CN117288860A - Analysis method of genotoxic impurities in flurbiprofen - Google Patents
Analysis method of genotoxic impurities in flurbiprofen Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 57
- SYTBZMRGLBWNTM-UHFFFAOYSA-N flurbiprofen Chemical compound FC1=CC(C(C(O)=O)C)=CC=C1C1=CC=CC=C1 SYTBZMRGLBWNTM-UHFFFAOYSA-N 0.000 title claims abstract description 40
- 229960002390 flurbiprofen Drugs 0.000 title claims abstract description 39
- 230000001738 genotoxic effect Effects 0.000 title claims abstract description 28
- 231100000024 genotoxic Toxicity 0.000 title claims abstract description 26
- 238000004458 analytical method Methods 0.000 title claims abstract description 15
- LQNUZADURLCDLV-UHFFFAOYSA-N nitrobenzene Chemical compound [O-][N+](=O)C1=CC=CC=C1 LQNUZADURLCDLV-UHFFFAOYSA-N 0.000 claims abstract description 24
- MVPPADPHJFYWMZ-UHFFFAOYSA-N chlorobenzene Chemical compound ClC1=CC=CC=C1 MVPPADPHJFYWMZ-UHFFFAOYSA-N 0.000 claims abstract description 23
- 238000001514 detection method Methods 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 20
- 239000012488 sample solution Substances 0.000 claims abstract description 12
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 claims abstract description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 75
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 60
- 239000000243 solution Substances 0.000 claims description 45
- 239000000523 sample Substances 0.000 claims description 12
- 239000007789 gas Substances 0.000 claims description 11
- 150000002500 ions Chemical class 0.000 claims description 11
- 238000012360 testing method Methods 0.000 claims description 11
- VITSNECNFNNVQB-UHFFFAOYSA-N 1,3-dichloro-2-nitrobenzene Chemical compound [O-][N+](=O)C1=C(Cl)C=CC=C1Cl VITSNECNFNNVQB-UHFFFAOYSA-N 0.000 claims description 10
- WFQDTOYDVUWQMS-UHFFFAOYSA-N 1-fluoro-4-nitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(F)C=C1 WFQDTOYDVUWQMS-UHFFFAOYSA-N 0.000 claims description 10
- QUIMTLZDMCNYGY-UHFFFAOYSA-N 2,4-dichloro-1-nitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(Cl)C=C1Cl QUIMTLZDMCNYGY-UHFFFAOYSA-N 0.000 claims description 10
- RJXOVESYJFXCGI-UHFFFAOYSA-N 2,4-difluoro-1-nitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(F)C=C1F RJXOVESYJFXCGI-UHFFFAOYSA-N 0.000 claims description 10
- RUBQQRMAWLSCCJ-UHFFFAOYSA-N 1,2-difluoro-4-nitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(F)C(F)=C1 RUBQQRMAWLSCCJ-UHFFFAOYSA-N 0.000 claims description 9
- SSNCMIDZGFCTST-UHFFFAOYSA-N 1,3-difluoro-2-nitrobenzene Chemical compound [O-][N+](=O)C1=C(F)C=CC=C1F SSNCMIDZGFCTST-UHFFFAOYSA-N 0.000 claims description 9
- BFCFYVKQTRLZHA-UHFFFAOYSA-N 1-chloro-2-nitrobenzene Chemical compound [O-][N+](=O)C1=CC=CC=C1Cl BFCFYVKQTRLZHA-UHFFFAOYSA-N 0.000 claims description 9
- PWKNBLFSJAVFAB-UHFFFAOYSA-N 1-fluoro-2-nitrobenzene Chemical compound [O-][N+](=O)C1=CC=CC=C1F PWKNBLFSJAVFAB-UHFFFAOYSA-N 0.000 claims description 9
- CZGCEKJOLUNIFY-UHFFFAOYSA-N 4-Chloronitrobenzene Chemical compound [O-][N+](=O)C1=CC=C(Cl)C=C1 CZGCEKJOLUNIFY-UHFFFAOYSA-N 0.000 claims description 9
- 238000002347 injection Methods 0.000 claims description 7
- 239000007924 injection Substances 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000002604 ultrasonography Methods 0.000 claims description 5
- 238000004817 gas chromatography Methods 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 238000001819 mass spectrum Methods 0.000 claims description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 2
- ALIVXCSEERJYHU-UHFFFAOYSA-N Flurbiprofen axetil Chemical compound FC1=CC(C(C)C(=O)OC(OC(C)=O)C)=CC=C1C1=CC=CC=C1 ALIVXCSEERJYHU-UHFFFAOYSA-N 0.000 claims description 2
- 239000012159 carrier gas Substances 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 claims description 2
- 229910001873 dinitrogen Inorganic materials 0.000 claims description 2
- 229950005941 flurbiprofen axetil Drugs 0.000 claims description 2
- 239000001307 helium Substances 0.000 claims description 2
- 229910052734 helium Inorganic materials 0.000 claims description 2
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 claims description 2
- 239000010453 quartz Substances 0.000 claims description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 9
- 229940079593 drug Drugs 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000000926 separation method Methods 0.000 abstract description 3
- 238000011161 development Methods 0.000 abstract description 2
- 239000011550 stock solution Substances 0.000 description 20
- 238000007865 diluting Methods 0.000 description 12
- 238000004090 dissolution Methods 0.000 description 11
- 239000013558 reference substance Substances 0.000 description 9
- 238000010790 dilution Methods 0.000 description 8
- 239000012895 dilution Substances 0.000 description 8
- 239000002994 raw material Substances 0.000 description 6
- 238000009210 therapy by ultrasound Methods 0.000 description 6
- 239000012490 blank solution Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000012085 test solution Substances 0.000 description 5
- 238000011084 recovery Methods 0.000 description 4
- 239000012088 reference solution Substances 0.000 description 3
- 208000002193 Pain Diseases 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 231100000025 genetic toxicology Toxicity 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 231100000219 mutagenic Toxicity 0.000 description 2
- 230000003505 mutagenic effect Effects 0.000 description 2
- 230000036407 pain Effects 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 206010002556 Ankylosing Spondylitis Diseases 0.000 description 1
- 229940089918 ansaid Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 238000012362 drug development process Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000000041 non-steroidal anti-inflammatory agent Substances 0.000 description 1
- 230000002352 nonmutagenic effect Effects 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000003637 steroidlike Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 230000000472 traumatic effect Effects 0.000 description 1
Classifications
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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
-
- 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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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention provides an analysis method of genotoxic impurities in flurbiprofen, which adopts a GC-MS/MS method to measure a sample solution; the analysis method can analyze a plurality of genotoxic impurities in the flurbiprofen bulk drug, so that the requirement on impurity separation degree is improved, a plurality of methods are not required to be developed to detect different impurities respectively, and the detection and method development cost is reduced; meanwhile, the analysis method greatly improves the detection sensitivity of impurities in the flurbiprofen, and can qualitatively and quantitatively detect nitrobenzene and chlorobenzene genotoxic impurities in the flurbiprofen at a trace level.
Description
Technical Field
The invention belongs to the technical field of medicine detection and analysis, and particularly relates to an analysis method of genotoxic impurities in flurbiprofen.
Background
Flurbiprofen is one of the excellent varieties in non-steroidal anti-inflammatory analgesics, and is mainly used for treating rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, traumatic pain and other pains, and the chemical structural formula of the flurbiprofen is as follows:
。
impurity studies are an important part of the drug development process, and are directly related to the safety and effectiveness of drugs. Genotoxic impurities (genotoxic impurities, GTIs) refer to impurities that can cause genotoxicity, including mutagenic impurities and other non-mutagenic impurities. It is mainly derived from the production process of crude drugs or preparations, such as starting materials, reactants, catalysts, reagents, solvents, intermediates, byproducts, degradation products and the like. According to ICH M7 guidelines, 11 nitrobenzene and 1 chlorobenzene impurities in flurbiprofen all contain genotoxicity warning structures, are introduced by starting materials, are irrelevant to the structures of raw materials, have no mutagenic data and belong to class 3, so that the acceptable limit of the flurbiprofen is set by a TTC (toxicology concern threshold) algorithm. With reference to FDA specifications of flurbiprofen axetil (trade name: ansaid), the daily maximum intake of the present preparation was 300mg, and the maximum intake of individual impurities per day was 1.5. Mu.g, giving an impurity limit of 5ppm. Because of the low impurity limit, the detection instruments such as common gas phase and high performance liquid chromatograph cannot meet the detection sensitivity requirement.
Disclosure of Invention
The invention aims to provide a method for detecting genotoxic impurities in flurbiprofen by a GC-MS/MS method, which can quantitatively detect nitrobenzene and chlorobenzene genotoxic impurities in flurbiprofen at a trace level.
According to one aspect of the invention, there is provided a method for analyzing genotoxic impurities in flurbiprofen, wherein a GC-MS/MS method is used for determining a sample solution;
the preparation method of the test sample solution comprises the following steps: and (3) treating flurbiprofen with sodium hydroxide solution, performing ultrasound, and fixing the volume with acetonitrile.
Further, the gas chromatography conditions were:
gas chromatographic column: VF-1701ms;
heating program: the initial temperature is 75-85 ℃, the temperature is maintained for 1 minute, the temperature is raised to 170 ℃ at the rate of 5 ℃ per minute, and then the temperature is raised to 250 ℃ at the rate of 10 ℃ per minute, and the temperature is maintained for 10 minutes;
sample inlet temperature: 245-255 ℃;
column flow rate: 0.9-1.1 ml/min; split sample injection, split ratio: 10:1; sample injection amount: 1 μl.
Further, the mass spectrometry conditions were:
EI source temperature: 230 ℃;
electron energy: 70eV;
solvent delay time: 3min;
scanning mode: multi-reactive ion detection (MRM).
Further, the genotoxic impurities include 3, 4-difluoronitrobenzene, 2, 6-difluoronitrobenzene, p-fluoronitrobenzene, 2, 4-difluoronitrobenzene, nitrobenzene, 1-fluoro-2-nitrobenzene, p-nitrochlorobenzene, o-nitrochlorobenzene, 2, 6-dichloronitrobenzene, 2, 4-dichloronitrobenzene, chlorobenzene.
Further, the qualitative and quantitative ion pairs and collision energies used for the multi-reaction ion detection are as follows:
。
further, in the preparation of the sample solution, the concentration of the sodium hydroxide solution is 5-10 mg/ml.
The invention has the beneficial effects that:
according to the analysis method of the flurbiprofen genotoxic impurities, the GC-MS/MS method is adopted to determine, and isomer impurities (such as 3, 4-difluoronitrobenzene, 2, 6-difluoronitrobenzene and 2, 4-difluoronitrobenzene, p-fluoronitrobenzene and 1-fluoro-2-nitrobenzene, p-nitrochlorobenzene and o-nitrochlorobenzene, 2, 6-dichloronitrobenzene and 2, 4-dichloronitrobenzene) can be separated through a gas phase module, and the impurities can be distinguished according to different gas phase peak positions; impurities with poor gas phase separation degree or consistent peak positions (such as impurities of 2, 4-difluoronitrobenzene and 1-fluoro-4-nitrobenzene) can be distinguished by different mass spectrum molecular weights; therefore, the analysis method greatly improves the requirement of impurity separation degree, can detect a plurality of impurities in the same method, does not need to develop a plurality of methods to detect different impurities respectively, and reduces the detection and method development cost.
The minimum detection limit of the analysis method of the flurbiprofen genotoxic impurities can reach about 0.04ng/ml, the detection sensitivity of the impurities in the flurbiprofen is greatly improved, nitrobenzene and chlorobenzene genotoxic impurities in the flurbiprofen can be qualitatively and quantitatively detected on a trace level, and each detection index accords with the regulation through methodological verification, the detection result is accurate and reliable, and the assurance is provided for monitoring the quality stability and clinical medication safety of the flurbiprofen.
Drawings
FIG. 1 is a chromatogram of a hollow white solution of example 1.
FIG. 2 is a chromatogram of the control solution of example 1; in the figure, 1, chlorobenzene; 2. 3, 4-difluoronitrobenzene; 3. 2, 6-difluoronitrobenzene; 4. 1-fluoro-4-nitrobenzene; 5.2, 4-difluoronitrobenzene; 6. nitrobenzene; 7. 1-fluoro-2-nitrobenzene; 8. p-nitrochlorobenzene; 9. o-chloronitrobenzene; 10. 2, 6-dichloronitrobenzene; 11. 2, 4-dichloronitrobenzene.
FIG. 3 is a chromatogram of a flurbiprofen (homemade) test solution in example 1.
FIG. 4 is a chromatogram of the sample solution with impurities in example 1; in the figure, 1, chlorobenzene; 2. 3, 4-difluoronitrobenzene; 3. 2, 6-difluoronitrobenzene; 4. 1-fluoro-4-nitrobenzene; 5.2, 4-difluoronitrobenzene; 6. nitrobenzene; 7. 1-fluoro-2-nitrobenzene; 8. p-nitrochlorobenzene; 9. o-chloronitrobenzene; 10. 2, 6-dichloronitrobenzene; 11. 2, 4-dichloronitrobenzene.
FIG. 5 is a chromatogram of the control solution of comparative example 1.
Detailed Description
The present application is described in further detail below by way of specific examples. The following examples are merely illustrative of the present application and should not be construed as limiting the present application.
The reagents and raw materials used in the invention are all commercially available.
Example 1
The instrument adopts an Agilent 8890-7010B GC/QT gas chromatograph-tandem mass spectrometer.
The gas chromatography conditions were as follows:
gas chromatographic column: VF-1701ms (30 m. Times.0.25 mm. Times.0.25 μm) quartz capillary column;
gas chromatography measurement conditions:
carrier gas: helium gas; flow rate: 1.0ml/min; collision gas: nitrogen gas.
The column temperature adopts a temperature rising program: the initial temperature is 80+/-5 ℃, the temperature is maintained for 1 minute, the temperature is increased to 170 ℃ at the rate of 5 ℃ per minute, and then the temperature is increased to 250 ℃ at the rate of 10 ℃ per minute, and the temperature is maintained for 10 minutes;
sample inlet temperature: 245+/-5 ℃; column flow rate: 1.0+/-0.1 ml/min; split sample injection, split ratio: 10:1; sample injection amount: 1 μl.
The mass spectrometry conditions were as follows:
ionization mode: an EI ion source; EI source temperature: 230 ℃; electron energy: 70eV; solvent delay time: 3min.
Scanning mode: multi-reactive ion detection (MRM), the qualitative and quantitative ion pairs and collision energies employed are as follows:
。
the detection steps are as follows:
sodium hydroxide solution (8 mg/ml): proper amount of sodium hydroxide is taken, precisely weighed, dissolved in water and diluted to prepare a solution containing about 8mg of sodium hydroxide per 1 ml.
Blank solution: precisely weighing 2ml of sodium hydroxide solution (8 mg/ml), placing into a 10ml measuring flask, adding acetonitrile for dissolving and diluting to a scale, shaking uniformly, and performing ultrasonic treatment for 30min.
Reference stock solution 1#: about 25mg of each of chlorobenzene, 3, 4-difluoronitrobenzene, 2, 6-difluoronitrobenzene, p-fluoronitrobenzene, 2, 4-difluoronitrobenzene, nitrobenzene, 1-fluoro-2-nitrobenzene, p-nitrochlorobenzene, o-nitrochlorobenzene, 2, 6-dichloronitrobenzene and 2, 4-dichloronitrobenzene reference substances are precisely weighed, a 50ml measuring flask is placed, acetonitrile is added for dissolution and dilution to scale, shaking is carried out, 1ml of the solution is measured, a 100ml measuring flask is placed, acetonitrile is added for dissolution and dilution to scale, and shaking is carried out.
Reference stock solution # 2: precisely measuring the reference stock solution 1#5ml, placing into a 50ml measuring flask, adding acetonitrile for dissolution, diluting to scale, and shaking uniformly.
A control solution; precisely measure 2#1ml of control stock solution, place in a 10ml measuring flask, precisely add 2ml of sodium hydroxide solution (8 mg/ml), sonicate for 30min, dissolve and dilute to scale with acetonitrile, shake well (about 50 ng/ml).
Test solution: about 100mg of flurbiprofen (self-made) is taken, precisely weighed, placed in a 10ml measuring flask, 2ml of sodium hydroxide solution (8 mg/ml) is precisely added, the solution is subjected to ultrasonic treatment for 30min, acetonitrile is added for dissolution, dilution is carried out to a scale, and shaking is carried out uniformly.
Impurity-added test solution: about 100mg of flurbiprofen (self-made) is taken, precisely weighed, placed in a 10ml measuring flask, 1.0ml of reference stock solution 2#is precisely added, 2ml of sodium hydroxide solution (8 mg/ml) is added, ultrasound is carried out for 30min, acetonitrile is added for dissolution and dilution to scale, and shaking is carried out uniformly.
Taking the blank solution, the reference solution, the sample solution and the impurity-added sample solution respectively, measuring according to the chromatographic conditions, recording chromatograms, and respectively showing detection results in figures 1, 2, 3 and 4.
Impurity content calculation: and calculating the content of genotoxic impurities in flurbiprofen by adopting an external standard method.
The analytical method of example 1 was subjected to methodological verification and the results are as follows.
Specialization of
Preparing each solution:
sodium hydroxide solution (8 mg/ml): proper amount of sodium hydroxide is taken, precisely weighed, dissolved in water and diluted to prepare a solution containing about 8mg of sodium hydroxide per 1 ml.
Blank solution: precisely weighing 2ml of sodium hydroxide solution (8 mg/ml), placing into a 10ml measuring flask, adding acetonitrile for dissolving and diluting to a scale, shaking uniformly, and performing ultrasonic treatment for 30min.
Stock solution 1#: 24.69mg of 2, 4-difluoronitrobenzene, 24.24mg of 1-fluoro-4-nitrobenzene, 23.97mg of 2, 6-difluoronitrobenzene, 25.08mg of 1-fluoro-2-nitrobenzene, 27.97mg of 3, 4-difluoronitrobenzene, 25.19mg of nitrobenzene, 24.61mg of chlorobenzene, 25.87mg of 2, 4-dichloronitrobenzene, 25.29mg of p-nitrochlorobenzene, 25.86mg of o-chloronitrobenzene and 25.92mg of 2, 6-dichloronitrobenzene are weighed respectively, put into different 50ml measuring bottles, and are dissolved and diluted to a scale by acetonitrile, and shaken uniformly.
Mixing the control solution stock solution: measuring 1#ml of each impurity reference substance solution stock solution, placing a 100ml measuring flask, adding acetonitrile for dissolving and diluting to scale, shaking uniformly, measuring 5ml of the solution, placing a 50ml measuring flask, adding acetonitrile for dissolving and diluting to scale, and shaking uniformly.
A control solution; precisely measuring 1ml of mixed control solution stock solution, placing into a 10ml measuring flask, precisely adding 2ml of sodium hydroxide solution (8 mg/ml), performing ultrasonic treatment for 30min, adding acetonitrile for dissolution, diluting to scale, and shaking uniformly.
Test solution: flurbiprofen (self-made) 101.57mg is weighed, placed in a 10ml measuring flask, 2ml of sodium hydroxide solution (8 mg/ml) is precisely added, the solution is subjected to ultrasonic treatment for 30min, acetonitrile is added for dissolution, dilution is carried out to a scale, and shaking is carried out uniformly.
Impurity control solutions stock solution 2#: the stock solution 1#0.2ml of each impurity reference substance solution is measured separately, put into 20ml measuring flask separately, add acetonitrile to dissolve and dilute to scale, shake well.
Each impurity localization solution: separately measuring 2#0.1ml of each impurity reference substance solution stock solution, placing into 10ml measuring flask, precisely adding 2ml of sodium hydroxide solution (8 mg/ml), performing ultrasonic treatment for 30min, adding acetonitrile for dissolution, diluting to scale, and shaking.
The blank solution, the reference solution, the sample solution, and the impurity localization solutions were measured under the chromatographic conditions in example 1, and the measurement results were as follows.
Table 1 specific results.
。
The results show that: the blank solution has no interference to the measurement of the sample, and the method has good specificity.
Limit of detection and limit of quantification
And (3) taking a control solution, gradually diluting, and then carrying out sample injection test, wherein the relative mass concentration is taken as a quantitative limit concentration, and the relative mass concentration is taken as a detection limit concentration, wherein the signal-to-noise ratio is about 10:1. The results are detailed in Table 2.
The limit and quantitative limit results are shown in Table 2.
。
The result shows that the analysis method of the flurbiprofen genotoxic impurities has high sensitivity.
Linear relationship
Mixing the stock solution with each impurity reference substance: 24.16mg of 2, 4-difluoronitrobenzene, 25.84mg of 1-fluoro-4-nitrobenzene, 25.02mg of 2, 6-difluoronitrobenzene, 25.11mg of 1-fluoro-2-nitrobenzene, 24.20mg of 3, 4-difluoronitrobenzene, 25.93mg of nitrobenzene, 27.30mg of chlorobenzene, 25.05mg of 2, 4-dichloronitrobenzene, 25.96mg of p-nitrochlorobenzene, 25.62mg of o-chloronitrobenzene and 24.47mg of 2, 6-dichloronitrobenzene are weighed respectively, 50ml measuring bottles are placed respectively, acetonitrile is added for dissolution, dilution is carried out to scale, and shaking is carried out uniformly.
Linear stock solution: measuring 1ml of each impurity reference substance solution stock solution respectively, placing a 100ml measuring flask together, adding acetonitrile for dissolving and diluting to a scale, shaking uniformly, measuring 5ml of the solution, placing a 50ml measuring flask, adding acetonitrile for dissolving and diluting to the scale, and shaking uniformly.
And precisely measuring a proper amount of the linear stock solution to prepare a series of reference substance solutions with the concentrations of about 10, 20, 40, 50, 80 and 100ng/ml of each object to be detected, wherein the test results of the linear relation of the reference solutions are shown in Table 3.
Table 3 linear relationship results.
。
From the above results, it can be seen that: the analysis method of the flurbiprofen genotoxic impurities is wide in linear range and good in linear correlation.
Recovery test
Stock solution 1#: 24.69mg of 2, 4-difluoronitrobenzene, 24.24mg of 1-fluoro-4-nitrobenzene, 23.97mg of 2, 6-difluoronitrobenzene, 25.08mg of 1-fluoro-2-nitrobenzene, 27.97mg of 3, 4-difluoronitrobenzene, 25.19mg of nitrobenzene, 24.61mg of chlorobenzene, 25.87mg of 2, 4-dichloronitrobenzene, 25.29mg of p-nitrochlorobenzene, 25.86mg of o-chloronitrobenzene and 25.92mg of 2, 6-dichloronitrobenzene are weighed respectively, put into different 50ml measuring bottles, and are dissolved and diluted to a scale by acetonitrile, and shaken uniformly.
Mixing the control solution stock solution: measuring 1#ml of each impurity reference substance solution stock solution, placing a 100ml measuring flask, adding acetonitrile for dissolving and diluting to scale, shaking uniformly, measuring 5ml of the solution, placing a 50ml measuring flask, adding acetonitrile for dissolving and diluting to scale, and shaking uniformly.
About 100mg of flurbiprofen raw material medicine is taken, 9 parts of flurbiprofen raw material medicine is precisely weighed and placed in a 10ml measuring flask respectively, 0.3 ml, 1ml, 1.5ml and 1.5ml of mixed reference substance solution stock solution are precisely added respectively, 2ml of sodium hydroxide solution (8 mg/ml) is added, ultrasound is carried out for 30min, acetonitrile is added for dissolution and dilution to scale, shaking is carried out, and the recovery rate test sample solution is obtained, and the results are shown in Table 4 in detail.
Table 4 recovery test results.
。
As can be seen from table 4: the analysis method of the flurbiprofen genotoxic impurities provided by the invention is used for measuring the genotoxic impurities in the flurbiprofen, and the average recovery rate is 87% -100%, so that the detection accuracy of the method is higher.
Precision test
Sample introduction and measurement are repeated 6 times according to the experimental condition of 'example 1', and the measurement results are recorded, and the results are shown in Table 5 in detail.
Table 5 precision test results.
。
Repeatability test
About 100mg of flurbiprofen raw material medicine is taken, 6 parts are precisely weighed, the obtained mixture is respectively placed in a 10ml measuring flask, 2ml of sodium hydroxide solution (8 mg/ml) is precisely added, ultrasound is carried out for 30min, acetonitrile is added for dissolution and dilution to scale, shaking is carried out, and the flurbiprofen raw material medicine repeatability test solution is obtained, and the results are shown in Table 6 in detail.
Table 6 repeatability test results.
。
The results show that: the detection of each impurity in 6 sample solutions is basically consistent, and the repeatability of the method is good.
Comparative example 1
The column in example 1 was replaced with VF-624ms (30 m. Times.0.25 mm. Times.1.40 μm) and the other detection conditions were unchanged. The control solution in example 1 was measured under the above chromatographic conditions, and the chromatogram was recorded, and the detection spectrum is shown in FIG. 5.
The results show that: the VF-624ms (30 m 0.25mm 1.40 μm) had the following drawbacks: the peaks of the 2, 4-dichloronitrobenzene and the 2, 6-dichloronitrobenzene are coincident, and the mass spectrum parent ions and the ion are also coincident. The peak separation of each component to be measured was better by using a VF-1701ms (30 m. Times.0.25 mm. Times.0.25 μm) capillary chromatographic column.
The foregoing is a further detailed description of the present application in connection with the specific embodiments, and it is not intended that the practice of the present application be limited to such descriptions. It should be understood that those skilled in the art to which the present application pertains may make several simple deductions or substitutions without departing from the spirit of the present application, and all such deductions or substitutions should be considered to be within the scope of the present application.
Claims (6)
1. The analysis method of genotoxic impurities in flurbiprofen is characterized in that a GC-MS/MS method is adopted to measure a sample solution;
the preparation method of the test sample solution comprises the following steps: and (3) treating flurbiprofen with sodium hydroxide solution, performing ultrasound, and fixing the volume with acetonitrile.
2. The method for analyzing genotoxic impurities in flurbiprofen according to claim 1, wherein,
the gas chromatography conditions were:
gas chromatographic column: VF-1701ms quartz capillary column;
carrier gas: helium, collision gas: nitrogen gas;
heating program: the initial temperature is 75-85 ℃, the temperature is maintained for 1 minute, the temperature is raised to 170 ℃ at the rate of 5 ℃ per minute, and then the temperature is raised to 250 ℃ at the rate of 10 ℃ per minute, and the temperature is maintained for 10 minutes;
sample inlet temperature: 245-255 ℃;
column flow rate: 0.9-1.1 ml/min; split sample injection, split ratio: 10:1; sample injection amount: 1 μl.
3. The method for analyzing genotoxic impurities in flurbiprofen according to claim 1 or 2, characterized in that,
the mass spectrum conditions are as follows:
EI source temperature: 230 ℃;
electron energy: 70eV;
solvent delay time: 3min;
scanning mode: multi-reactive ion detection (MRM).
4. The method for analyzing genotoxic impurities in flurbiprofen according to any one of claims 1 to 3, wherein the genotoxic impurities comprise 3, 4-difluoronitrobenzene, 2, 6-difluoronitrobenzene, p-fluoronitrobenzene, 2, 4-difluoronitrobenzene, nitrobenzene, 1-fluoro-2-nitrobenzene, p-nitrochlorobenzene, o-nitrochlorobenzene, 2, 6-dichloronitrobenzene, 2, 4-dichloronitrobenzene, chlorobenzene.
5. The method for analyzing genotoxic impurities in flurbiprofen according to any one of claims 1 to 4, wherein the qualitative and quantitative ion pairs and collision energies used for the multi-reaction ion detection are as follows:
。
6. the method for analyzing flurbiprofen axetil according to claim 1, wherein,
the concentration of the sodium hydroxide solution is 5-10 mg/ml.
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