CN116735761A - UPLC-MS/MS detection method of brivaracetam in human plasma - Google Patents
UPLC-MS/MS detection method of brivaracetam in human plasma Download PDFInfo
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- MSYKRHVOOPPJKU-BDAKNGLRSA-N brivaracetam Chemical compound CCC[C@H]1CN([C@@H](CC)C(N)=O)C(=O)C1 MSYKRHVOOPPJKU-BDAKNGLRSA-N 0.000 title claims abstract description 72
- 229960002161 brivaracetam Drugs 0.000 title claims abstract description 71
- 238000001514 detection method Methods 0.000 title claims abstract description 24
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 title claims abstract description 23
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 78
- 238000000034 method Methods 0.000 claims abstract description 29
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000000243 solution Substances 0.000 claims abstract description 24
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 14
- 235000019253 formic acid Nutrition 0.000 claims abstract description 14
- 238000004458 analytical method Methods 0.000 claims abstract description 8
- 230000006920 protein precipitation Effects 0.000 claims abstract description 7
- 239000007864 aqueous solution Substances 0.000 claims abstract description 3
- 239000000523 sample Substances 0.000 claims description 53
- 239000012224 working solution Substances 0.000 claims description 41
- 239000011550 stock solution Substances 0.000 claims description 28
- 238000003908 quality control method Methods 0.000 claims description 23
- 239000013062 quality control Sample Substances 0.000 claims description 12
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 10
- 238000007865 diluting Methods 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
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- 238000010828 elution Methods 0.000 claims description 7
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- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000012417 linear regression Methods 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 239000007921 spray Substances 0.000 claims description 3
- 238000004704 ultra performance liquid chromatography Methods 0.000 claims description 3
- 229940090047 auto-injector Drugs 0.000 claims description 2
- 238000012937 correction Methods 0.000 claims description 2
- 238000010790 dilution Methods 0.000 claims description 2
- 239000012895 dilution Substances 0.000 claims description 2
- 239000000945 filler Substances 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 239000013558 reference substance Substances 0.000 claims description 2
- 238000011084 recovery Methods 0.000 abstract description 9
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000003672 processing method Methods 0.000 abstract description 2
- 210000002381 plasma Anatomy 0.000 description 27
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
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- 239000002904 solvent Substances 0.000 description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
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- 239000012086 standard solution Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 206010010904 Convulsion Diseases 0.000 description 1
- 108010052164 Sodium Channels Proteins 0.000 description 1
- 102000018674 Sodium Channels Human genes 0.000 description 1
- 101710084141 Synaptic vesicle glycoprotein 2A Proteins 0.000 description 1
- 230000003556 anti-epileptic effect Effects 0.000 description 1
- 239000001961 anticonvulsive agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 230000001419 dependent effect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000001037 epileptic effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
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- 230000002401 inhibitory effect Effects 0.000 description 1
- HPHUVLMMVZITSG-ZCFIWIBFSA-N levetiracetam Chemical class CC[C@H](C(N)=O)N1CCCC1=O HPHUVLMMVZITSG-ZCFIWIBFSA-N 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
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- 238000002203 pretreatment Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
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- 230000003977 synaptic function Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The application relates to the technical field of biological analysis, in particular to a UPLC-MS/MS detection method of brivaracetam in human plasma. The application discloses a UPLC-MS/MS detection method of brivaracetam in human plasma, which is characterized in that a to-be-detected human plasma sample is processed by a protein precipitation method and then is analyzed and measured by adopting a mobile phase containing formic acid. Preferably, the mobile phase comprises mobile phase a, which is an aqueous solution containing 0.1% formic acid, and mobile phase B, which is a methanol solution containing 0.1% formic acid. The sample processing method adopts a protein precipitation method, the recovery rate is close to 100%, the requirement of the recovery rate can be met, and the detection efficiency can be improved; high sensitivity, strong specificity and good stability.
Description
Technical Field
The application relates to the technical field of biological analysis, in particular to a UPLC-MS/MS detection method of brivaracetam in human plasma.
Background
Brivaracetam, also known as levetiracetam derivatives. Brivaracetam can be combined with central synaptic vesicle protein 2A (SV 2A) in a strong selectivity and reversibility manner, and plays an anti-epileptic left b role by influencing synaptic function; and inhibiting voltage-dependent sodium ion channels, reducing the duration and frequency of epileptic discharges, thereby reducing seizures.
At present, no quantitative detection technology of brivaracetam in a subject and application of a clinical first-stage bioequivalence test have been reported in China, and methods reported in foreign documents are liquid-liquid extraction or solid-phase extraction technologies, so that time and labor are wasted, operation is troublesome, and practicality is poor. In order to accelerate the research and development of imitation pharmacy in China and solve the situation of medicine shortage in China at present, a high-sensitivity and high-flux brivaracetam quantitative analysis method needs to be established.
Disclosure of Invention
The application aims to provide a UPLC-MS/MS detection method for brivaracetam in human plasma, which extracts brivaracetam from human plasma by a sample processing mode of protein precipitation, and performs liquid-solid combined analysis on the extracted sample, thereby solving the problems of time and labor waste, troublesome operation and poor practicability caused by adopting a liquid-liquid extraction or solid-phase extraction technology in the prior art.
In order to solve the technical problems, the application adopts the following scheme:
a UPLC-MS/MS detection method for brivaracetam in human plasma is characterized by that the human plasma sample to be detected is treated by protein precipitation method, and then the mobile phase containing formic acid is used for UPLC-MS/MS analysis and determination.
Further, formic acid is contained in the mobile phase system, and under the premise of ensuring that impurity peaks do not influence main peaks, the volume of the formic acid is preferably 0.1% of the total volume of the mobile phase, so that the ionization efficiency of the mass spectrum is improved.
Preferably, the mobile phase comprises mobile phase a, which is an aqueous solution containing 0.1% formic acid, and mobile phase B, which is a methanol solution containing 0.1% formic acid.
Further, the mobile phase organic phase is a methanol system, and if acetonitrile is adopted in the mobile phase, the mobile phase is kept on a chromatographic column to be weak, and the response is also reduced, so that methanol is eliminated from being used in acetonitrile.
Preferably, the method comprises the following steps:
s1, preparing a brivaracetam standard curve working solution, a quality control working solution and an internal standard working solution, and further preparing a standard curve sample and a quality control sample;
s2, taking an equal volume standard curve sample, a quality control sample and a human plasma sample to be tested, and adding an internal standard working solution to mix uniformly; taking blank plasma, adding 50% methanol solution, adding methanol to precipitate protein, and centrifuging to obtain supernatant;
s3, adding methanol-water solution with the volume ratio of 36:64 into the supernatant in the step S2 for dilution, carrying out vortex shaking, measuring by using UPLC-MS/MS, taking the peak area ratio of brivaracetam and internal standard brivaracetam as an ordinate, taking the concentration of an object to be measured in a standard curve sample as an abscissa, and carrying out linear regression;
and S4, calculating the human plasma sample to be detected treated in the step S2 according to the standard curve equation obtained in the step S3 to obtain the concentration of the brivaracetam in the human plasma sample to be detected.
Preferably, the step of preparing the standard working solution is: weighing a brivaracetam reference substance, adding methanol for dissolution, preparing a brivaracetam stock solution with the concentration of 1.000mg/mL, and preparing at least two stock solutions, one serving as a standard curve stock solution and one serving as a quality control stock solution; diluting the brivaracetam standard curve stock solution with a methanol-water solution with the volume ratio of 50:50 to obtain a standard curve working solution with the gradient of 400.000~36000.000 ng/mL; diluting the brivaracetam quality control stock solution with a methanol-water solution with the volume ratio of 50:50 to obtain a quality control working solution with the gradient of 400.000-24000.000 ng/mL; adding methanol into an internal standard to prepare internal standard stock solution with the concentration of 1.000mg/mL, diluting the internal standard stock solution with methanol-water solution with the volume ratio of 50:50 to obtain 800.000ng/mL internal standard working solution, and storing all stock solution and working solution at-20 ℃ for later use after subpackaging; and (3) taking blank plasma of healthy people, respectively adding the brivaracetam working solution, and uniformly mixing by vortex to prepare a standard curve sample with the concentration range of 20.000-1800.000 ng/mL and a quality control sample with the concentration range of 20.000~1200.000 ng/mL.
Preferably, the brivaracetam working solution concentration is 400.000 ng/mL, 1000.000 ng/mL, 2000.000ng/mL, 4000.000ng/mL, 8000.000 ng/mL, 16000.000ng/mL, 30000.000 ng/mL and 36000.000 ng/mL; the concentration of the brivaracetam quality control working solution is as follows: 400.000 ng/mL, 1200.000ng/mL, 3600.000 ng/mL, 12000.000ng/mL, 24000.000 ng/mL; the calibration standard sample and the quality control sample are freshly prepared; the concentration of the correction standard sample is as follows: 20.000 ng/mL, 50.000 ng/mL, 100.000 ng/mL, 200.000ng/mL, 400.000 ng/mL, 800.000ng/mL, 1500.000 ng/mL, 1800.000 ng/mL; the concentration of the quality control sample is as follows: 20.000 ng/mL, 60.000. 60.000ng/mL, 180.000 ng/mL, 600.000ng/mL, 1200.000ng/mL.
Preferably, the internal standard is brivaracetam-d 7.
Preferably, in the step S2, the time of vortex mixing is 5min; the centrifugation conditions are as follows: the temperature during centrifugation is 4 ℃, the centrifugal speed is 2120 g, and the centrifugal time is 10 min.
Preferably, in step S3, the process of the present application,
the chromatographic conditions are as follows: chromatographic column: ACQUITY UPLC BEH C18, 50X 2.1 mm, filler particle size 1.7 μm; the flow rate is 0.3mL/min; column temperature: 40 ℃; sample injection volume: 1. mu L; autoinjector temperature: 8 ℃.
Preferably, in step S3, the mass spectrometry conditions are: an ion source ESI source adopts a positive ion mode and a multi-reaction monitoring MRM mode; electrospray voltage 5500V; the temperature of the ion source is 500 ℃; the air curtain gas is 35 psi; the collision gas is 9 psi; the spray gas was 45 psi; the auxiliary heating gas was 45 psi.
Preferably, the elution is set as:
the elution set up was: the volume flow is 0.3ml/min; the gradient elution conditions were:
in 0.00-1.50 min, the mobile phase A is 60% -5%, and the mobile phase B is 40% -95%;
at 1.51-2.50 min, mobile phase A is 5% and mobile phase B is 95%;
in 2.51-2.60 min, the mobile phase A is 5-60%, and the mobile phase B is 95-40%;
and the mobile phase A is 60% and the mobile phase B is 40% within 2.61-3.50 min.
The beneficial effects of the application are as follows: 1. sample treatment optimization: the sample processing method adopts a protein precipitation method, the recovery rate is close to 100%, the requirement of the recovery rate can be met, and the detection efficiency can be improved. 2. The sensitivity is high: the lower limit of the brivaracetam quantification is 20.000 ng/mL. 3. The specificity is strong: the brivaracetam-d 7 is adopted as an isotope internal standard, which is completely consistent with the brivaracetam chromatograph, so that the influence of matrix effect on the detection result is reduced. 4. The stability is good: the working fluid and plasma samples were stable when placed at room temperature and-20 ℃.
Drawings
FIG. 1 is brivaracetam [ M+H ]] + Is a product ion scan of (2);
FIG. 2 shows brivaracetam-d7 [ M+H] + Is a product ion scan of (2);
FIG. 3 is a typical chromatogram of brivaracetam in a blank plasma sample;
FIG. 4 is a typical chromatogram of brivaracetam in a lower limit sample of quantification;
FIG. 5 is a typical chromatogram of brivaracetam-d 7 in a blank plasma sample;
FIG. 6 is a typical chromatogram of brivaracetam-d 7 in a lower limit sample of quantification.
Detailed Description
In order to more clearly demonstrate the objects, technical solutions and advantages of the present application, the present application will be further described with reference to the following examples.
Example 1
Embodiment 1 of the present application is a method for detecting brivaracetam in human plasma by UPLC-MS/MS, as shown in fig. 1 and 2, comprising the steps of:
1. preparation of solutions and samples
1.1 Standard series samples: precisely weighing a brivaracetam standard substance, converting, adding a certain volume of methanol for dissolution and fixing the volume, preparing a brivaracetam stock solution with the concentration of 1.000-mg/mL, preparing at least two standard substance stock solutions, wherein one standard substance stock solution is used as a standard curve stock solution, and the other standard curve stock solution is used as a quality control stock solution, diluting the brivaracetam standard curve stock solution with a blank solvent (methanol-water solution with the volume ratio of 50:50) to obtain a standard curve working solution with the gradient of 400.000-36000.000 ng/mL, and drawing a standard curve.
1.2 quality control samples: and diluting the brivaracetam quality control stock solution prepared in the standard series samples by using a blank solvent (methanol-water solution with the volume ratio of 50:50) to obtain a quality control working solution with the gradient of 400.000~24000.000 ng/mL.
1.3 internal standard solution: precisely weighing a proper amount of brivaracetam-d 7 standard substance, adding a certain volume of methanol to prepare an internal standard stock solution with the concentration of 1.000mg/mL, diluting the internal standard stock solution with a blank solvent (methanol-water solution with the volume ratio of 50:50) to obtain an internal standard working solution with the concentration of 800.000 ng/mL.
1.4 preparation process of brivaracetam standard curve working solution and quality control working solution: see tables 1 and 2, respectively.
Table 1 preparation of brivaracetam standard curve working solution
Table 2 preparation of brivaracetam quality control working solution
1.5 calibration curve samples and quality control samples: to 190. Mu.L of blank plasma, 10. Mu.L of the corresponding working solution (standard curve working solution or quality control working solution) was added and mixed uniformly. The preparation volume can be adjusted proportionally according to the actual situation. The concentrations of the standard samples containing the brivaracetam are 20.000 ng/mL, 50.000 ng/mL, 100.000 ng/mL, 200.000ng/mL, 400.000 ng/mL, 800.000ng/mL, 1500.000 ng/mL and 1800.000 ng/mL; the concentration of the quality control sample is 20.000 ng/mL, 60.000ng/mL, 180.000 ng/mL, 600.000ng/mL and 1200.000ng/mL.
2. Sample pretreatment
Mixing samples thawed at room temperature or newly prepared samples by vortex, adding 40.0 mu L of standard curve samples, quality control samples and samples to be tested into holes of a 96-well plate, and then adding 20.0 mu L of internal standard working solution for mixing uniformly; taking 40.0 mu L of blank plasma sample, adding 20.0 mu L of 50% methanol sample, adding 380 mu L of methanol precipitated protein into each sample hole, and centrifuging for 10min after vortex, wherein the centrifugation conditions are as follows: the temperature during centrifugation is 4 ℃, the centrifugal speed is 2120 g, and the centrifugal time is 10 min. After centrifugation, 40. Mu.L of the supernatant was removed and 500. Mu.L of methanol was used: water (36:64, v/v) was diluted, vortexed and homogenized for UPLC-MS/MS determination.
3. Detection instrument and analysis conditions
3.1 drugs and reagents: brivaracetam (TLC), brivaracetam-d 7 (TLC), methanol (LCMS; FISHER), formic acid (LCMS; FISHER).
3.2 major equipment instruments
LC-30AD ultra high performance liquid chromatograph; TQ5500 triple quadrupole mass spectrometer.
3.3 analysis conditions
3.3.1 chromatography conditions:
the column was ACQUITY UPLC BEH C (50X 2.1 mm,1.7 μm);
mobile phase a: (water/formic acid=100/0.1), mobile phase B: (methanol/formic acid=100/0.1);
column oven temperature: 40 ℃;
flow rate: 0.3mL/min;
sample volume 1 uL;
the automatic injector cleaning solution is as follows: methanol/water=80/20;
soaking time is 2 s when the automatic sampling needle is cleaned;
automatic injector cleaning mode: cleaning before and after sample injection;
the temperature of the sample tray is 8 ℃;
the elution procedure settings are shown in table 3.
TABLE 3 elution procedure parameter settings
3.3.2 Mass Spectrometry conditions: the ion source is an ESI source, and adopts a positive ion mode and a multi-reaction monitoring (MRM) mode; electrospray voltage 5500V; the temperature of the ion source is 500 ℃; the air curtain gas is 35 psi; the collision gas is 9 psi; the spray gas was 45 psi; the auxiliary heating gas is 45 psi; the mass spectrometry MRM parameter settings are shown in table 4.
Table 4 mass spectrometry MRM parameter settings
Example 2
Example 2 of the present application is a methodological verification of the detection method of the present application.
1. Selectivity of
Samples were treated as in example 1 with 40 μl (n=3) of blank plasma from 6 different sources, and mass spectrometry was performed on the samples (without internal standard) to obtain a chromatogram of the blank plasma sample. A standard curve minimum concentration plasma sample of 40 μl was prepared from 6 blank plasmas (n=3) from different sources, and the samples were treated as in example 1, and mass spectrometry was performed to investigate the interference of endogenous substances in the conventional matrix.
The results show that endogenous substances do not interfere with the determination of brivaracetam, brivaracetam-d 7. Typical blank plasma sample chromatograms and typical LLOQ chromatograms are shown in fig. 3-6.
2. Standard curve
The concentration of the object to be measured in the standard curve sample is taken as an abscissa (x), the area ratio of the object to be measured to the internal standard is taken as an ordinate (y), and linear regression calculation is performed (weight factor w=1/x 2 ) A typical regression equation for brivaracetam is y=0.002222x+0.000651 (R 2 =0.9999), brivaracetam is well-linearly related at 20.000 ng to 1800.000 ng/mL.
3. Precision and accuracy
The method validated three analytical batches, each of which was tested for 6 samples of lower limit of quantitation (LLOQ: 20.000 ng/mL), low (LQC: 60.000 ng/mL), medium (MQC: 600.000 ng/mL), and high (HQC: 1200.000 ng/mL) level QC. The precision and accuracy within and between batches were calculated.
Table 5 results of precision and accuracy of three analytical batches
The results show that: the method has acceptable precision and accuracy for measuring the brivaracetam, and the lowest quantitative lower limit of the brivaracetam is 20.000 ng/mL.
4. Extraction recovery and matrix effect
Extraction recovery rate: analyzing 6 low, medium and high quality control samples, simultaneously taking 120 mu L of blank plasma, and processing according to a plasma sample pretreatment method; after the treatment, 380 mu L of the extracted blank matrix solution is respectively taken, and 20 mu L of low-concentration (1200.000 ng/mL), medium-concentration (12000.000 ng/mL) and high-concentration (24000.000 ng/mL) quality control working solutions are added and uniformly mixed by vortex shaking. 40. Mu.L of the mixture (n=6) was added to 20. Mu.L of the internal standard working solution (800.000 ng/mL), and after mixing, 380. Mu.L of the extracted blank matrix supernatant was added and mixed. And then 40 mu L of the mixture is diluted by 500 mu L of 36% methanol solution to prepare low, medium and high quality control sample concentration, and sample injection analysis is carried out. The ratio of the brivaracetam peak area in the sample is the extraction recovery rate of brivaracetam. The results show that the extraction recovery rates of three concentration levels of brivaracetam are 99.25%, 102.5% and 102.79% respectively; the recovery of the internal standard brivarenical-d 7 was 103.50%.
Normal plasma matrix effects: extraction of 6 source substrates: taking 120 mu L of blank plasma, adding 60 mu L of 50% methanol, shaking for 1 minute, adding 1140 mu L of methanol, shaking for 5 minutes, centrifuging at a low temperature of 4 ℃ for 10 minutes, and taking supernatant for later use, wherein the centrifugal force is 2120 g.
Pretreatment of matrix effect samples (matrix samples): and (3) respectively taking 190 mu L of the extracted blank matrix solution, adding 10 mu L of low-concentration (1200.000 ng/mL), medium-concentration (12000.000 ng/mL) and high-concentration (24000.000 ng/mL) quality control working solution, and uniformly mixing by vortex shaking. 40. Mu.L of the mixture (n=3) was added to 20. Mu.L of the internal standard working solution (800.000 ng/mL), and after mixing, 380. Mu.L of the extracted blank matrix supernatant was added and mixed. Centrifuging at 4deg.C for 10min at low temperature with a centrifugal force of 2120 g.
Pretreatment of reference samples (standard solutions): taking 10 mu L of low-concentration (1200.000 ng/mL), medium-concentration (12000.000 ng/mL) and high-concentration (24000.000 ng/mL) quality control working solution, adding 100 mu L of internal standard working solution, adding 1900 mu L of methanol, adding 190 mu L of ultrapure water, and uniformly mixing.
500 μl of 36% methanol solution was added to a new 96-well plate, and 40 μl of the substrate effect sample supernatant and the reference sample solution were added to the corresponding wells, respectively, and vortexed and mixed.
The mean value of the internal standard normalized matrix effector of brivaracetam is HQC 1.032, MQC 1.025 and LQC 1.045; the internal standard normalized matrix effector variation coefficients are HQC0.70%, MQC 0.84% and LQC 1.42% respectively. It was shown that under the present test conditions the effect of the matrix effect on the brivaracetam assay can be neglected.
5. Residue of
Brivaracetam residue: not more than 3.39% of the average response of the LLOQ sample analyte; brivaracetam-d 7 residue: not more than 0.10% of the average response of the LLOQ sample internal standard.
6. Mutual interference between object to be measured and internal standard
When BRI was added to the sample alone at ULOQ concentration (no internal standard was added), the sample internal standard response maximum was 0.02% of the average response of the internal standard of the batch standard curve LLOQ sample, proving that the test substance had no interference with the internal standard substance assay. When BRI-d7 is added to the sample (no analyte is added) at the concentration specified by the method, the maximum value of the response of the analyte is 4.84% of the average response value of the analyte in the LLOQ sample of the batch standard curve, and the internal standard substance is proved to have no interference on the measurement of the analyte.
7. Stability verification results
As shown in table 6.
TABLE 6 stability validation results
The experimental results show that: the method provided by the application is verified by methodology, and the established method has the advantages of high sensitivity, good selectivity, accuracy, precision, good stability and good linearity.
The results of the examples show that the method of the application can extract the brivaracetam by a protein precipitation method, can realize the rapid and high-sensitivity detection of the brivaracetam in the blood plasma, and can apply the research of the pharmacokinetics and bioequivalence of the brivaracetam Yu Buli.
The foregoing description of the preferred embodiment of the application is not intended to limit the application in any way, but rather to cover all modifications, equivalents, improvements and alternatives falling within the spirit and principles of the application.
Claims (10)
1. A UPLC-MS/MS detection method of brivaracetam in human plasma is characterized in that after a human plasma sample to be detected is processed by a protein precipitation method, a mobile phase containing formic acid is adopted for UPLC-MS/MS analysis and determination.
2. The method for UPLC-MS/MS detection of brivaracetam in human plasma according to claim 1, wherein mobile phase comprises mobile phase a and mobile phase B, mobile phase a being an aqueous solution containing 0.1% formic acid, mobile phase B being a methanol solution containing 0.1% formic acid.
3. A method for the UPLC-MS/MS detection of brivaracetam in human plasma according to claim 1, comprising the steps of:
s1, preparing a brivaracetam standard curve working solution, a quality control working solution and an internal standard working solution, and further preparing a standard curve sample and a quality control sample;
s2, taking an equal volume standard curve sample, a quality control sample and a human plasma sample to be tested, and adding an internal standard working solution to mix uniformly; taking a blank human plasma sample, adding 50% methanol solution, adding methanol to precipitate protein, and centrifuging to obtain supernatant;
s3, adding methanol-water solution with the volume ratio of 36:64 into the supernatant in the step S2 for dilution, carrying out vortex shaking, measuring by using UPLC-MS/MS, taking the peak area ratio of brivaracetam and an internal standard as an ordinate, and taking the concentration of an object to be detected in a standard curve sample as an abscissa to prepare a standard curve, and carrying out linear regression;
and S4, calculating the human plasma sample to be detected treated in the step S2 according to the standard curve equation obtained in the step S3 to obtain the concentration of the brivaracetam in the human plasma sample to be detected.
4. A method for the UPLC-MS/MS detection of brivaracetam in human plasma according to claim 3, characterized in that the step of preparing a standard working solution is: weighing a brivaracetam reference substance, adding methanol for dissolution, preparing a brivaracetam stock solution with the concentration of 1.000mg/mL, and preparing at least two stock solutions, one serving as a standard curve stock solution and one serving as a quality control stock solution; diluting the brivaracetam standard curve stock solution with a methanol-water solution with the volume ratio of 50:50 to obtain a standard curve working solution with the gradient of 400.000~36000.000 ng/mL; diluting the brivaracetam quality control stock solution with a methanol-water solution with the volume ratio of 50:50 to obtain a quality control working solution with the gradient of 400.000~24000.000 ng/mL; adding methanol into an internal standard to prepare internal standard stock solution with the concentration of 1.000mg/mL, diluting the internal standard stock solution with methanol-water solution with the volume ratio of 50:50 to obtain 800.000ng/mL internal standard working solution, and storing all stock solution and working solution at-20 ℃ for later use after subpackaging; and (3) taking blank plasma of healthy people, respectively adding a brivaracetam working solution, and uniformly mixing by vortex to prepare a standard curve sample with the concentration range of 20.000-1800.000 ng/mL and a quality control sample with the concentration range of 20.000~1200.000 ng/mL.
5. The method for UPLC-MS/MS detection of brivaracetam in human plasma according to claim 4, wherein said brivaracetam working solution concentration is 400.000 ng/mL, 1000.000 ng/mL, 2000.000ng/mL, 4000.000ng/mL, 8000.000 ng/mL, 16000.000ng/mL, 30000.000 ng/mL and 36000.000 ng/mL; the concentration of the brivaracetam quality control working solution is as follows: 400.000 ng/mL, 1200.000ng/mL, 3600.000 ng/mL, 12000.000ng/mL, 24000.000 ng/mL; the calibration standard sample and the quality control sample are freshly prepared; the concentration of the correction standard sample is as follows: 20.000 ng/mL, 50.000 ng/mL, 100.000 ng/mL, 200.000ng/mL, 400.000 ng/mL, 800.000ng/mL, 1500.000 ng/mL, 1800.000 ng/mL; the concentration of the quality control sample is as follows: 20.000 ng/mL, 60.000ng/mL, 180.000 ng/mL, 600.000ng/mL, 1200.000ng/mL.
6. The method for UPLC-MS/MS detection of brivaracetam in human plasma according to claim 4, wherein said internal standard is brivaracetam-d 7.
7. A method for UPLC-MS/MS detection of brivaracetam in human plasma according to claim 3, characterized in that in step S2 the vortex mixing time is 5min; the centrifugation conditions are as follows: the temperature during centrifugation is 4 ℃, the centrifugal speed is 2120 g, and the centrifugal time is 10 min.
8. A method for the UPLC-MS/MS detection of brivaracetam in human plasma according to claim 3, characterized in that in step S3 the chromatographic conditions are: chromatographic column: ACQUITY UPLC BEH C18, 50X 2.1 mm, filler particle size 1.7 μm; the flow rate is 0.3mL/min; column temperature: 40 ℃; sample injection volume: 1. mu L; autoinjector temperature: 8 ℃.
9. A method for the UPLC-MS/MS detection of brivaracetam in human plasma according to claim 3, characterized in that in step S3 the mass spectrometry conditions are: an ion source ESI source adopts a positive ion mode and a multi-reaction monitoring MRM mode; electrospray voltage 5500V; the temperature of the ion source is 500 ℃; the air curtain gas is 35 psi; the collision gas is 9 psi; the spray gas was 45 psi; the auxiliary heating gas was 45 psi.
10. A method for the UPLC-MS/MS detection of brivaracetam in human plasma according to claim 8, characterized in that the elution is set to: the volume flow is 0.3ml/min; the gradient elution conditions were:
in 0.00-1.50 min, the mobile phase A is 60% -5%, and the mobile phase B is 40% -95%;
at 1.51-2.50 min, mobile phase A is 5% and mobile phase B is 95%;
in 2.51-2.60 min, the mobile phase A is 5-60%, and the mobile phase B is 95-40%;
and the mobile phase A is 60% and the mobile phase B is 40% within 2.61-3.50 min.
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