CN117825567A - HPLC-MS/MS method, kit and application for detecting Carfilzomib content in blood plasma - Google Patents
HPLC-MS/MS method, kit and application for detecting Carfilzomib content in blood plasma Download PDFInfo
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- BLMPQMFVWMYDKT-NZTKNTHTSA-N carfilzomib Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)[C@]1(C)OC1)NC(=O)CN1CCOCC1)CC1=CC=CC=C1 BLMPQMFVWMYDKT-NZTKNTHTSA-N 0.000 title claims abstract description 103
- 229960002438 carfilzomib Drugs 0.000 title claims abstract description 103
- 108010021331 carfilzomib Proteins 0.000 title claims abstract description 103
- 210000002381 plasma Anatomy 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 32
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 title claims abstract description 28
- 238000001514 detection method Methods 0.000 claims abstract description 40
- 150000002500 ions Chemical class 0.000 claims abstract description 14
- IYIKLHRQXLHMJQ-UHFFFAOYSA-N amiodarone Chemical compound CCCCC=1OC2=CC=CC=C2C=1C(=O)C1=CC(I)=C(OCCN(CC)CC)C(I)=C1 IYIKLHRQXLHMJQ-UHFFFAOYSA-N 0.000 claims abstract description 13
- 229960005260 amiodarone Drugs 0.000 claims abstract description 13
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims abstract description 10
- 238000001819 mass spectrum Methods 0.000 claims abstract description 6
- 238000012544 monitoring process Methods 0.000 claims abstract description 6
- 238000006243 chemical reaction Methods 0.000 claims abstract description 5
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 claims abstract description 5
- SVWLIIFHXFGESG-UHFFFAOYSA-N formic acid;methanol Chemical compound OC.OC=O SVWLIIFHXFGESG-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 66
- 239000000243 solution Substances 0.000 claims description 60
- 239000012086 standard solution Substances 0.000 claims description 57
- 238000003908 quality control method Methods 0.000 claims description 29
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 16
- 238000012360 testing method Methods 0.000 claims description 9
- 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 8
- 235000019253 formic acid Nutrition 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 238000002347 injection Methods 0.000 claims description 7
- 239000007924 injection Substances 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000011084 recovery Methods 0.000 claims description 5
- 239000000126 substance Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 238000013112 stability test Methods 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 3
- 238000004949 mass spectrometry Methods 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 238000011088 calibration curve Methods 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 239000011550 stock solution Substances 0.000 claims description 2
- 238000010200 validation analysis Methods 0.000 claims 2
- 239000003814 drug Substances 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract 2
- 239000000523 sample Substances 0.000 description 27
- 239000000047 product Substances 0.000 description 9
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 8
- 239000005695 Ammonium acetate Substances 0.000 description 8
- 235000019257 ammonium acetate Nutrition 0.000 description 8
- 229940043376 ammonium acetate Drugs 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- 239000012153 distilled water Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000004885 tandem mass spectrometry Methods 0.000 description 3
- 102000004245 Proteasome Endopeptidase Complex Human genes 0.000 description 2
- 108090000708 Proteasome Endopeptidase Complex Proteins 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 108020002908 Epoxide hydrolase Proteins 0.000 description 1
- 102000005486 Epoxide hydrolase Human genes 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 102000004005 Prostaglandin-endoperoxide synthases Human genes 0.000 description 1
- 108090000459 Prostaglandin-endoperoxide synthases Proteins 0.000 description 1
- 229940079156 Proteasome inhibitor Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
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- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 239000012452 mother liquor Substances 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 239000003207 proteasome inhibitor Substances 0.000 description 1
- 239000013062 quality control Sample Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000002604 ultrasonography Methods 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
-
- 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
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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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
- G01N30/86—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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- 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
- G01N2030/042—Standards
- G01N2030/045—Standards internal
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Abstract
The invention relates to an HPLC-MS/MS method for detecting the content of Carfilzomib in blood plasma, a kit and application, wherein Amiodarone is used as an internal standard to detect the content of the Carfilzomib in the blood plasma; the liquid chromatography conditions were: the chromatographic column is Ultimate XB-C18, the mobile phase A is 0.1% formic acid methanol solution, and the mobile phase B is 2mmol/L ammonium acetate-0.1% formic acid water solution; 0.0 to 0.5min, 50 percent of A phase, 0.5 to 0.6min, 50 to 85 percent of A phase, 1.4min,2.0 to 2.1min, 85 to 50 percent of A phase, 0.9min and stopping; the mass spectrum detection conditions are as follows: electrospray ion source, ion ejection voltage: 5500V, ion source temperature: 450 ℃, GS1:60psi, GS2:55psi,Curtain gas:30psi, the monitoring mode employed was multiplex reaction detection mode MRM. The invention is not interfered by common medicines and combined medicines, and has high accuracy and precision.
Description
Technical Field
The invention belongs to the technical field of Carfilzomib detection, and particularly relates to an HPLC-MS/MS method, a kit and application for detecting the content of Carfilzomib in blood plasma.
Background
Carfilzomib (Carfilzomib) is a cyclooxygenase proteasome inhibitor, which can be selectively and irreversibly combined with structural proteasome and immunoproteasome, thereby avoiding toxic and side effects caused by inhibiting the constitutive proteasome in non-malignant cells, and can be used for clinically treating multiple myeloma and reducing toxic and side effects. Carfilzomib is metabolized primarily by peptidases and epoxide hydrolases. Individual differences in Carfilzomib were large and their AUC and Cmax did not increase proportionally with increasing amounts of Carfilzomib. Therefore, the clinical pharmacokinetics of the Carfilzomib is studied, and the clinical dose of the Carfilzomib is adjusted according to the monitored concentration, so that the method has important significance in improving the effective rate of the clinical treatment of the Carfilzomib and reducing side effects.
However, at present, although some detection methods of Carfilzomib, such as liquid chromatography, exist, the detection method of Carfilzomib in the synthesis process is aimed at, the detection limit is high, the time is long, when the method is applied to the detection of the Carfilzomib with extremely low content in pharmacokinetics, on one hand, the accuracy is poor, the conventional pharmaceutical ingredients in the plasma interfere with the detection result, on the other hand, the detection time is long, the reaction of the Carfilzomib with other substances is easy to cause, and the accurate detection of the Carfilzomib is affected, so that the detection method conventionally applied to the synthesis process of the Carfilzomib is not suitable for the detection of the plasma Carfilzomib in the pharmacokinetics study, and therefore, it is very important to establish an HPLC-MS/MS method for specifically, sensitively and accurately detecting the plasma Carfilzomib.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides an HPLC-MS/MS method for detecting the Carfilzomib content in blood plasma, a kit and application thereof, wherein the method is simple in pretreatment, small in sample consumption, short in analysis time and high in sensitivity and specificity.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
an HPLC-MS/MS method for detecting the content of Carfilzomib in blood plasma adopts an HPLC-MS/MS method, and uses Amiodarone as an internal standard to detect the content of the Carfilzomib in the blood plasma; the internal standard liquid is as follows: 1000ng/mL of Amiodarone methanol standard solution;
the liquid chromatography conditions for HPLC-MS/MS detection are: the chromatographic column is Ultimate XB-C18, with specification of 4.6mm×50mm, and particle diameter of 5 μm; mobile phase: mobile phase A is 0.1% formic acid methanol solution with volume concentration, mobile phase B is 2mmol/L ammonium acetate-0.1% formic acid water solution with volume concentration; gradient elution was used, and specific gradient elution conditions are as follows:
| Time | mobile phase a | Mobile phase B |
| 0.01min | 50％ | 50％ |
| 0.5min | 50％ | 50％ |
| 0.6min | 85％ | 15％ |
| 2.0min | 85％ | 15％ |
| 2.1min | 50％ | 50％ |
| 3.0min | 50％ | 50％ |
Column temperature: 60 ℃; flow rate: 0.8mL/min; sample injection volume: 1 μl;
the mass spectrum detection conditions of the HPLC-MS/MS detection are as follows: electrospray ion source, ion ejection voltage: 5500V, ion source temperature: 450 ℃, GS1:60psi, GS2:55psi,Curtain gas:30psi, the monitoring mode employed was multiplex reaction detection mode MRM.
As a further technical scheme, the mass spectrum conditions of the MRM are as follows;
as a further technical scheme, the plasma pretreatment method also comprises the pretreatment of plasma samples;
pretreatment of the plasma sample, comprising: adding the internal standard solution and the sample treatment solution into the plasma to be detected according to the volume ratio of the plasma, the internal standard solution and the sample treatment solution of 1:3:1, oscillating and mixing uniformly, centrifuging, and reserving supernatant for later use.
As a further technical scheme, the method further comprises the step of drawing a standard curve;
the drawing of the standard curve comprises the following steps: and (3) respectively adding the Carfilzomib standard solution with different concentration gradients into the internal standard solution and the sample treatment solution, then carrying out sample injection detection, and drawing a calibration curve by taking the concentration of the Carfilzomib standard solution as an abscissa and the ratio of the measured peak area of the Carfilzomib standard solution to the peak area of the internal standard solution as an ordinate.
As a further technical scheme, the sample treatment solution is as follows: 2mmol/L ammonium acetate-0.1% strength by volume aqueous formic acid solution.
As a further technical scheme, the preparation method of the Carfilzomib standard solution with different concentration gradients comprises the following steps: stock solutions of the standard were prepared with methanol and then diluted with methanol to 7 concentration gradients of 0ng/mL, 5000ng/mL, 10000ng/mL, 15000ng/mL, 25000ng/mL, 50000ng/mL, 75000ng/mL, respectively.
As a further technical scheme, the method also comprises a quality control product, wherein the quality control product is used for a verification test of a detection method, and the verification test of the detection method comprises a precision test, a recovery rate test and a stability test.
As a further technical scheme, the quality control product comprises a single level of low, medium and high concentration Carfilzomib quality control solution;
the low concentration level quality control solution adopts 7500ng/mL of Carfilzomib standard solution;
the medium concentration level quality control solution adopts a standard solution of Carfilzomib with the concentration of 15000 ng/mL;
the high concentration level quality control solution was 60000ng/mL of Carfilzomib standard solution.
A kit for use in the HPLC-MS/MS method for detecting the Carfilzomib content in blood plasma, comprising a quality control material, a calibrator, an internal standard solution, a sample processing solution, and a mobile phase, wherein the calibrator comprises 0ng/mL, 5000ng/mL, 10000ng/mL, 15000ng/mL, 25000ng/mL, 50000ng/mL, 75000ng/mL of a Carfilzomib standard solution.
Use of said kit in plasma Carfilzomib detection.
Compared with the prior art, the invention has the beneficial effects that:
according to the method, amiodarone is used as an internal standard, and the detection limit and the quantitative limit of Amiodarone in blood plasma are greatly reduced by optimizing the detection conditions of HPLC-MS/MS, so that the detection time is shortened, the interference of common medicaments and combined medicaments is avoided, the accuracy and the precision of Carfilzomib detection are improved, and finally, the method for accurately, simply, sensitively and selectively measuring the concentration of Carfilzomib in the blood plasma is obtained; in addition, the invention has the characteristics of simple pretreatment method of the plasma sample, small plasma sample amount and the like, and can be used for monitoring the clinical blood concentration of the Carfilzomib and analyzing and researching the pharmacokinetics.
Drawings
FIG. 1 is a graph of the two-level scanned fragment ions of Carfilzomib in the present invention;
FIG. 2 is a graph of the secondary scanned fragment ions of Amiodarone in accordance with the present invention;
FIG. 3 is a graph of a Carfilzomib chromatogram of the present invention;
Detailed Description
The technical solutions of the present invention will be clearly and fully described below with reference to specific embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A kit for an HPLC-MS/MS method for detecting the content of Carfilzomib in blood plasma, which comprises a calibrator, a quality control product, a sample treatment fluid, an internal standard fluid and a mobile phase;
1) The calibrator was 7 different concentration levels of Carfilzomib standard solutions;
concentration level 6:75000ng/mL of Carfilzomib standard solution;
concentration level 5:50000ng/mL of Carfilzomib standard solution;
concentration level 4:25000ng/mL of Carfilzomib standard solution;
concentration level 3:15000ng/mL of Carfilzomib standard solution;
concentration level 2:10000ng/mL of Carfilzomib standard solution;
concentration level 1:5000ng/mL of Carfilzomib standard solution;
concentration level 0: methanol.
Taking 5mg of Carfilzomib standard substance, dissolving the standard substance with methanol, fixing the volume to a 10mL volumetric flask, carrying out ultrasonic mixing uniformly to obtain 500 mug/mL of Carfilzomib mother liquor, and sub-packaging for later use.
Concentration level 6: 150 mu L of methanol is added from 500 mu g/mL of Carfilzomib standard solution, and the mixture is mixed with shaking to obtain 75000ng/mL of Carfilzomib standard solution;
concentration level 5: taking 100 mu L from 500 mu g/mL of Carfilzomib standard solution, adding 900 mu L of methanol, and shaking and mixing uniformly to obtain 50000ng/mL of Carfilzomib standard solution;
concentration level 4: 50 mu L of methanol is added from 500 mu g/mL of Carfilzomib standard solution, and the mixture is mixed with shaking to obtain 25000ng/mL of Carfilzomib standard solution;
concentration level 3: 200 mu L of solution with the concentration level of 6 is taken, 800 mu L of methanol is added, and the mixture is uniformly mixed by shaking, thus obtaining 15000ng/mL of Carfilzomib standard solution;
concentration level 2: 200 mu L of solution with the concentration level of 5 is taken, 800 mu L of methanol is added, and the solution is mixed uniformly by shaking, thus obtaining 10000ng/mL of Carfilzomib standard solution;
concentration level 1: taking 100 mu L of solution with the concentration level of 5, adding 900 mu L of methanol, and shaking and uniformly mixing to obtain 5000ng/mL of Carfilzomib standard solution;
concentration level 0: methanol.
2) The quality control product is a Carfilzomib quality control solution with low, medium and high concentration levels;
low concentration level of Carfilzomib quality control solution: 7500ng/mL of Carfilzomib standard solution; taking 100 mu L of the standard solution of the Carfilzomib from 75000ng/mL, adding 900 mu L of methanol, and shaking and mixing uniformly to obtain the standard solution of the Carfilzomib from 7500 ng/mL;
medium concentration level of Carfilzomib quality control solution: 15000ng/mL of Carfilzomib standard solution; 200 mu L of methanol is added from 75000ng/mL of Carfilzomib standard solution, and the mixture is mixed with shaking to obtain 15000ng/mL of Carfilzomib standard solution;
high concentration level of Carfilzomib quality control solution: 60000ng/mL of Carfilzomib standard solution; 120 mu L of methanol is added from 500 mu g/mL of Carfilzomib standard solution, and the mixture is mixed with shaking to obtain 60000ng/mL of Carfilzomib standard solution.
3) The sample treatment solution adopts 2mmol/L ammonium acetate-0.1% formic acid aqueous solution with volume concentration, and the preparation method comprises the following steps:
step 1: weighing 7.7g of ammonium acetate, dissolving the ammonium acetate in distilled water, fixing the volume to a 100mL volumetric flask, and uniformly mixing to obtain 100mmol/L ammonium acetate solution;
step 2: taking 1mL of formic acid solution, 2mL of 100mmol/L ammonium acetate solution, using distilled water to fix the volume to a 1000mL volumetric flask, and uniformly mixing to obtain 2mmol/L ammonium acetate-0.1% formic acid aqueous solution.
4) The internal standard solution adopts an Amiodarone solution with the concentration of 1000ng/mL, and the preparation method comprises the following steps:
dissolving 10.2mg of Amiodarone standard substance with methanol, fixing the volume with methanol to a 10mL volumetric flask to obtain 1mg/mL Amiodarone solution, mixing with ultrasound, and packaging;
mu.L of 1mg/mL of Amiodarone solution is taken, the volume is fixed to a 250mL volumetric flask by methanol, and the mixture is evenly mixed by ultrasonic, so as to obtain 1000ng/mL of Amiodarone solution.
5) Mobile phase includes mobile phase a and mobile phase B; mobile phase a was 0.1% strength by volume methanolic formate solution and mobile phase B was 2mmol/L ammonium acetate-0.1% strength by volume aqueous formic acid solution.
Wherein, mobile phase A is prepared by: 1mL of formic acid solution is taken, methanol is used for constant volume to 1000mL, and the mixture is uniformly mixed to obtain the formic acid methanol solution with the volume concentration of 0.1 percent.
Preparation of mobile phase B: weighing 7.7g of ammonium acetate, dissolving the ammonium acetate in distilled water, fixing the volume to 100mL, and uniformly mixing to obtain 100mmol/L ammonium acetate solution;
1mL of formic acid solution, 2mL of 100mmol. L was taken -1 And (3) the ammonium acetate solution is fixed to 1000mL by distilled water, and is uniformly mixed to obtain 2mmol/L ammonium acetate-0.1% formic acid water solution by volume.
Example 2
An HPLC-MS/MS method for detecting the level of Carfilzomib in plasma comprising:
1) Preparation of calibrator solution, quality control solution, plasma sample:
taking 10 mu L of each solution in the calibrator and the quality control product in the kit of the embodiment 1, respectively adding 90 mu L of blank plasma, 100 mu L of sample treatment solution and 300 mu L of internal standard solution, vibrating and mixing uniformly, centrifuging, and taking supernatant to obtain calibrator solutions and quality control product solutions with different concentrations for later use;
taking 100 mu L of plasma to be measured, adding 100 mu L of sample treatment liquid and 300 mu L of internal standard liquid, vibrating and mixing uniformly, centrifuging, and taking supernatant to obtain a plasma sample to be measured for later use.
2) The detection conditions of the high performance liquid chromatography tandem mass spectrometry are as follows:
the liquid chromatography conditions for HPLC-MS/MS detection are: the chromatographic column is Ultimate XB-C18, with specification of 4.6mm×50mm, and particle diameter of 5 μm; mobile phase: mobile phase A is 0.1% formic acid methanol solution with volume concentration, mobile phase B is 2mmol/L ammonium acetate-0.1% formic acid water solution with volume concentration; gradient elution is adopted, and specific gradient elution conditions are shown in table 1;
TABLE 1
| Time | Mobile phase a | Mobile phase B |
| 0.01min | 50％ | 50％ |
| 0.5min | 50％ | 50％ |
| 0.6min | 85％ | 15％ |
| 2.0min | 85％ | 15％ |
| 2.1min | 50％ | 50％ |
| 3.0min | 50％ | 50％ |
Column temperature: 60 ℃; flow rate: 0.8mL/min; sample injection volume: 1 μl;
the mass spectrum detection conditions of the HPLC-MS/MS detection are as follows: electrospray ion source, ion ejection voltage: 5500V, ion source temperature: 450 ℃, GS1:60psi, GS2:55psi,Curtain gas:30psi, the monitoring mode employed was multiplex reaction detection mode MRM.
The mass spectrometry conditions of the MRM are shown in Table 2;
table 2 mass spectrometry conditions
3) Drawing a standard curve
Detecting different concentration gradient calibrator solutions, using a mass spectrum internal calibration method, taking the concentration of a Carfilzomib standard cone as an abscissa, taking the ratio of the measured peak area of the Carfilzomib standard to the peak area of an internal standard as an ordinate, establishing a standard curve, and calculating the concentration of Carfilzomib in a sample to be detected according to the standard curve;
drawing a standard curve: and (3) taking 6 standard solutions with the concentration of Carfilzomib as an abscissa (X), taking the ratio of the measured peak area of the 6 standard solutions with the concentration of Carfilzomib to the peak area of each internal standard as an ordinate (Y), carrying out linear fitting on X-line weighted square regression by Y, and drawing a standard curve to obtain a linear regression equation of Y=0.00677X+0.507, wherein r=0.9989.
4) And before detecting the sample every day, firstly adopting a quality control solution to verify the method, then detecting the plasma sample to be detected, and obtaining the content of Carfilzomib in the plasma sample by using a standard curve.
Example 3: and (3) verifying a detection method:
taking 6 parts of Carfilzomib quality control solution with each concentration in the quality control product of example 1, taking 10 mu L of each part, respectively adding 90 mu L of blank plasma, 100 mu L of sample treatment liquid and 300 mu L of internal standard liquid, shaking, mixing uniformly, centrifuging, and taking supernatant for later use; and then carrying out sample injection analysis according to the detection conditions of the high performance liquid chromatography tandem mass spectrometry in the embodiment 2, and obtaining the measured concentration of the quality control sample according to the standard curve of the same day, and continuously measuring for 3 days. Based on the measurement results, RSD was calculated on the day and day, and the ratio of the measured concentration to the standard concentration was used as the relative recovery rate, and the results are shown in table 3.
Table 3Carfilzomib precision and recovery assay (n=6,)
2. stability test
Taking 6 parts of Carfilzomib quality control solution with each concentration in the quality control product of example 1, taking 10 mu L of each part, respectively adding 90 mu L of blank plasma, 100 mu L of sample treatment liquid and 300 mu L of internal standard liquid, respectively refrigerating and standing at 4 ℃, -20 ℃ for 72 hours and standing at room temperature for 72 hours, respectively, carrying out sample injection analysis according to the detection conditions of high performance liquid chromatography tandem mass spectrometry in example 2, respectively measuring the concentration of each sample, examining the stability, and comparing the measured concentration with the standard concentration, wherein the result is shown in Table 4;
table 4: carfilzomib stability test (n=6,)
in conclusion, the test shows that the kit has the advantages of simple operation, accurate result, good precision, relative recovery rate up to 85% -115%, good accuracy, RSD lower than 10%, and precision and stability meeting the monitoring requirements of clinical batch samples.
The above-described embodiments are merely preferred examples of the present invention and are not intended to be examples of possible implementations of the invention. Any obvious modifications thereof, which would be apparent to those skilled in the art without departing from the principles and spirit of the present invention, should be considered to be included within the scope of the appended claims.
Claims (10)
1. An HPLC-MS/MS method for detecting the content of Carfilzomib in blood plasma is characterized in that the content of the Carfilzomib in the blood plasma is detected by adopting an HPLC-MS/MS method and taking Amiodarone as an internal standard; the internal standard liquid is as follows: 1000ng/mL of Amiodarone methanol standard solution;
the liquid chromatography conditions for HPLC-MS/MS detection are: the chromatographic column is Ultimate XB-C18, with specification of 4.6mm×50mm, and particle diameter of 5 μm; mobile phase: mobile phase A is 0.1% formic acid methanol solution with volume concentration, mobile phase B is 2mmol/L ammonium acetate-0.1% formic acid water solution with volume concentration; gradient elution was used, and specific gradient elution conditions are as follows:
Column temperature: 60 ℃; flow rate: 0.8mL/min; sample injection volume: 1 μl;
the mass spectrum detection conditions of the HPLC-MS/MS detection are as follows: electrospray ion source, ion ejection voltage: 5500V, ion source temperature: 450 ℃, GS1:60psi, GS2:55psi,Curtain gas:30psi, the monitoring mode employed was multiplex reaction detection mode MRM.
2. An HPLC-MS/MS method for detecting the level of Carfilzomib in blood plasma according to claim 1,
the mass spectrometry conditions of the MRM are shown in the following table;
3. an HPLC-MS/MS method for detecting the level of Carfilzomib in plasma according to claim 1, further comprising a pretreatment of the plasma sample;
pretreatment of the plasma sample, comprising: adding the internal standard solution and the sample treatment solution into the plasma to be detected according to the volume ratio of the plasma, the internal standard solution and the sample treatment solution of 1:3:1, oscillating and mixing uniformly, centrifuging, and reserving supernatant for later use.
4. A HPLC-MS/MS method for detecting Carfilzomib content in plasma according to claim 3, further comprising plotting a standard curve;
the drawing of the standard curve comprises the following steps: and (3) respectively adding the Carfilzomib standard solution with different concentration gradients into the internal standard solution and the sample treatment solution, then carrying out sample injection detection, and drawing a calibration curve by taking the concentration of the Carfilzomib standard solution as an abscissa and the ratio of the measured peak area of the Carfilzomib standard solution to the peak area of the internal standard solution as an ordinate.
5. An HPLC-MS/MS method for detecting Carfilzomib content in plasma according to claim 4, wherein the sample treatment fluid is: 2mmol/L ammonium acetate-0.1% strength by volume aqueous formic acid solution.
6. The method for detecting the concentration of Carfilzomib in blood plasma by HPLC-MS/MS according to claim 4, wherein the preparation method of the standard solution of the Carfilzomib with different concentration gradients is as follows: stock solutions of the standard were prepared with methanol and then diluted with methanol to 7 concentration gradients of 0ng/mL, 5000ng/mL, 10000ng/mL, 15000ng/mL, 25000ng/mL, 50000ng/mL, 75000ng/mL, respectively.
7. An HPLC-MS/MS method for detecting the level of Carfilzomib in plasma according to claim 1, further comprising a quality control for use in a validation test of the detection method, the validation test of the detection method comprising a precision test, a recovery test and a stability test.
8. An HPLC-MS/MS method for detecting the level of Carfilzomib in plasma according to claim 7, wherein the quality control comprises a single level of low, medium, high concentration of Carfilzomib quality control solution;
the low concentration level quality control solution adopts 7500ng/mL of Carfilzomib standard solution;
the medium concentration level quality control solution adopts a standard solution of Carfilzomib with the concentration of 15000 ng/mL;
the high concentration level quality control solution was 60000ng/mL of Carfilzomib standard solution.
9. A kit for use in an HPLC-MS/MS method for detecting the level of Carfilzomib in plasma according to any one of claims 1 to 8, comprising a quality control substance, a calibrator, an internal standard solution, a sample processing solution and a mobile phase, wherein the calibrator comprises a standard solution of Carfilzomib at 0ng/mL, 5000ng/mL, 10000ng/mL, 15000ng/mL, 25000ng/mL, 50000ng/mL, 75000ng/mL.
10. Use of a kit according to claim 9 in plasma Carfilzomib detection.
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