CN112198244A - Method for determining concentration of abiraterone in blood plasma - Google Patents
Method for determining concentration of abiraterone in blood plasma Download PDFInfo
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- CN112198244A CN112198244A CN202010967078.6A CN202010967078A CN112198244A CN 112198244 A CN112198244 A CN 112198244A CN 202010967078 A CN202010967078 A CN 202010967078A CN 112198244 A CN112198244 A CN 112198244A
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- G—PHYSICS
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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
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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
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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
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- G—PHYSICS
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/045—Standards internal
Abstract
The invention belongs to the technical field of medicine detection, and particularly discloses a method for determining concentration of abiraterone in plasma, wherein the concentration of the abiraterone in the plasma is determined and analyzed by an LC-MS/MS method, deuterated abiraterone is used as an internal standard, a Waters XTERRA MS C18 column is used for elution, and ESI tandem mass spectrometry is used for detection, so that the extraction recovery rate of the method is more than 99%, and the matrix effect, the hyperlipemia matrix effect and the hemolysis matrix effect are free of interference, and the method has high accuracy and precision and small dosage; compared with the existing testing method which mainly adopts a high performance liquid chromatography, the lower detection limit of the method can meet the requirement of content determination in organisms with smaller dosage, the signal-to-noise ratio in the detection process meets the standard requirement, the interference of impurity peaks is avoided, and the content accuracy of the determined abiraterone is high.
Description
Technical Field
The invention relates to the technical field of medicine detection, in particular to a method for determining abiraterone concentration in blood plasma.
Background
Abiraterone is an antiandrogen drug, is a CYP17 enzyme inhibitor in the androgen metabolism process, belongs to the field of endocrine therapy, reduces the testosterone level by blocking key enzymes of androgen biosynthesis, and can reduce the androgen synthesis in the microenvironment inside the prostate tumor besides testosterone from testis and adrenal gland. Abiraterone can strongly inhibit 17 alpha-hydroxylase/C17, 20-lyase (CYP17), so that the biosynthesis of androgen is remarkably inhibited, and the inhibition activity of abiraterone on CYP17 in a pharmacokinetic model of a mouse is far higher than that of ketoconazole. Phase I clinical test results show that abiraterone not only can well inhibit testosterone levels in a castration environment, but also has good tolerance and does not show toxic reaction like ketoconazole. Open, single-center and dose escalation clinical trials have shown that continuous abiraterone treatment reduces prostate cancer-specific antigen in the serum of 50-57% of patients, and that the phase II clinical recommended treatment dose is 1000mg/d, at which glucocorticoid and androgen synthesis is inhibited simultaneously. The chemical structural formula of the abiraterone is shown as follows.
At present, no relevant literature reports at home and abroad exist about the quantitative detection and analysis of abiraterone in beagle plasma.
Patent application number CN201710159438.8 discloses a quantitative determination method of abiraterone in whole blood, which takes whole blood as an object to be detected and sequentially carries out the following steps: adding ethyl acetate into the whole blood lysate, oscillating and whirling, centrifuging, drying an ethyl acetate layer by nitrogen flow, redissolving by using 20% acetonitrile water solution, centrifuging, and absorbing supernatant I; weighing alkaline diatomite, placing the alkaline diatomite into a glass chromatographic column, carrying out vibration treatment, adding supernatant I, eluting with pure water, 20% methanol water solution and chromatographic methanol in sequence, drying eluent nitrogen flow, redissolving the eluent with 20% acetonitrile water solution, centrifuging the eluent, absorbing supernatant II, and carrying out high performance liquid chromatography system analysis to obtain an abiraterone medicine peak area Y, substituting the formula Y which is 27524X +1956.6, wherein X is the abiraterone concentration of the supernatant II; and then the abiraterone concentration in the whole blood to be detected is obtained through conversion.
Synthesis and quality detection research of abiraterone acetate are carried out by Wuqingsong and the like, the pharmaceutical engineering academy of the southwest university of transportation, and the synthesis and quality detection research of the abiraterone acetate are carried out by adopting a Luna C18 chromatographic column (250mm multiplied by 4.6mm, 5 mu m), the flow rate is 1.0ml/min, the detection wavelength is 220nm, the column temperature is 40 ℃, the mobile phase A is methanol-acetonitrile (80-20 percent), the mobile phase B is water, and the content of the abiraterone acetate and the abiraterone existing as impurities is quantitatively analyzed by gradient elution.
Foreign journal reports that Abiraterone in rat plasma is quantitatively analyzed by adopting RP-HPLC (reverse phase high performance liquid chromatography) method by Kumar in India, Betasil C18 chromatographic column is adopted, ethyl acetate is used for extraction, and the Abiraterone content in rat plasma is quantitatively detected under room temperature environment. The recovery rate is above 70%, and the mobile phase is acetonitrile-water-0.01M potassium dihydrogen phosphate (pH3.0) ═ 55-5-40 (V/V/V).
At present, high performance liquid chromatography is mainly used, the lower detection limit cannot meet the requirement of content determination in organisms with small dosage, the signal-to-noise ratio is low in the detection process, the interference of a solvent peak exists, and the content accuracy of the determined abiraterone is insufficient, so that the method has important significance for solving the problems.
Disclosure of Invention
The invention aims to provide a method for measuring the concentration of abiraterone in blood plasma, which overcomes the defect that the detection method in the prior art cannot detect the accurate quantitative detection of the lower concentration level in the blood plasma; the method has the advantages of simplifying the sample pretreatment process, avoiding matrix effect, having excellent chromatographic peak shape, realizing the rapid, sensitive and specific analysis of the drugs in the blood plasma, providing a simple and convenient analysis method for the detection of the pharmacokinetics in vivo, and realizing the accurate quantification of the lower concentration level in the blood plasma; can meet the requirement of analyzing a large quantity of clinical samples.
In order to solve the technical problems, the invention adopts the following technical scheme:
in one aspect, the invention provides a method for determining abiraterone concentration in blood plasma, comprising the following steps:
1) preparation of standard series working solution: weighing abiraterone, putting the abiraterone into a volumetric flask, and dissolving the abiraterone into methanol to prepare a standard reference substance stock solution; diluting the standard reference substance stock solution with 50% acetonitrile water solution to prepare standard series abiraterone working solution;
2) preparation of standard curve plasma samples: taking standard series of abiraterone working solution, diluting the standard series of abiraterone working solution into blank plasma, and preparing an abiraterone standard curve plasma sample with the concentration of 1-500 ng/ml;
3) and (3) drawing a standard curve regression equation: adding an internal standard working solution into a standard curve plasma sample, carrying out vortex, adding an acetonitrile precipitator for precipitation, carrying out vortex and centrifugation, and then carrying out sample injection on an LC-MS/MS system to obtain a chromatogram; calculating the concentration of the abiraterone in the plasma according to the peak area of the chromatographic peak in the obtained chromatogram and the peak area of the internal standard, and making a standard curve regression equation;
wherein, the liquid phase conditions in the LC-MS/MS system are as follows:
liquid phase conditions
A chromatographic column: waters XTERRA MS C18; mobile phase A, 10mM ammonium acetate-0.1% formic acid aqueous solution and mobile phase B, 0.1% formic acid methanol solution;
conditions of Mass Spectrometry
ESI source is adopted, ionization mode: electrospray ionizing positive ions; the monitoring mode is as follows: MRM; polarity: a positive ion; fragmentation voltage: 240V, collision energy: 65V
Abiraterone-d4(ABTL-d4): the parent ion m/z is 354.2, and the daughter ion m/z is 159.9;
abiraterone (ABTL): the parent ion m/z was 350.2 and the daughter ion m/z was 155.9.
Further, the concentration of the standard reference substance stock solution is 1 mg/mL-1；
Further, the concentration range of the standard series of abiraterone working solution is 20-10000 ng/mL;
further, the concentration range of the abiraterone standard curve plasma sample is 1-500 ng/mL.
Further, the plasma is derived from a mammal such as beagle dog, rabbit, mouse or human.
Further, the standard curve regression equation is obtained as follows: taking 50 mu L of each standard curve plasma sample, adding 20 mu L of internal standard working solution, carrying out vortex for 30s, adding 300 mu L of acetonitrile precipitator for precipitation, carrying out vortex for 3min, centrifuging 2490g, and carrying out centrifugation at 4 ℃ for 10 min; 5 mu L of sample injection LC-MS/MS system to obtain a chromatogram; and calculating the concentration of the abiraterone in the plasma according to the peak area of the chromatographic peak in the obtained chromatogram and the peak area of the internal standard, and making a standard curve regression equation.
Further, the preparation process of the internal standard working solution is as follows: weighing Abiraterone-d4Dissolving in methanol in a volumetric flask to prepare 1 mg/mL-1Abiraterone-d of4An internal standard control stock solution;
abiraterone-d4Diluting the stock solution of the internal standard reference substance with methanol to obtain a solution with a concentration of 100ng/mL-1The internal standard working solution of (4).
Further, the conditions of the LC-MS/MS system are as follows:
chromatographic conditions
A chromatographic column: waters XTERRA MS C18 (2.1X 50mm, 5 microns); mobile phase A, 10mM ammonium acetate-0.1% formic acid aqueous solution and mobile phase B, 0.1% formic acid methanol solution; flow rate: 0.5 mL/min-1(ii) a Sample introduction amount: 5 mu L of the solution; column temperature: 30 ℃; needle washing liquid: methanol; washing the needle washing liquid: 5 s; sample introduction amount: 5 mu L of the solution; column temperature: 30 ℃; stopping time: 6.00 min;
the mobile phase ratio was set as follows:
| Time | A | B | Flow | Max |
|
| 1 | 0.00min | 40.0％ | 60.0％ | 0.500mL/min | 400.00 |
| 2 | 1.00min | 5.0％ | 95.0％ | 0.500mL/min | 400.00 |
| 3 | 4.80min | 5.0％ | 95.0％ | 0.500mL/min | 400.00 |
| 4 | 4.81min | 40.0％ | 60.0％ | 0.500mL/min | 400.00 |
| 5 | 6.00min | 40.0％ | 60.0％ | 0.500mL/min | 400.00bar |
conditions of Mass Spectrometry
ESI source is adopted, ionization mode: electrospray ionizing positive ions; the monitoring mode is as follows: MRM; polarity: a positive ion; fragmentation voltage: 240V, collision energy: 65V; abiraterone-d4(ABTL-d4): the parent ion m/z is 354.2, and the daughter ion m/z is 159.9; abiraterone (ABTL): the m/z of the parent ion is 350.2, and the m/z of the daughter ion is 155.9;
the mass spectrum switching valve is set as follows:
| serial number | Start time (min) | Switching |
| 1 | 0.8 | |
| 2 | 2.8 | Cutting waste liquid |
The ion source parameters were set as follows: the temperature of the capillary blowback drying gas is 300 ℃; capillary blowback dry air flow: 10 l/min; atomizer pressure 45 psi; the temperature of sheath gas flow is 350 ℃; sheath flow rate: 11ml/min, capillary voltage: 3500V.
Further, after the LC-MS/MS system detects, the abiraterone peaks at about 2.16 min;
the regression equation for the standard curve is as follows: the regression equation of the standard curve of the measured abiraterone in the beagle plasma is that y is 0.026236x-0.001218, R20.99956482; the linear range of the abiraterone is 1 ng/mL-500 ng/mL, and the lowest quantitative lower limit of the blood concentration of the abiraterone is 1 ng/mL.
In another aspect, the present invention provides a method for determining abiraterone concentration in plasma, comprising the steps of:
preparing a sample solution: adding an internal standard working solution into a plasma sample to be detected, carrying out vortex, adding an acetonitrile precipitator for precipitation, and then carrying out vortex and centrifugation;
detection of sample solution: injecting the prepared sample solution into an LC-MS/MS system to obtain a chromatogram;
calculation of the concentration in the sample solution: substituting the peak area ratio of the target drug in the plasma sample to be measured to the peak area ratio of the internal standard into the standard curve regression equation obtained by the method of the invention, and calculating the corresponding concentration of the measured drug.
Further, the sample solution was prepared as follows: adding 20 μ L of internal standard working solution into 50 μ L of plasma sample to be detected, adding 300 μ L of acetonitrile precipitant, precipitating, vortexing for 3min, centrifuging 2490g, and centrifuging at 4 deg.C for 10 min.
Further, the detection conditions of the LC-MS/MS system are as follows:
chromatographic conditions
A chromatographic column: waters XTERRA MS C18 (2.1X 50mm, 5 microns); mobile phase A, 10mM ammonium acetate-0.1% formic acid aqueous solution and mobile phase B, 0.1% formic acid methanol solution; flow rate: 0.5 mL/min-1(ii) a Sample introduction amount: 5 mu L of the solution; column temperature: 30 ℃; needle washing liquid: methanol; washing the needle washing liquid: 5 s; sample introduction amount: 5 mu L of the solution; column temperature: 30 ℃; stopping time: 6.00 min;
the mobile phase ratio was set as follows:
| Time | A | B | | Max Pressure | ||
| 1 | 0.00min | 40.0％ | 60.0％ | 0.500mL/min | 400.00 |
|
| 2 | 1.00min | 5.0％ | 95.0％ | 0.500mL/min | 400.00 |
|
| 3 | 4.80min | 5.0％ | 95.0％ | 0.500mL/min | 400.00 |
|
| 4 | 4.81min | 40.0％ | 60.0％ | 0.500mL/min | 400.00 |
|
| 5 | 6.00min | 40.0％ | 60.0％ | 0.500mL/min | 400.00bar |
conditions of Mass Spectrometry
Detection was performed using an ESI source, positive ion MRM mode.
The mass spectrum switching valve is set as follows:
| StartTime(min) | |
|
| 1 | 0.8 | |
| 2 | 2.8 | ToWaste |
the ion source parameters were set as follows:
| Parameter | Value(+) |
| Gas Temp(℃) | 300 |
| Gas Flow(l/min) | 10 |
| Nebulizer(psi) | 45 |
| |
350 |
| SheathGasFlow | 11 |
| Capillary(V) | 3500 |
| |
500 |
further, the preparation process of the internal standard working solution is as follows: weighing Abiraterone-d4Dissolving in methanol in a volumetric flask to prepare 1 mg/mL-1Abiraterone-d of4An internal standard control stock solution;
abiraterone-d4Diluting the stock solution of the internal standard reference substance with methanol to obtain a solution with a concentration of 100ng/mL-1The internal standard working solution of (4).
The qualitative method comprises the following steps: the retention time of the drug to be detected is consistent with that of the drug standard, and the drug to be detected is determined as the target drug by combining the qualitative and quantitative ion pairs in the mass spectrum parameters.
The quantitative method comprises the following steps: and (3) performing linear least square regression calculation on the theoretical concentration of the analyte in the standard curve by comparing the peak area of the analyte with the peak area of the internal standard, and calculating the actually measured concentration of the analyte in the sample by using the obtained regression equation.
The invention adopts a liquid chromatogram-mass spectrum combined method for detection, combines the capability of a liquid chromatograph for effectively separating compounds and the strong component identification capability of a mass spectrometer, can effectively separate and identify the abiraterone in the blood plasma, and carries out quantitative detection.
The method can obtain high recovery rate, effectively reduce the interference of impurity peaks, avoid the influence of the traditional method caused by solvent effect, and avoid the situations of complexity and uncontrolled conditions of the traditional method.
The invention adopts deuterated abiraterone as an internal standard substance, so that the measured abiraterone concentration in the blood plasma has good reproducibility and high accuracy;
the method has system applicability, no selectivity to blank plasma from different sources, capability of specifically measuring abiraterone and good separation degree.
The method has small dosage, only needs 5 mu L of blood plasma sample test each time, and can accurately determine the compounds.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a simple determination method for determining abiraterone concentration in plasma by a pretreatment method, which adopts beagle plasma as demonstration and a protein precipitation method, wherein under the chromatographic condition adopted by the experiment, the abiraterone retention time is about 2.16min, the peak shape is good, the determination is free from the interference of a miscellaneous peak, and the baseline is stable; the method has high specificity, can accurately determine the concentration of the abiraterone in the blood plasma, has high sensitivity, has the minimum quantitative limit of the blood plasma of the abiraterone of 1ng/mL, is quick and accurate, has high sensitivity, is simple and convenient to operate, and provides a basis for determining the blood concentration of the abiraterone. The linear range of the plasma standard curve of the abiraterone in the method is 1-500 ng/mL, and the precision RSD in batches and among batches is less than +/-10%. The method has good reproducibility and high accuracy, is not interfered by a substrate, a hyperlipemia substrate and a hemolysis substrate, and has no interference phenomenon of blood plasma of different individuals to the method.
The existing testing method is mainly high performance liquid chromatography. Compared with the prior art, the method has the advantages that the lower detection limit can meet the content determination requirement in organisms with smaller dosage, the signal-to-noise ratio in the detection process meets the standard requirement, the interference of impurity peaks is avoided, and the content accuracy of the determined abiraterone is high.
The method can meet the detection requirement of the biological sample, and can be applied to preclinical and clinical evaluation research of medicaments.
Drawings
FIG. 1 is a standard graph of Abiraterone in beagle plasma measured by LC-MS/MS;
FIG. 2 determination of Abiraterone and Abiraterone-d in plasma of beagle dog by LC-MS/MS method4Liquid mass diagram of (1);
FIG. 3 is a graph of an Abiraterone positive ion multi-reaction detection scan mass spectrum;
FIG. 4 Abiraterone-d4Scanning mass spectrogram by positive ion multi-reaction detection;
FIG. 5 is a graph showing the Abiraterone blood concentration-time curve of Beagle dogs after fasting oral administration of Abiraterone acetate
Detailed Description
The invention will be further illustrated with reference to the following specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention.
1. Main instrument
An Agilent G6470 model triple quadrupole lc Mass spectrometer equipped with a liquid chromatograph (including an autosampler), an electrospray ionization ion source (ESI), and an Agilent Mass Hunter Software data processing system (Agilent, usa); the METTLER TOLEDO AB 135-model S one hundred thousandth of a balance (METTLER, switzerland).
2. Chromatographic conditions
A chromatographic column: waters XTERRA MS C18 (2.1X 50mm, 5 microns); mobile phase A, 10mM ammonium acetate-0.1% formic acid aqueous solution and mobile phase B, 0.1% formic acid methanol solution; flow rate: 0.5 mL/min-1(ii) a Sample introduction amount: 5 mu L of the solution; column temperature: 30 ℃; needle washing liquid: methanol; washing the needle washing liquid: 5 s; sample introduction amount: 5 mu L of the solution; column temperature: 30 ℃; stopping time: 6.00 min.
The mobile phase ratio was set as follows:
| Time | A | B | | Max Pressure | ||
| 1 | 0.00min | 40.0％ | 60.0％ | 0.500mL/min | 400.00 |
|
| 2 | 1.00min | 5.0％ | 95.0％ | 0.500mL/min | 400.00 |
|
| 3 | 4.80min | 5.0％ | 95.0％ | 0.500mL/min | 400.00 |
|
| 4 | 4.81min | 40.0％ | 60.0％ | 0.500mL/min | 400.00 |
|
| 5 | 6.00min | 40.0％ | 60.0％ | 0.500mL/min | 400.00bar |
3. conditions of Mass Spectrometry
Detection was performed using an ESI source, positive ion MRM mode.
The mass spectrum switching valve is set as follows:
| Start Time(min) | |
|
| 1 | 0.8 | To |
| 2 | 2.8 | To Waste |
the ion source parameters were set as follows:
the ion source parameters were set as follows: the temperature of capillary blowback drying Gas (Gas Temp) is 300 deg.C; dry Gas Flow reverse capillary Flow (Gas Flow): 10 l/min; atomizer pressure (Nebulizer) 45 psi; sheath Gas temperature (Sheath Gas Heater) 350 ℃; sheath Flow rate (Sheath Gas Flow): 11ml/min, Capillary voltage (Capillary): 3500V.
4. Anticoagulant: EDTA-K2
5. Internal standard: abiraterone-d4
6. Data processing
The retention time and peak area of the chromatogram were collected and processed by Agilent Mass Hunter Workstation Software and analyzed quantitatively, using Agilent ECM network system. And (3) performing linear least square regression calculation on the theoretical concentration of the analyte in the standard curve by comparing the peak area of the analyte with the peak area of the internal standard, and calculating the actually measured concentration of the analyte in the sample by using the obtained regression equation. The measured concentration of the analyte in the sample is calculated by the instrument using the following regression equation:
y＝ax+b
wherein y is the analyte/internal standard peak area ratio; a is the slope of the standard curve; x is drug concentration/internal standard concentration; b is the intercept of the standard curve; (weight factor is 1/x)2)
The decimal point number used for all calculations and statistical analyses is consistent with the software output. All data related to concentration retain 3 bits after the decimal point, chromatographic peak retention time retains 3 bits after the decimal point, percentage retains 1-bit decimal, chromatographic peak area retains an integer, and peak area retains 6 bits after the decimal point.
RSD values, deviations, mean values, etc. of the data were calculated using Microsoft Office Excel 2013.
7. The pretreatment method before the detection of the sample comprises the following steps:
taking whole blood, adding an anticoagulant EDTA-K2, treating by a protein precipitation method to prepare blank plasma, refrigerating for sample dilution or preparation, and unfreezing before use.
Adding 20 mu L of internal standard working solution before 50 mu L of standard curve plasma sample is used, vortexing for 30s, adding 300 mu L of acetonitrile, vortexing for 3min, centrifuging, 2490g, 4 ℃, and 10min for sample injection.
Adding 20 mu L of internal standard working solution before 50 mu L of SST is used, whirling for 30s, adding 300 mu L of acetonitrile, whirling for 3min, centrifuging, 2490g, 4 ℃, and 10min for sample injection.
Adding 20 mu L of internal standard working solution into 50 mu L of quality control plasma sample before use, performing vortex for 30s, adding 300 mu L of acetonitrile, performing vortex for 3min, performing centrifugation, 2490g, performing 4 ℃, and performing 10min for sample injection.
mu.L of methanol 20. mu.L before use, vortexed for 30s, added 300. mu.L of acetonitrile, vortexed for 3min, centrifuged, 2490g, 4 ℃,10 min, ready for injection, and recorded as: double blank or Carryover samples.
Or adding 20 μ L of internal standard working solution before 50 μ L of blank plasma is used, vortexing for 30s, adding 300 μ L of acetonitrile, vortexing for 3min, centrifuging, 2490g, 4 ℃,10 min, and recording as: blank sample.
Before 50 mul of ultrapure water is used, 20 mul of methanol is added, vortex is carried out for 30s, 300 mul of acetonitrile is added, vortex is carried out for 3min, centrifugation is carried out, 2490g, 4 ℃ and 10min are carried out, and after sample injection, the sample injection is recorded as: reagent and Materials blank samples.
Remarking: double blank for Double blank, carryover for residual investigation, blank for single blank with internal standard only, Reagent and Materials blank for Reagent consumables acceptance.
Example 1
1. Solution preparation
Mobile phase a (10mM ammonium acetate-0.1% aqueous formic acid): about 0.7708g of ammonium acetate was weighed into a suitable solvent bottle, 1000mL of ultrapure water and 1mL of formic acid were added, and the mixture was sonicated.
Mobile phase B (0.1% formic acid methanol): 1000mL of methanol is weighed and transferred into a proper solvent bottle, 1mL of formic acid is added, and the mixture is uniformly mixed and subjected to ultrasound.
Diluted solution (50% acetonitrile in water): 200mL of acetonitrile and 200mL of ultrapure water are respectively taken and transferred into a proper solvent bottle, and the mixture is uniformly mixed for standby.
2. Preparing a standard solution:
preparation of standard reference substance stock solution
Precisely weighing 10.04mg of abiraterone reference substance in 10mL volumetric flask, respectively, sucking 10mL of methanol with 10mL pipette to obtain a solution with a concentration of 1 mg/mL-1The abiraterone standard reference substance stock solution is stored at the temperature of minus 20 ℃.
Preparation of standard series working solution:
taking a proper amount of standard reference substance stock solution, diluting with 50% acetonitrile water solution to obtain an abiraterone working solution: 20. 40, 100, 200, 1000, 5000, 8000, 10000, 100000 ng/mL.
Preparation of Standard Curve plasma samples
Taking 10 mu L of each standard series working solution, diluting the standard series working solutions into blank plasma to ensure that the total volume is 0.2mL, and preparing standard curve plasma samples with the abiraterone concentration values as follows: 1.2, 5, 10, 50, 250, 400, 500 ng/mL.
SST samples: taking 10 mu L of standard series working solution, diluting the standard series working solution into blank plasma to ensure that the total volume is 0.2mL, and preparing an SST sample with the concentration value of abiraterone as follows: 1 ng/mL.
3. Preparation of internal standard solution
Internal standard stock solution preparation
Abiraterone-d with the specification of 1.02mg4Methanol was added to the control (content 98.8%) and shaken well. To obtain the compound containing abiraterone-d4The concentration is 1 mg/mL-1The internal standard reference stock solution of (4) is stored at-20 ℃.
Preparation of internal standard working solution (IS Spike,100 ng. mL)-1)
4. Preparation of quality control standard solution
Preparing a quality control reference substance stock solution:
respectively weighing 10.04mg of abiraterone reference substance, sucking 10mL of methanol by a 10mL pipette, adding into a 10mL volumetric flask, and keeping the concentration of the substance to be detected (abiraterone) at 1 mg/mL-1The quality control reference substance stock solution is stored at the temperature of minus 20 ℃.
Preparing a quality control working solution:
and respectively taking quality control reference substance stock solutions, diluting the stock solutions into 50% acetonitrile water solution, and preparing the abiraterone quality control working solution with the concentration of 20, 60, 750, 7500 and 75000 ng/mL.
Preparing a quality control plasma sample:
respectively taking the quality control working solution, diluting the quality control working solution into blank plasma, and preparing abiraterone quality control plasma sample solution with the concentration of 3750, 375, 37.5, 3 and 1 ng/mL; respectively corresponding to AQL, HOQ QC, MOQ QC, LOQ QC and LLOQ.
5. Methodology validation
5.1 Linear Range and lower quantitative limits
Taking 10 mu L of each standard series working solution, diluting the standard series working solutions into blank plasma to ensure that the total volume is 0.2mL, and preparing standard curve plasma samples with the abiraterone concentration values as follows: 1.2, 5, 10, 50, 250, 400, 500 ng/mL.
Standard curve plasma samples, Blank plasma Double Blank samples, 5. mu.L of each sample were injected into the chromatographic system for analysis.
And (4) conclusion: the linear curve of the obtained standard curve sample is shown in figure 1, wherein the standard curve equation of abiraterone in beagle plasma measured by LC-MS/MS method is as follows: 0.026236x-0.001218, R20.99956482; average determination 8 times, R2Has an SD of 0.0015 and a CV of 0.1; the slope SD was 0.0004 and CV was 1.7; the linear range of the abiraterone is 1 ng/mL-500 ng/mL, the lowest quantitative lower limit of the blood concentration of the abiraterone is 1ng/mL, and the linear relation in the linear range is good.
5.2 precision and accuracy
Taking four quality control plasma samples with different concentrations, wherein the concentration of the abiraterone quality control plasma sample liquid is 375, 37.5, 3 and 1 ng/mL; the concentrations of HOQ QC, MOQ QC, LOQ QC and LLOQ are respectively corresponded to and detected. As a review of accuracy and precision within and between batches. Each concentration was prepared in 6 replicates and tested at least three times. The mean values were used to assess batch accuracy and precision. The results are given in the following table:
table 1: LC-MS/MS method for determining precision and accuracy of abiraterone in and among batches in plasma
The results show that: the plasma sample precision and accuracy deviation of the abiraterone in batches and among batches are less than 15%. The measured values of the low, medium and high concentration standard plasma samples should be in the range of 85.0-115.0% of the standard values, and the batch-to-batch precision (RSD) is less than 15.0%. The method has good precision and accuracy in detecting the caprostatins in the plasma.
5.3 System applicability:
taking a pretreated abiraterone quality control plasma sample, wherein the concentration is 1 ng/mL; 5 mu L of sample injection detection, and repeated analysis for 8 times;
taking a Blank plasma Blank sample after pretreatment, carrying out sample injection detection by 5 mu L, and repeatedly analyzing for 8 times;
and respectively recording chromatographic peak area values and retention time of the object to be detected and the internal standard, and calculating the chromatographic peak area ratio of the object to be detected and the internal standard and the variation coefficient of the chromatographic peak retention time. The specific results are shown in table 2 below:
table 2: system applicability of abiraterone
And (4) conclusion: the signal to noise ratio (S/N is not less than 5) of the substance to be detected, and the variation coefficient of the ratio of the area of the substance to be detected to the area of the internal standard chromatographic peak obtained by 8 times of repeated analysis is less than 5%; the variation coefficient of the retention time of the sample to be tested and the internal standard chromatographic peak obtained by 8 times of repeated analysis is less than 0.5 percent. The retention time variation coefficient of the object to be measured is 0.0-1.2%, the retention time variation coefficient of the internal standard is 0.0-1.3%, and the variation coefficient of the area ratio is 0.5-5.7%. The method is applicable to the determination of abiraterone in the detection method of the medicine in the blood plasma.
5.4 Selectivity
Selectivity refers to the ability to distinguish interference in a biological matrix when an analyte is measured by chromatographic methods. Before the method is validated, at least 6 different batches of blank substrates need to be screened for method validation.
Taking blank plasma of 6 beagle dogs from different sources, pretreating the blank plasma to obtain a Carryover sample, and then injecting 5 mu L of the sample into a chromatographic system for analysis;
taking 6 parts of blank plasma from different sources, preparing abiraterone quality control plasma sample solution with the concentration of 1ng/mL, pretreating, and feeding 5 mu L of sample into a chromatographic system for analysis;
6 portions of Blank plasma from different sources were pre-treated to Blank samples and 5. mu.L of sample was injected into the chromatography system for analysis.
The results of each blank sample and the standard plasma sample at the lower limit concentration level were recorded separately as follows.
Table 3: comparison table for selective investigation of blank healthy beagle plasma from six different sources on analytes and internal standards (Abiraterone)
And (4) conclusion: the peak area of the chromatographic peak at the retention time of the analyte in at least 6 blank matrix samples from different sources is lower than 20.0% of the peak area of the to-be-measured substance at the corresponding blank matrix LLOQ concentration from different sources, and the peak area of the internal standard at the retention time of the internal standard is lower than 5.0% of the peak area of the internal standard at the corresponding blank matrix LLOQ concentration from different sources. The measured value of the LLOQ concentration prepared by 6 blank matrixes from different sources is in the range of 80.0-120.0% of the theoretical value. The method provided by the invention aims at the interference range of the analytes in the blank matrix samples from different sources to be 0-1.5%, and the internal standard interference response value is 0-0.1%. Therefore, blank blood plasma of different human bodies does not interfere the detection result of the abiraterone, the method can be used for detecting the abiraterone in the blood plasma of different human bodies, and the blood plasma of different people has no influence on the detection method.
5.5 recovery
5.5.1 extraction recovery of analytes
Analyte recovery sample
Taking abiraterone quality control plasma sample solutions with the concentrations of 375, 37.5 and 3ng/mL respectively, preparing 6 parts in parallel, adding no 100ng/mL internal standard solution, taking a supernatant solution with a proper volume after precipitation, adding 100ng/mL internal standard solution with the same volume as the supernatant, carrying out vortex mixing, and then measuring.
Recovery reference sample:
the extraction procedure was the same as for the analyte recovery samples, but 100ng/mL of internal standard solution was added after precipitation and the supernatant vortexed for assay.
5.5.2 extraction recovery of internal standard
Extraction recovery sample of internal standard
6 parts of blank plasma samples are prepared in parallel, 100ng/mL of internal standard solution is added, after precipitation, supernatant solution with proper volume is taken, and standard solution with concentration equivalent to that of MOQ QC quality control samples is added.
Recovery of internal standard reference sample:
the extraction process is the same as the extraction operation of the internal standard extraction recovery sample, but the MOQ QC quality control sample and the internal standard solution are added after precipitation.
And (4) conclusion: 1) the extraction recovery rates of the abiraterone to be detected in the high, middle and low concentration ranges are 110.3%, 107.1% and 101.5% respectively, and the corresponding coefficient of variation CV values are 0.7%, 1.7% and 1.5%; the total recovery rate is 106.3%, and the variation CV value of the total recovery rate is 3.7%. 2) The extraction recovery rate of the internal standard substance abiraterone in the method is 106.2%, and the CV value of the recovery rate is 4.1%.
5.6 matrix Effect
1) Matrix effects refer to the inhibition or enhancement of the ionization of an analyte by a component present in a biological matrix. Taking 6 blank beagle plasma of different batches respectively, preparing one portion of the blank beagle plasma in each batch, and adding a standard solution and an internal standard solution which have the same concentrations as the processed low-concentration and high-concentration quality control samples respectively for analysis after pretreatment.
The analysis was carried out in 6 replicates of pure solutions corresponding to the concentrations of the low-concentration and high-concentration quality control samples, in the absence of matrix.
2) Preparation of hemolysis matrix: taking 100 mu L of blank whole blood, carrying out ultrasonic destruction on blood cells, taking 20 mu L of blank whole blood, adding 980 mu L of normal blank plasma, and uniformly mixing to obtain 2% of hemolyzed plasma subjected to ultrasonic destruction, wherein severe hemolysis is considered.
Two samples of the analyte at two concentration levels (LOQ QC, HOQ QC) were prepared 6 times using simulated hemolyzed plasma, processed and then injected into a chromatographic system for analysis.
3) The low and high concentration level (LOQ QC, HOQ QC) samples of the test substance are prepared 6 parts each by using the blood plasma sample with high blood fat, and the processed samples are injected into a chromatographic system for analysis.
And (4) conclusion: coefficient of variation CV% of matrix factor normalized by internal standard: 2.2-3.5%; mean deviation in accuracy of the lysomatrix effect range: -7.5 to-1.6%; mean accuracy deviation range for the hyperlipidemia matrix effect: -9.0-0.6%; the method of the invention has no obvious matrix effect, and has no obvious hemolysis and hyperlipemia matrix effect.
5.7 stability
The stability of the beagle plasma quality control samples with low and high concentrations (LOQ QC and HOQ QC) of the object to be detected is investigated under the following conditions:
1) repeated freeze thawing at-70 deg.C for 3 times for stabilization.
2) The mixture is placed at room temperature for 17 hours to be stable.
3) Is stable at 31 days under the condition of minus 20 ℃.
4) Whole blood stability: and respectively inspecting the change of the ratio of peak areas of analytes containing low and high concentration levels (LOQ QC and HOQ QC) placed in ice-water bath in whole blood for (0h) and (0.5-1.5 h), after placing corresponding inspection time points, centrifugally separating the whole blood sample into plasma, adding an internal standard, and processing according to a corresponding plasma sample processing operation method, wherein each concentration level is parallel to 6 parts.
The samples after stability investigation and the samples prepared now are tested simultaneously, and the comparison result of each concentration is as follows:
table 4 stability at room temperature for 17 h-short term stability results:
table 5-20 ℃ stability at 31 d-long term stability results:
table 6-70 ℃ repeated freeze-thaw 3 times stability results:
table 7 whole blood stability results:
the CV value of the concentration of each drug in the plasma detected by the method under the investigation condition is less than 5%, which indicates that the method has stronger stability for detecting the abiraterone in the plasma under the investigation condition.
5.8 beagle plasma sample detection
50uL of a plasma sample with an Abiraterone concentration of 1ng/ml was added precisely to a 96-well plate, 20uL of IS Spike was added, 300uL of acetonitrile was added to the 96-well plate, vortex mixing was performed for 5min, centrifugation was performed at 2490g for 10min at 4 ℃, and 5uL of sample was injected to a chromatography system for LC-MS/MS analysis, the results of which are shown in FIG. 2.
And (4) conclusion: proved by methodology, the method has the advantages of high sensitivity, strong applicability, good precision and accuracy and good stability, the extraction recovery rate of the medicine is within an acceptable range, no obvious matrix effect is seen, and the method also has the advantages of high speed and high flux.
5.9 clinical applications
250mg (1 tablet) abiraterone acetate tablets were orally administered to 9 male Beagle dogs on an empty stomach at a single time. Collecting 1-2 mL of forelimb venous blood at 0h before administration and 0.25h, 0.5h, 0.75h, 1h, 1.25h, 1.5h, 1.75h, 2h, 2.5h, 3h, 3.5h, 4h and 6h after administration. The concentration of abiraterone in the plasma samples was determined using the LC-MS/MS method. Specific numerical values are shown in table 8 and fig. 5.
TABLE 8 Abiraterone major pharmacokinetic parameters after Beagle canine fasting oral administration of Abiraterone acetate tablets
| Pharmacokinetic parameters | Numerical value |
| Tmax(h) | 1.31±0.58 |
| Cmax(ng/mL) | 102.256±72.255 |
| AUC0-t(ng·h/mL) | 183.41±158.82 |
| AUC0-∞(ng·h/mL) | 200.07±172.75 |
| λz(h-1) | 0.38±0.11 |
| T1/2z(h) | 1.95±0.55 |
| AUC-％Extrap | 8.73±2.31 |
And (4) conclusion: the method can be used for quickly, stably and efficiently detecting the concentration of the abiraterone in the plasma sample, and can be applied to the research of clinical pharmacokinetics.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for determining abiraterone concentration in plasma is characterized by comprising the following steps:
1) preparation of standard series working solution: weighing abiraterone, putting the abiraterone into a volumetric flask, and dissolving the abiraterone into methanol to prepare a standard reference substance stock solution; diluting the standard reference substance stock solution with 50% acetonitrile water solution to prepare standard series abiraterone working solution;
2) preparation of standard curve plasma samples: taking standard series of abiraterone working solution, diluting the standard series of abiraterone working solution into blank plasma, and preparing an abiraterone standard curve plasma sample with the concentration of 1-500 ng/ml;
3) and (3) drawing a standard curve regression equation: adding an internal standard working solution into a standard curve plasma sample, carrying out vortex, adding an acetonitrile precipitator for precipitation, carrying out vortex and centrifugation, and then carrying out sample injection on an LC-MS/MS system to obtain a chromatogram; calculating the concentration of the abiraterone in the plasma according to the peak area of the chromatographic peak in the obtained chromatogram and the peak area of the internal standard, and making a standard curve regression equation;
wherein, the liquid phase conditions in the LC-MS/MS system are as follows:
liquid phase conditions
A chromatographic column: waters XTERRA MS C18; mobile phase A, 10mM ammonium acetate-0.1% formic acid aqueous solution and mobile phase B, 0.1% formic acid methanol solution;
conditions of Mass Spectrometry
ESI source is adopted, ionization mode: electrospray ionizing positive ions; the monitoring mode is as follows: MRM; polarity: a positive ion; fragmentation voltage: 240V, collision energy: 65V
Abiraterone-d4(ABTL-d4): the parent ion m/z is 354.2,the m/z of the daughter ion was 159.9;
abiraterone (ABTL): the parent ion m/z was 350.2 and the daughter ion m/z was 155.9.
2. The method for determining abiraterone concentration of plasma of claim 1, wherein the concentration of the standard control stock solution is 1 mg-mL-1；
The concentration range of the standard series of abiraterone working solution is 20-10000 ng/mL;
the concentration range of the abiraterone standard curve plasma sample is 1-500 ng/mL;
the plasma is from beagle dog, rabbit, mouse or human.
3. The method for determining abiraterone concentration in plasma of claim 1, wherein the standard curve regression equation is obtained by the following steps: taking 50 mu L of each standard curve plasma sample, adding 20 mu L of internal standard working solution, carrying out vortex for 30s, adding 300 mu L of acetonitrile precipitator for precipitation, carrying out vortex for 3min, centrifuging 2490g, and carrying out centrifugation at 4 ℃ for 10 min; 5 mu L of sample injection LC-MS/MS system to obtain a chromatogram; and calculating the concentration of the abiraterone in the plasma according to the area of the chromatographic peak in the obtained chromatogram, and formulating a standard curve regression equation.
4. The method for determining abiraterone concentration in plasma of claim 1, wherein the internal standard working solution is prepared by the following process: weighing Abiraterone-d4Dissolving in methanol in a volumetric flask to prepare 1 mg/mL-1Abiraterone-d of4An internal standard control stock solution;
abiraterone-d4Diluting the stock solution of the internal standard reference substance with methanol to obtain a solution with a concentration of 100ng/mL-1The internal standard working solution of (4).
5. The method for determining abiraterone concentration in plasma of claim 1, wherein the conditions of the LC-MS/MS system are as follows:
chromatographic stripA piece: a chromatographic column: waters XTERRA MS C18 (2.1X 50mm, 5 microns); mobile phase A, 10mM ammonium acetate-0.1% formic acid aqueous solution and mobile phase B, 0.1% formic acid methanol solution; flow rate: 0.5 mL/min-1(ii) a Sample introduction amount: 5 mu L of the solution; column temperature: 30 ℃; needle washing liquid: methanol; washing the needle washing liquid: 5 s; sample introduction amount: 5 mu L of the solution; column temperature: 30 ℃; stopping time: 6.00 min;
the mobile phase ratio was set as follows:
mass spectrum conditions: ESI source is adopted, ionization mode: electrospray ionizing positive ions; the monitoring mode is as follows: MRM; polarity: a positive ion; fragmentation voltage: 240V, collision energy: 65V; abiraterone-d4(ABTL-d4): the parent ion m/z is 354.2, and the daughter ion m/z is 159.9; abiraterone (ABTL): the m/z of the parent ion is 350.2, and the m/z of the daughter ion is 155.9;
the mass spectrum switching valve is set as follows:
The ion source parameters were set as follows: the temperature of the capillary blowback drying gas is 300 ℃; capillary blowback dry air flow: 10 l/min; atomizer pressure 45 psi; the temperature of sheath gas flow is 350 ℃; sheath flow rate: 11ml/min, capillary voltage: 3500V.
6. The method for determining abiraterone concentration in blood plasma of claim 1, wherein after detection by the LC-MS/MS system, abiraterone peaks at about 2.16 min;
the regression equation for the standard curve is as follows:
the regression equation of the standard curve of the measured abiraterone in the beagle plasma is that y is 0.026236x-0.001218, R20.99956482; the linear range of the abiraterone is 1 ng/mL-500 ng/mL, and the lowest quantitative lower limit of the blood concentration of the abiraterone is 1 ng/mL.
7. A method for determining abiraterone concentration in plasma is characterized by comprising the following steps:
preparing a sample solution: adding an internal standard working solution into a plasma sample to be detected, carrying out vortex, adding an acetonitrile precipitator for precipitation, and then carrying out vortex and centrifugation;
detection of sample solution: injecting the prepared sample solution into an LC-MS/MS system to obtain a chromatogram;
calculation of the concentration in the sample solution: substituting the peak area of the target drug in the plasma sample to be measured and the peak area ratio of the internal standard into the standard curve regression equation obtained by the method of any one of claims 1-6, and calculating the corresponding concentration of the measured drug.
8. The method for determining abiraterone concentration in plasma of claim 7, wherein the sample solution is formulated as follows: adding 20 μ L of internal standard working solution into 50 μ L of plasma sample to be detected, adding 300 μ L of acetonitrile precipitant, precipitating, vortexing for 3min, centrifuging 2490g, and centrifuging at 4 deg.C for 10 min.
9. The method for determining abiraterone concentration in blood plasma of claim 7, wherein the detection conditions of the LC-MS/MS system are as follows:
chromatographic conditions
A chromatographic column: waters XTERRA MS C18 (2.1X 50mm, 5 microns); mobile phase A, 10mM ammonium acetate-0.1% formic acid aqueous solution and mobile phase B, 0.1% formic acid methanol solution; flow rate: 0.5 mL/min-1(ii) a Sample introduction amount: 5 mu L of the solution; column temperature: 30 ℃; needle washing liquid: methanol; washing the needle washing liquid: 5 s; sample introduction amount: 5 mu L of the solution; column temperature: 30 ℃; stopping time: 6.00 min;
the mobile phase ratio was set as follows:
conditions of Mass Spectrometry
ESI source is adopted, ionization mode: electrospray ionizing positive ions; the monitoring mode is as follows: MRM; polarity: a positive ion; fragmentation voltage: 240V, collision energy: 65V
Abiraterone-d4(ABTL-d4): the parent ion m/z is 354.2, and the daughter ion m/z is 159.9;
abiraterone (ABTL): the parent ion m/z is 350.2, and the daughter ion m/z is 155.9
The mass spectrum switching valve is set as follows:
The ion source parameters were set as follows: the temperature of the capillary blowback drying gas is 300 ℃; capillary blowback dry air flow: 10 l/min; atomizer pressure 45 psi; the temperature of sheath gas flow is 350 ℃; sheath flow rate: 11ml/min, capillary voltage: 3500V.
10. The method for determining abiraterone concentration in plasma of claim 7, wherein the internal standard working solution is prepared by the following process: weighing Abiraterone-d4Dissolving in methanol in a volumetric flask to prepare 1 mg/mL-1Abiraterone-d of4An internal standard control stock solution;
abiraterone-d4Diluting the stock solution of the internal standard reference substance with methanol to obtain a solution with a concentration of 100ng/mL-1The internal standard working solution of (4).
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| CN114740120A (en) * | 2022-04-26 | 2022-07-12 | 武汉宏韧生物医药股份有限公司 | Method for simultaneously and quantitatively determining abiraterone and five metabolites thereof and application |
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| US20160168191A1 (en) * | 2013-07-29 | 2016-06-16 | Industriale Chimica S.R.L. | Process for the preparation of abiraterone or abiraterone acetate |
| CN107014915A (en) * | 2017-03-17 | 2017-08-04 | 中国计量大学 | The quantitative detecting method of abiraterone in whole blood |
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| US20160168191A1 (en) * | 2013-07-29 | 2016-06-16 | Industriale Chimica S.R.L. | Process for the preparation of abiraterone or abiraterone acetate |
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| CN114740120A (en) * | 2022-04-26 | 2022-07-12 | 武汉宏韧生物医药股份有限公司 | Method for simultaneously and quantitatively determining abiraterone and five metabolites thereof and application |
| CN114740120B (en) * | 2022-04-26 | 2024-02-02 | 武汉宏韧生物医药股份有限公司 | Method for simultaneously and quantitatively determining abiraterone and five metabolites thereof and application |
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