.9900;
(2) and (3) calculating the recovery rate: the measured concentration of each analyte in the quality control product can be obtained from a calibration curve according to the peak area ratio of each analyte in the quality control product to the corresponding internal standard, and the calculation formula of the recovery rate is as follows: the recovery rate (%) is that the determination concentration/indication concentration is multiplied by 100, and the recovery rate (%) is in the range of 85-115%;
(3) calculation of sample results: the measured concentration of each analyte in the sample can be obtained from the calibration curve based on the ratio of the peak areas of each analyte in the sample and the corresponding internal standard.
FIG. 1 is a chemical structural formula of 5 antiepileptic drugs of phenobarbital, phenytoin, carbamazepine, oxcarbazepine and lamotrigine of the present invention; FIG. 2 is a chromatogram of 5 antiepileptic drugs of phenobarbital, phenytoin, carbamazepine, oxcarbazepine and lamotrigine of the present invention and an internal standard solution; FIG. 3 is a chromatogram of a blank serum sample of the present invention; FIG. 4 is a chromatogram of an actual measurement serum sample of 5 antiepileptic drugs of phenobarbital, phenytoin, carbamazepine, oxcarbazepine and lamotrigine.
The detection results of the embodiments 1-5 are basically consistent, and the analysis performance of the kit of the embodiment 1 is described by combining the related drawings as follows:
1. standard curve versus linearity: figure 5 lamotrigine standard curve linear range: (1.00-40.00) mu g/mL. FIG. 6 Linear Range of Phenobarbital Standard Curve: (2.00-80.00) mu g/mL. Figure 7 linear range of oxcarbazepine standard curve: (1.50-60.00) mu g/mL. Fig. 8 carbamazepine standard curve linear range: (1.50-60.00) mu g/mL. Figure 9 linear range of phenytoin standard curve: (2.00-80.00) mu g/mL. As can be seen from the figure, the correlation coefficients r of lamotrigine, phenobarbital, oxcarbazepine, carbamazepine and phenytoin are all greater than 0.99, and the linearity is good.
2. Accuracy and precision
The results of the measurement of quality control 1 and quality control 2 were as follows:
TABLE 3 measurement results of quality control 1 and quality control 2
According to the detection results, the kit and the liquid chromatography detection method have good accuracy, precision and linearity, can completely meet the clinical detection requirements, and can be used for processing serum samples to obtain very clean processing liquid without complicated purification and purification steps, so that high sensitivity can be obtained by concentration, the required analysis time is short, 5 antiepileptic drugs and internal standards can be separated within 5-9min, and the detection speed is improved; the gradient elution mode is adopted, so that the efficiency of the chromatographic column can be better protected, and the chromatographic column can be matched with a liquid chromatograph for use and is suitable for the inspection of clinical samples; meanwhile, the product can simultaneously detect the concentration of 5 antiepileptic drugs including phenobarbital, phenytoin, carbamazepine, oxcarbazepine and lamotrigine in serum, achieves the function of single-sample multi-index synchronous detection, and has the characteristics of high accuracy, short detection time, less reagent consumption, convenient operation, low cost and the like.
Materials, reagents and experimental equipment related to the embodiment of the invention are all commercial products meeting the inspection and quarantine field if no special description is provided.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, modifications and decorations can be made without departing from the core technology of the present invention, and these modifications and decorations shall also fall within the protection scope of the present invention. Any changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.
Claims (10)
1. A kit for accurately determining the blood concentration of a plurality of antiepileptic drugs in human serum is characterized in that: the kit mainly comprises:
(1) mother liquor of a calibrator: using methanol or acetonitrile as a diluent to prepare a mother liquor of a calibrator comprising 6 concentration points, wherein the 6 concentration points of the mother liquor of the calibrator are as follows in sequence:
lamotrigine: 10.00. mu.g/mL, 20.00. mu.g/mL, 50.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL;
phenobarbital: 20.00. mu.g/mL, 40.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL, 800.00. mu.g/mL;
oxcarbazepine: 15.00. mu.g/mL, 30.00. mu.g/mL, 75.00. mu.g/mL, 150.00. mu.g/mL, 300.00. mu.g/mL, 600.00. mu.g/mL;
carbamazepine: 15.00. mu.g/mL, 30.00. mu.g/mL, 75.00. mu.g/mL, 150.00. mu.g/mL, 300.00. mu.g/mL, 600.00. mu.g/mL;
phenytoin: 20.00. mu.g/mL, 40.00. mu.g/mL, 100.00. mu.g/mL, 200.00. mu.g/mL, 400.00. mu.g/mL, 800.00. mu.g/mL;
the mother liquor of the calibrator is a mixed solution of lamotrigine, phenobarbital, oxcarbazepine, carbamazepine and phenytoin; the concentration of each component in the total mixed solution is the concentration of each component in the six points; the concentration of the calibrator working solution is multiplied by 40/200;
(2) internal standard: the internal standard solution is ketoprofen methanol solution, and the concentration range is 10-1000 mug/mL;
(3) quality control product: comprises a quality control product 1 and a quality control product 2, wherein
The concentration range and the components of the quality control material 1 comprise: lamotrigine: 3.95-5.93 mu g/mL; phenobarbital: 8.76-11.80 mug/mL; oxcarbazepine: 5.59-9.32 mu g/mL; carbamazepine: 3.86-5.78 mu g/mL; phenytoin: 4.22-5.72 mu g/mL;
the concentration range and the components of the quality control material 2 comprise: lamotrigine: 11.40-17.00 mu g/mL; phenobarbital: 38.00-57.00 mu g/mL; oxcarbazepine: 15.80-23.70 mu g/mL; carbamazepine: 11.70-17.60 mu g/mL; phenytoin: 23.10-31.30 mu g/mL;
the quality control product 1 and the quality control product 2 are both a mixture of lamotrigine, phenobarbital, oxcarbazepine, carbamazepine and phenytoin; the concentrations of the respective components are the respective concentrations in the mixture of the quality control material 1 or the quality control material 2;
(4) matrix correction fluid: the matrix correction fluid is blank serum freeze-dried powder;
(5) extracting liquid: the extract is tert-butyl methyl ether solution;
(6) compounding the solution: the complex solution is a mixture of methanol or acetonitrile and water, and the volume percentage content of the methanol or the acetonitrile in the mixture is 40-100%;
(7) mobile phase: the mobile phase A is aqueous solution, the mobile phase B is acetonitrile solution, and both the mobile phase A and the mobile phase B contain 0.05-0.2% of formic acid by volume percentage.
2. The kit for accurately determining the blood concentration of multiple antiepileptic drugs in human serum according to claim 1, wherein: the diluent in the step (1) is a methanol solution.
3. The kit for accurately determining the blood concentration of multiple antiepileptic drugs in human serum according to claim 1, wherein: in the step (3), the quality control product 1 and the quality control product 2 are both composed of a mixture of lamotrigine, phenobarbital, oxcarbazepine, carbamazepine and phenytoin, and the quality control product is in the form of freeze-dried powder, a serum matrix or a methanol solution matrix.
4. The kit for accurately determining the blood concentration of multiple antiepileptic drugs in human serum according to claim 1, wherein: and (4) the volume percentage content of the composite solution in the step (6) is 50% of methanol aqueous solution.
5. The kit for accurately determining the blood concentration of multiple antiepileptic drugs in human serum according to claim 1, wherein: in the step (7), the content of formic acid in the mobile phase A is 0.1% by volume, and the content of formic acid in the mobile phase B is 0.05% by volume.
6. The kit for accurately determining the blood concentration of multiple antiepileptic drugs in human serum according to claim 1, wherein: the kit also comprises an instruction manual.
7. A detection method of a kit for accurately determining the concentration of an anti-epileptic drug in blood in human serum is characterized in that: the method comprises the following steps: adding methanol solution of internal standard ketoprofen into human serum, mixing, adding the extract, mixing and centrifuging, removing supernatant, drying with nitrogen, adding redissolution for redissolving, mixing and centrifuging, taking supernatant, detecting by using a liquid chromatograph-PDA detector, and quantifying by using an internal standard curve method.
8. The method for detecting a kit for accurately measuring the plasma concentration of an anti-epileptic drug in human serum according to claim 7, characterized in that: the method comprises the following specific steps:
(1) test sample processing
(1.1) calibrator: accurately sucking 40 mu L of calibrator mother liquor into a centrifuge tube at room temperature, drying the calibrator mother liquor by nitrogen at 45 ℃, adding 200 mu L of matrix correction fluid, mixing in a vortex manner, adding 100 mu L of internal standard with known concentration, mixing in a vortex manner for 1min, adding 800 mu L of extract liquor, mixing in a vortex manner for 5min to completely mix the internal standard with the matrix correction fluid, centrifuging at 15000r/min for 2min, and drying the supernatant by nitrogen at 45 ℃; adding 150 μ L of redissolution for redissolution, mixing with vortex, centrifuging at 15000r/min for 5min, collecting supernatant 120 μ L, and adding into sample bottle for chromatographic analysis;
(1.2) quality control/serum sample: accurately sucking 200 μ L of quality control product/serum sample into a centrifuge tube at room temperature, adding 100 μ L of internal standard with known concentration, mixing for 1min by vortex, adding 800 μ L of extract, mixing for 5min by vortex, centrifuging at 15000r/min for 2min, collecting supernatant, and blow-drying with 45 deg.C nitrogen gas; adding 150 μ L of redissolution for redissolution, mixing with vortex, centrifuging at 15000r/min for 5min, collecting supernatant 120 μ L, and adding into sample bottle for chromatographic analysis;
(2) sample detection assay
(2.1) chromatographic conditions
Column chromatography: c18 column or equivalent;
the mobile phase: mobile phase a and mobile phase B;
detection wavelength: lamotrigine (265 nm); phenobarbital and phenytoin (230 nm); oxcarbazepine (240 nm); carbamazepine (284 nm);
flow rate: 0.5 mL/min;
gradient elution: the following conditions were followed;
in the gradient elution process in the above condition, the volume ratio of the mobile phase A and the mobile phase B in the mobile phase is continuously adjusted and changed along with the time, and the proportion change of the mobile phase B is reflected;
column temperature: 40 ℃;
sample injector temperature: 8 ℃;
sample size: 8 mu L of the solution;
(2.2) injecting 8 mu L of each of the processed calibrator, the quality control material and the sample into a liquid chromatograph, and recording a chromatogram;
(2.3) calculation of detection result
(2.3.1) plotting and fitting of calibration curves: general liquid chromatography analysis software can automatically draw a calibration curve according to a calibration result, linear regression is carried out by using a least square method by taking the concentration ratio of 6 calibrator working solutions to an internal standard as an abscissa x and the ratio of actual measurement peak areas of the 6 calibrator working solutions to respective internal standard peak areas as an ordinate y, and a regression equation can be obtained by automatically drawing a standard curve: y is ax + b, wherein y is a vertical coordinate, x is a horizontal coordinate, a is a slope, b is an intercept, and the r value is calculated and is more than or equal to 0.9900;
(2.3.2) calculation of recovery: the measured concentration of each analyte in the quality control product can be obtained from a calibration curve according to the peak area ratio of each analyte in the quality control product to the corresponding internal standard, and the calculation formula of the recovery rate is as follows: the recovery rate is measured concentration/marked concentration multiplied by 100, and the recovery rate percent is in the range of 85 to 115 percent;
(2.3.3) calculation of sample results: the measured concentration of each analyte in the sample can be obtained from the calibration curve based on the ratio of the peak areas of each analyte in the sample and the corresponding internal standard.
9. The method for detecting a kit for accurately measuring the plasma concentration of an anti-epileptic drug in human serum according to claim 8, wherein the kit comprises: the vortex mixing speed in step (1) was 2000rpm, and the volume of the extract added in step (1) was 800. mu.L.
10. The method for detecting a kit for accurately measuring the plasma concentration of an anti-epileptic drug in human serum according to claim 8, wherein the kit comprises: adding the extract and the redissolution in the step (1), mixing and centrifuging at low temperature and high speed, namely centrifuging at 15000rpm at 5-10 ℃, preferably 8 ℃.
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| CN115774067A (en) * | 2022-11-22 | 2023-03-10 | 浙江迪赛思诊断技术有限公司 | Detection kit for detecting antiepileptic drug in serum by high performance liquid chromatography tandem mass spectrometry and detection method thereof |
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