CN115541729A - Method for detecting concentration of perampanel in human plasma based on HPLC-MS/MS - Google Patents
Method for detecting concentration of perampanel in human plasma based on HPLC-MS/MS Download PDFInfo
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- PRMWGUBFXWROHD-UHFFFAOYSA-N perampanel Chemical compound O=C1C(C=2C(=CC=CC=2)C#N)=CC(C=2N=CC=CC=2)=CN1C1=CC=CC=C1 PRMWGUBFXWROHD-UHFFFAOYSA-N 0.000 title claims abstract description 73
- 229960005198 perampanel Drugs 0.000 title claims abstract description 73
- 238000000034 method Methods 0.000 title claims abstract description 38
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 title claims abstract description 12
- 229910052805 deuterium Inorganic materials 0.000 claims abstract description 21
- 238000001514 detection method Methods 0.000 claims abstract description 15
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 11
- 238000012544 monitoring process Methods 0.000 claims abstract description 7
- 239000001961 anticonvulsive agent Substances 0.000 claims abstract description 6
- 229960003965 antiepileptics Drugs 0.000 claims abstract description 6
- 210000002381 plasma Anatomy 0.000 claims description 75
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 54
- 239000012071 phase Substances 0.000 claims description 39
- 239000012224 working solution Substances 0.000 claims description 30
- 238000003908 quality control method Methods 0.000 claims description 25
- 239000000243 solution Substances 0.000 claims description 17
- 239000011550 stock solution Substances 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 10
- 239000011159 matrix material Substances 0.000 claims description 9
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 7
- 238000002203 pretreatment Methods 0.000 claims description 7
- XBJFCYDKBDVADW-UHFFFAOYSA-N acetonitrile;formic acid Chemical compound CC#N.OC=O XBJFCYDKBDVADW-UHFFFAOYSA-N 0.000 claims description 5
- 238000012417 linear regression Methods 0.000 claims description 5
- 238000011002 quantification Methods 0.000 claims description 5
- 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 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 235000019253 formic acid Nutrition 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 238000004949 mass spectrometry Methods 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000000132 electrospray ionisation Methods 0.000 claims description 3
- 238000004811 liquid chromatography Methods 0.000 claims description 3
- 239000012074 organic phase Substances 0.000 claims description 3
- 230000036470 plasma concentration Effects 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 claims description 2
- 238000010813 internal standard method Methods 0.000 claims description 2
- 238000003260 vortexing Methods 0.000 claims description 2
- 239000000523 sample Substances 0.000 abstract description 35
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- 229940079593 drug Drugs 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 5
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 abstract description 5
- YZCKVEUIGOORGS-OUBTZVSYSA-N Deuterium Chemical compound [2H] YZCKVEUIGOORGS-OUBTZVSYSA-N 0.000 abstract description 3
- 239000012472 biological sample Substances 0.000 abstract description 3
- 238000004885 tandem mass spectrometry Methods 0.000 abstract description 3
- 230000006920 protein precipitation Effects 0.000 abstract 1
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
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- 102000003678 AMPA Receptors Human genes 0.000 description 2
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- 206010010904 Convulsion Diseases 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
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- 238000004587 chromatography analysis Methods 0.000 description 2
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- 102100022742 Lupus La protein Human genes 0.000 description 1
- 206010061334 Partial seizures Diseases 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
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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
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- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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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
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- G01N2030/042—Standards
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Abstract
The invention discloses a method for detecting the concentration of an antiepileptic drug, namely, perampanel in human plasma based on an HPLC-MS/MS technology. The detection method only needs 100 mu L of plasma sample, and after protein precipitation treatment, quantitative detection is carried out by using high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) with the Perampanel deuterium 5 as an internal standard. The detection method disclosed by the invention has the advantages that the sample pretreatment is rapid and convenient, the linear relation of the perampanel in 0.5-500ng/mL is good, the accuracy and the precision are good, the specificity is strong, the stability is high, the analysis requirements of biological samples are met, the detection requirements of the perampanel in human plasma are met, and the method is suitable for monitoring the conventional clinical treatment drugs of the perampanel.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a method for detecting the concentration of perampanel in human plasma based on a high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) technology.
Background
Perampanel is a novel antiepileptic drug developed by the Japanese toilet paper company, and exerts an antiepileptic effect by selectively and non-competitively inhibiting AMPA receptors. Perampanel is the only antagonist that effectively inhibits all AMPA receptor subtypes and can be used as an adjunct therapy in the treatment of patients with partial seizures over the age of 12 years, with or without secondary generalized seizures. Perampanel is the first antiepileptic drug approved by the FDA to have the mechanism of action.
The treatment scheme of the antiepileptic drug needs individuation, and when the perampanel is clinically applied, the plasma concentration of the perampanel of a patient is measured by a biological analysis method, so that important help and guidance significance are provided for monitoring the curative effect and safety of the perampanel and the interaction among drugs and designing the individuation administration scheme. Therefore, an accurate and rapid biological analysis method for clinically detecting the concentration of the perampanel in the human plasma is needed in clinic.
The current methods for quantifying the concentration of perampanel in human plasma include ultraviolet spectrophotometry, high performance liquid chromatography, and the like. The methods have the problems of complex and time-consuming pretreatment steps, inaccurate qualitative determination, insensitive quantification and the like. High performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) technology is increasingly widely applied to a plurality of fields such as medicine, food, environment, clinical detection and the like. The HPLC-MS/MS has the characteristics of high sensitivity and accuracy, strong selectivity and specificity and the like, and the detection capability of the HPLC-MS/MS is superior to that of the traditional immune and chemical detection method when the HPLC-MS/MS is applied to clinical detection.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for detecting the concentration of perampanel in human blood plasma based on an LC-MS/MS technology; the detection method provided by the invention has the advantages of rapid and convenient sample pretreatment, high sensitivity and high accuracy, and is suitable for detecting the Perampanel in a clinical routine manner.
In order to achieve the purpose, the invention adopts the technical scheme that:
providing a method for determining the concentration of perampanel in blood plasma based on HPLC-MS/MS, wherein the perampanel is an antiepileptic drug, and the internal standard of the perampanel is perampanel-deuterium 5;
the method comprises the following steps:
detecting the concentration of the perampanel in the pre-treated plasma sample by adopting HPLC-MS/MS, separating the perampanel from a plasma sample substrate by liquid chromatography, carrying out internal standard method quantification by using stable isotopes, drawing a standard curve by taking the ratio of the peak area of the perampanel to the peak area of an internal standard perampanel-deuterium 5 as a vertical coordinate and the concentration of the perampanel in the plasma sample of the standard curve as a horizontal coordinate, and calculating the concentration of the perampanel in the plasma.
Further, the liquid chromatography conditions of the high performance liquid chromatography-tandem mass spectrometry are as follows:
the high performance liquid chromatography conditions include:
a chromatographic column: ZORBAX Eclipse XDB-C18,3.5 μm, 2.1X 100mm;
flow rate: 0.3-0.4mL/min;
mobile phase: the water phase is 0.1-1% formic acid water solution; the organic phase is 0.1-1% formic acid acetonitrile solution;
column temperature: 40 ℃;
mixing a mobile phase A and a mobile phase B in different volumes, and performing gradient elution, wherein the mobile phase A is a formic acid aqueous solution, and the mobile phase B is a formic acid acetonitrile solution;
sample injection amount: 3 mu L of the solution;
the gradient elution process is as follows: in 0-2 minutes, the volume ratio of mobile phase a to mobile phase B is 95; the volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 95 to 5 to 50 in 2 to 8 minutes; the volume ratio of mobile phase a to mobile phase B was maintained at 50; the volume ratio of mobile phase a to mobile phase B was uniformly graded from 50.
Further, the mass spectrum conditions of the high performance liquid tandem mass spectrum are as follows:
in an electrospray ionization detection mode, multi-reaction monitoring and positive ion mode scanning are adopted; the ion pair of perampanel is: m/z 350.2 → 218.7, ion pair of the Perampanel-deuterium 5 internal standard is: m/z 354.8 → 220.1, scanning time of 200ms, capillary voltage of 5500V; the ion source temperature is 550 ℃; atomizing Gas (Gas 1) 15psi; turbine Gas (Gas 2) 15psi; air curtain air (CUR) was 20psi.
Further, the plasma sample pretreatment step is performed according to the following method:
taking 100 mu L of a plasma sample, putting the plasma sample into a 1.5mL centrifuge tube, adding 400-900 mu L of acetonitrile and 1.5-3 mu L of deuterated internal standard working solution, vortexing for 2min, centrifuging for 15min at 14000rpm at 4 ℃, taking supernatant, and analyzing by sample injection.
Further, the internal standard working solution is prepared according to the following method: putting 1.04mg of Perampanel-deuterium 5 reference substance into a 1.5mL centrifuge tube, adding 1.04mL of acetonitrile solution to dissolve the reference substance to prepare Perampanel-deuterium 5 stock solution with the concentration of 1mg/mL, adding 100 mu L of the stock solution into 900 mu L of acetonitrile, uniformly mixing to prepare 100 mu g/mL of deuterium substituted internal standard working solution, and storing the working solution in a refrigerator at the temperature of-20 ℃ for later use.
Further, the standard curve plasma sample was prepared as follows: diluting Perampanel stock solution with acetonitrile, preparing standard curve working solution and quality control working solution with multiple concentrations, taking 100 μ L of standard curve working solution and quality control working solution, adding 900 μ L of blank plasma matrix, and mixing to obtain solutions with concentrations of 0.5,5, 10, 50, 100, 200 and 500ng/mL -1 The standard curve of (1) plasma samples and concentrations were 4, 40, 400 ng.mL -1 The standard curve is established by using the peak area ratio of the Perampanel and the internal standard and the concentration of the standard curve sample to obtain a corresponding linear regression equation.
Further, the standard curve linear regression equation is as follows: the regression equation of the standard curve of the measured Perampanel in human plasma is as follows: y =0.00116x +0.0116, correlation coefficient R 2 Is 0.9994; the concentration range of the Perampanel working solution of the standard series is 1 ng/mL-1000 ng/mL; the linear range of the Perampanel is 0.5 ng/mL-500 ng/mL, and the lowest quantitative lower limit of the plasma concentration of the Perampanel in the method is 0.5ng/mL.
Compared with the prior art, the invention has the following technical effects:
the detection method provided by the invention can be simple, convenient and fast,Accurately and sensitively detecting the concentration of Perampanel in the plasma. The lower limit of the quantification of the invention can reach 0.5ng/mL, the quantification range can reach 0.5-500ng/mL, the detection range is wide, and the sensitivity is high. Coefficient of correlation (r) 2 ) More than 0.999, the accuracy deviation between the three quality control concentrations in the batch and between the batches is less than 13.0 percent, and the precision RSD is less than 6.81 percent. The recovery rate is 101-127%, and RSD is less than 9.80%. The samples were allowed to stand at room temperature for 24 hours, at-20 ℃ for 7 days, and freeze-thaw repeated 3 times to maintain stability. Therefore, the specificity, precision, accuracy, linearity, stability and the like of the method meet the analysis requirements of biological samples, the sensitivity is high, and the method can be used for monitoring the clinical treatment drugs of Perampanel.
Drawings
FIG. 1 is a chromatogram of a blank plasma in an embodiment of the present invention;
FIG. 2 is a chromatogram of a control of Perampanel-deuterium 5 (internal standard, concentration 100 ng/mL) added to blank plasma in accordance with an embodiment of the present invention;
FIG. 3 is a chromatogram of a control of Perampanel and Perampanel-deuterium 5 (both at 100 ng/mL) added to blank plasma in accordance with an embodiment of the present invention;
fig. 4 is a chromatogram of plasma from a patient taking perampanel according to an embodiment of the invention, on which: a total ion flow diagram of Perampanel and an internal standard Perampanel deuterium 5; the method comprises the following steps: extracting an ion flow diagram of Perampanel; the following: an extracted ion flow diagram of Perampanel-deuterium 5;
FIG. 5 is a Perampanel fragment ion scan;
FIG. 6 is a fragment ion scan of Perampanel-deuterium 5 (internal standard).
Detailed Description
The present invention will now be described in detail and specifically with reference to the following examples so as to provide a better understanding of the present invention, but the following examples are not intended to limit the scope of the present invention.
The following instruments and reagents were used in the specific embodiment of the invention:
1. main instrument
Shimadzu HPLC system, SCIEX QQQ 5500 Mass Spectrometry System, analyst chromatography workstation (Version 1.7.1), agilent ZORBAX Eclipse XDB-C18 (3.5 μm, 2.1X 100 mm) chromatography column;
a low temperature high speed refrigerated centrifuge;
a vortex mixer;
an analytical balance;
refrigerator (Thermo Fisher);
millipore ultrapure water device (Direct Q, millipore Ltd, molsheim, france)
2. Blank plasma, reagent and reference substance
The blank plasma used by the invention is from the blood center of Shanghai city; methanol (HPLC grade, merck), acetonitrile (HPLC grade, merck), formic acid (HPLC grade, sigma-Aldrich), water as ultrapure water, isopropanol (HPLC grade, merck); perampanel (TLC, P181001), perampanel-deuterium 5 (TLC, P181002).
3. Chromatographic conditions
A chromatographic column: agilent ZORBAX Eclipse XDB-C18 (3.5 μm, 2.1X 100 mm);
flow rate: 0.4mL/min;
column temperature: 40 ℃;
mobile phase: phase A: 0.1% aqueous formic acid, phase B: 0.1% formic acid acetonitrile solution;
mixing mobile phase A and mobile phase B in different volumes, and performing gradient elution. The gradient elution procedure is shown in Table 1, with each sample being taken for 16 minutes and a sample volume of 3. Mu.L.
TABLE 1 mobile phase gradient elution conditions
| Time (min) | Organic phase (B phase,%) | Aqueous phase (A phase,%) |
| 0 | 5 | 95 |
| 2 | 5 | 95 |
| 8 | 50 | 50 |
| 12 | 50 | 50 |
| 14 | 95 | 5 |
| 16 | Stop | Stop |
4. Conditions of Mass Spectrometry
Under an electrospray ionization detection mode, adopting multi-reaction monitoring and positive ion mode scanning; the corresponding mass spectral parameters of the test substance and its internal standard are shown in Table 2, and the fragment ion scan is shown in FIGS. 4 and 5. The scanning time of the mass spectrum is 200ms, and the capillary voltage is 5500V; the ion source temperature is 550 ℃; the first ion source gas is 15psi; the second ion source gas is 15psi; the air curtain air is 20psi.
TABLE 2 Mass Spectrometry parameters of Perampanel and the internal standard Perampanel-deuterium 5
[ example 1 ] Experimental procedure
1. Preparation of standard curve working solution and quality control working solution
(1) Preparing a Perampanel stock solution:
and precisely weighing se:Sup>A Perampanel reference substance, dissolving the reference substance by using se:Sup>A proper amount of acetonitrile, and preparing stock solution SS-A with the concentration of 1.00 mg/mL.
20 mu L of SS-A is added into 1980 mu L of acetonitrile, and the SS-A and the acetonitrile are uniformly mixed to prepare stock solution SS-B with the concentration of 10000 ng/mL.
200 mu L of SS-A is added into 1800 mu L of acetonitrile, and the SS-C stock solution with the concentration of 1000ng/mL is prepared by even mixing.
(2) Acetonitrile was used to prepare standard curve working solution and quality control working solution from different stock solutions according to the following table.
Table 3: preparation of standard curve working solution
Table 4: preparation of quality control working solution
2. Preparing internal standard stock solution and working solution:
accurately weighing the internal standard Perampanel-d 5, and dissolving the internal standard Perampanel-d 5 in acetonitrile to obtain an internal standard stock solution with the concentration of 1 mg/mL.
And adding 900 mu L of acetonitrile solution into 100 mu L of the internal standard stock solution, and uniformly mixing to prepare 100 mu g/mL of internal standard working solution.
3. Preparation of standard curve plasma sample and quality control plasma sample:
collecting 100 μ L of standard curve working solution and quality control working solution with multiple concentrations, adding 900 μ L of blank plasma, and mixing to obtain solutions with concentrations of 0.5,5, 10, 50, 100, 200 and 500ng/mL -1 The standard curve of (1) is that of the plasma sample and the concentration is 4, 40, 400 ng.mL -1 Quality control of plasma samples.
Table 5: preparation method of standard curve plasma sample
Table 6: preparation method of quality control plasma sample
4. Pretreatment of plasma samples
Taking 100 mu L of plasma sample in a 1.5mL centrifuge tube (taking 100 mu L of blank plasma for blank sample and internal standard blank sample), adding 400 mu L of acetonitrile and 1.5 mu L of internal standard working solution (10 mu L of acetonitrile is added for replacing blank sample), after vortex for 2min, centrifuging for 15min at 4 ℃,14000rpm, taking supernatant, and analyzing by LC-MS/MS sample introduction.
[ example 2 ] methodological verification
1. Linearity
And taking the peak area ratio of the perampanel to the perampanel-deuterium 5 as a vertical coordinate, and performing weighted linear regression on the concentration (x, ng/mL) of the perampanel in the plasma sample with the standard curve. The correlation coefficient r of the regression equation is greater than 0.999, as shown in the graph 7, perampanel has good linearity in the range of 0.5-500ng/mL, and meets the quantitative requirement.
TABLE 7 Linear Range, linear coefficient and Linear equation for Perampanel
| Name (R) | Linear range (ng/mL) | Coefficient of linearity r 2 | Linear equation of equations |
| Perampanel | 0.5-500 | 0.9994 | y＝0.0016x+0.016 |
2. Specificity
Blank plasma (without any control and internal standard) 100 μ L was pre-processed and then assayed according to the plasma sample pre-treatment and assay method of example 1, and the chromatogram of the blank plasma was compared with blank plasma with 100ng/mL of perampanel, respectively, and it was confirmed that there was no interference of endogenous impurities in the blank plasma, see fig. 1-3.
3. Accuracy and precision
Preparing 5 batches of quality control plasma samples according to a quality control plasma sample preparation method, respectively taking 100 mu L of LLOQ, LQC, MQC and HQC quality control plasma samples into a 1.5mL centrifuge tube, performing pretreatment on the quality control plasma samples according to the plasma sample pretreatment and determination method in the embodiment 2, and then determining, and calculating the average value, the standard deviation and the relative standard deviation according to the result.
The results show that: the accuracy deviation of the quality control sample of the Perampanel under the three concentrations is less than 13.0 percent, and the precision RSD between batches is less than 6.81 percent, which is shown in a table 7. The results show that: the method has good precision and accuracy, and meets the analysis requirements.
TABLE 7 Perampanel batch and batch to batch accuracy and precision
4. Extraction recovery rate
And (3) respectively taking LQC, MQC and HQC quality control plasma samples, carrying out pretreatment and determination according to the plasma sample pretreatment and determination method in the embodiment 1, and then, taking standard working solution with corresponding concentration for sample injection and determination. And calculating the extraction yield according to the ratio of the peak area of the drug after the blood sample treatment to the peak area of the drug injected by the corresponding concentration control solution. Referring to Table 8 below, the mean extraction recovery of quality control plasma samples at each concentration of Perampanel was 101-127% with RSD% < 9.8%.
Table 8 recovery of perampanel (n = 5)
5. Stability of
(1) Room temperature stability test
The quality control plasma samples LQC and HQC were taken and left at room temperature for 24 hours, and the quality control plasma samples were pretreated and then measured according to the plasma sample pretreatment and measurement method in example 1, and the deviation of the measurement of the quality control plasma samples at each concentration after left at room temperature for 24 hours was less than 3.00% as compared with the results of the immediate measurement, as shown in table 9.
(2) Freezing and thawing experiment
The quality control plasma samples LQC and HQC are placed at-80 ℃ and melted at normal temperature after 24 hours, the process is repeated for 3 times, and the quality control plasma samples are pretreated and measured according to the plasma sample pretreatment and measurement method in the embodiment 1 and then are compared with the instant measurement result. The results show a deviation of < 7.30%, see table 9.
(3) Short term stability test
The quality control plasma samples LQC and HQC were taken and left at-20 ℃ for 7 days, and then pre-treated according to the plasma sample pre-treatment and measurement method in example 1, and then measured, and the deviation of the quality control plasma sample measurement of each concentration was less than 10.0% compared with the results of the immediate measurement, as shown in table 9.
(4) Matrix effect
Taking a blank plasma sample, pretreating the blank plasma sample according to the plasma sample pretreatment and measurement method in the example 1 to obtain a blank plasma matrix, preparing samples containing 4ng/mL and 400 ng/mL of Perampanel by using the matrix, and simultaneously measuring the samples with corresponding concentrations by using a control solution prepared by using a mobile phase. The results show a matrix effect of < 15.0%. See table 9.
TABLE 9 stability and matrix Effect of Perampanel
In conclusion, the invention provides the method for detecting the concentration of the perampanel in the human plasma by adopting the LC-MS/MS technology, the specificity, the precision, the accuracy, the linearity, the stability, the recovery rate, the matrix effect and the like of the method all meet the analysis requirements of biological samples, and the method has the advantages of high sensitivity, simple and convenient pretreatment method and low cost, and can be used for monitoring the clinical treatment drugs of the perampanel.
The embodiments of the present invention have been described in detail, but the embodiments are only examples, and the present invention is not limited to the above-described embodiments. Any equivalent modifications and substitutions for the present invention are within the scope of the present invention for those skilled in the art. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Claims (8)
1. A method for determining the concentration of perampanel in blood plasma based on HPLC-MS/MS is characterized in that the perampanel is an antiepileptic drug, and the internal standard of the perampanel is perampanel-deuterium 5;
the method comprises the following steps:
detecting the concentration of the perampanel in the pre-treated plasma sample by HPLC-MS/MS, separating the perampanel from a plasma sample matrix by liquid chromatography, performing internal standard method quantification by using stable isotopes, drawing a standard curve by taking the ratio of the peak area of the perampanel to the peak area of an internal standard perampanel-deuterium 5 as an ordinate and the concentration of the perampanel in the plasma sample of the standard curve as an abscissa, and calculating the concentration of the perampanel in the plasma.
2. The method of claim 1, wherein the high performance liquid chromatography conditions comprise:
a chromatographic column: ZORBAX Eclipse XDB-C18,3.5 μm, 2.1X 100mm;
flow rate: 0.3-0.4mL/min;
mobile phase: the water phase is 0.1-1% formic acid water solution; the organic phase is 0.1-1% formic acid acetonitrile solution;
column temperature: 40 ℃;
mixing a mobile phase A and a mobile phase B in different volumes, and performing gradient elution, wherein the mobile phase A is a formic acid aqueous solution, and the mobile phase B is a formic acid acetonitrile solution;
sample introduction amount: 3 mu L of the solution;
the gradient elution procedure was as follows: in 0-2 minutes, the volume ratio of mobile phase a to mobile phase B is 95; the volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 95; the volume ratio of mobile phase a to mobile phase B was maintained at 50; the volume ratio of mobile phase a to mobile phase B was uniformly graded from 50 to 95 in 12-14 minutes, maintaining the volume ratio of mobile phase a to mobile phase B at 95 in 14-16 minutes, and each sample collection time was 16 minutes.
3. The method of claim 1, wherein the mass spectrometry conditions comprise: in an electrospray ionization detection mode, multi-reaction monitoring and positive ion mode scanning are adopted; the ion pair of perampanel is: m/z 350.2 → 218.7, ion pair of the Perampanel-deuterium 5 internal standard is: m/z 354.8 → 220.1, scanning time of 200ms, capillary voltage of 5500V; the ion source temperature is 550 ℃; the first ion source gas is 15psi; the second ion source gas is 15psi; the air curtain air is 20psi.
4. The method according to claim 1, wherein the pre-treatment of human plasma comprises the following steps: taking 100 mu L of a plasma sample, putting the plasma sample into a 1.5mL centrifuge tube, adding 400-900 mu L of acetonitrile and 1.5-3 mu L of deuterated internal standard working solution, vortexing for 2min, centrifuging for 15min at 14000rpm at 4 ℃, taking supernatant, and analyzing by sample injection.
5. The method as claimed in claim 4, wherein the deuterated internal standard working solution is Perampanel-deuterium 5.
6. The method according to claim 5, wherein the internal standard working solution is prepared according to the following method: putting 1.04mg of a perampanel-deuterium 5 standard substance into a 1.5mL centrifuge tube, adding 1.04mL of acetonitrile solution to dissolve the standard substance to prepare a perampanel-deuterium 5 stock solution with the concentration of 1mg/mL, adding 100 mu L of the stock solution into 900 mu L of acetonitrile, uniformly mixing to prepare a 100 mu g/mL deuterated internal standard working solution, and storing in a refrigerator at the temperature of-20 ℃ for later use.
7. The method of claim 1, wherein the standard curve plasma sample is prepared according to the following method: diluting Perampanel stock solution with acetonitrile, preparing standard curve working solution and quality control working solution with multiple concentrations, taking 100 μ L of standard curve working solution and quality control working solution, adding 900 μ L of blank plasma matrix, and mixing to obtain solutions with concentrations of 0.5,5, 10, 50, 100, 200 and 500ng/mL -1 The standard curve of (1) plasma samples and concentrations were 4, 40, 400 ng.mL -1 The standard curve is established by using the peak area ratio of the Perampanel and the internal standard and the concentration of the standard curve sample to obtain a corresponding linear regression equation.
8. The method of claim 7, wherein the standard curve linear regression equation is as follows: the regression equation of the standard curve of the measured Perampanel in human plasma is as follows: y =0.00116x +0.0116 and correlation coefficient R 2 Is 0.9994; the concentration range of the standard series of the Perampanel working solution is 1 ng/mL-1000 ng/mL; the linear range of the Perampanel is 0.5 ng/mL-500 ng/mL, and the lowest quantitative lower limit of the plasma concentration of the Perampanel in the method is 0.5ng/mL.
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Non-Patent Citations (1)
| Title |
|---|
| HAO-RAN DAI ET AL.: "LC-MS/MS assay for the therapeutic drug monitoring of perampanel in children with drug-resistant epilepsy", 《ACTA CHROMATOGRAPHICA》, 11 April 2022 (2022-04-11), pages 149 - 160 * |
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