CN116879452A - Method for simultaneously detecting various aniline genotoxic impurities in pirenzenenaphthalene - Google Patents
Method for simultaneously detecting various aniline genotoxic impurities in pirenzenenaphthalene Download PDFInfo
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- CN116879452A CN116879452A CN202310929452.7A CN202310929452A CN116879452A CN 116879452 A CN116879452 A CN 116879452A CN 202310929452 A CN202310929452 A CN 202310929452A CN 116879452 A CN116879452 A CN 116879452A
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- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 title claims abstract description 112
- 238000000034 method Methods 0.000 title claims abstract description 65
- 239000012535 impurity Substances 0.000 title claims abstract description 61
- 231100000024 genotoxic Toxicity 0.000 title claims abstract description 35
- 230000001738 genotoxic effect Effects 0.000 title claims abstract description 35
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 100
- RZXMPPFPUUCRFN-UHFFFAOYSA-N p-toluidine Chemical compound CC1=CC=C(N)C=C1 RZXMPPFPUUCRFN-UHFFFAOYSA-N 0.000 claims abstract description 79
- 239000000243 solution Substances 0.000 claims abstract description 74
- RNVCVTLRINQCPJ-UHFFFAOYSA-N o-toluidine Chemical compound CC1=CC=CC=C1N RNVCVTLRINQCPJ-UHFFFAOYSA-N 0.000 claims abstract description 68
- 239000013558 reference substance Substances 0.000 claims abstract description 40
- AFBPFSWMIHJQDM-UHFFFAOYSA-N N-methyl-N-phenylamine Natural products CNC1=CC=CC=C1 AFBPFSWMIHJQDM-UHFFFAOYSA-N 0.000 claims abstract description 38
- 238000001514 detection method Methods 0.000 claims abstract description 29
- 238000007865 diluting Methods 0.000 claims abstract description 27
- 239000012488 sample solution Substances 0.000 claims abstract description 26
- 239000011550 stock solution Substances 0.000 claims abstract description 23
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 17
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 17
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 3
- 238000010828 elution Methods 0.000 claims description 18
- 239000000523 sample Substances 0.000 claims description 18
- 238000005303 weighing Methods 0.000 claims description 13
- 229940079593 drug Drugs 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 8
- 238000010812 external standard method Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 238000003908 quality control method Methods 0.000 claims description 2
- 230000002829 reductive effect Effects 0.000 claims description 2
- 238000012360 testing method Methods 0.000 description 17
- 238000000926 separation method Methods 0.000 description 14
- 239000012085 test solution Substances 0.000 description 13
- 239000000047 product Substances 0.000 description 12
- 238000010790 dilution Methods 0.000 description 7
- 239000012895 dilution Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 239000000543 intermediate Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 238000004090 dissolution Methods 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 239000012467 final product Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- JFDZBHWFFUWGJE-UHFFFAOYSA-N benzonitrile Chemical compound N#CC1=CC=CC=C1 JFDZBHWFFUWGJE-UHFFFAOYSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 3
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 239000012088 reference solution Substances 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 206010010904 Convulsion Diseases 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 230000000711 cancerogenic effect Effects 0.000 description 2
- 231100000315 carcinogenic Toxicity 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 231100000219 mutagenic Toxicity 0.000 description 2
- 230000003505 mutagenic effect Effects 0.000 description 2
- PRMWGUBFXWROHD-UHFFFAOYSA-N perampanel Chemical compound O=C1C(C=2C(=CC=CC=2)C#N)=CC(C=2N=CC=CC=2)=CN1C1=CC=CC=C1 PRMWGUBFXWROHD-UHFFFAOYSA-N 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- DWXSYDKEWORWBT-UHFFFAOYSA-N 2-(2-bromophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC=C1Br DWXSYDKEWORWBT-UHFFFAOYSA-N 0.000 description 1
- -1 6 '-oxo-1' -phenyl-1 ',6' -dihydro- [2,3 '-bipyridyl ] -5' -yl Chemical group 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 206010061334 Partial seizures Diseases 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 238000009098 adjuvant therapy Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 150000003931 anilides Chemical class 0.000 description 1
- 150000001448 anilines Chemical class 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 239000001961 anticonvulsive agent Substances 0.000 description 1
- 229960003965 antiepileptics Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940088679 drug related substance Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229940024040 fycompa Drugs 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000036963 noncompetitive effect Effects 0.000 description 1
- 229960005198 perampanel Drugs 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- 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
- G01N2030/047—Standards external
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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)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The application provides a method for simultaneously detecting various aniline genotoxic impurities in pirenzenenaphthalene. The method employs high performance liquid chromatography, which includes: taking potassium dihydrogen phosphate solution as mobile phase A, acetonitrile as mobile phase B, acetonitrile for dissolving and diluting the pirenzenenaphthalene to prepare a sample solution, acetonitrile-mobile phase A solution for dissolving and diluting the reference substance to prepare a reference substance solution stock solution, and acetonitrile for diluting the reference substance solution stock solution to prepare a reference substance solution; wherein the plurality of aniline genotoxic impurities are aniline, o-toluidine and p-toluidine. The method has wide linear range, can reach a detection limit as low as 0.0336 mug/mL, has a quantitative limit as low as 0.001%, has incomparable advantages in repeatability and durability, and has higher precision and accuracy.
Description
Technical Field
The application relates to the field of chemical drug analysis and detection, in particular to a method for simultaneously detecting various aniline genotoxic impurities in pirenzenenaphthalene.
Background
Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.
Pirenzenenaphthalene (Perampanel, trade name Fycompa) chemical name [2- (6 '-oxo-1' -phenyl-1 ',6' -dihydro- [2,3 '-bipyridyl ] -5' -yl) benzonitrile ] tri-quarterhydrate, is a non-competitive alpha-amino-3 hydroxy-5 methyl-4 isoxazole receptor (AMPAR) antagonist, is an antiepileptic drug developed by japan sanitary pharmacy (EISAI) company, and is used for the adjuvant treatment of partial seizures in children aged 12 years and older (with or without secondary global seizures).
The process route of the pirenzenenaphthalene is to take o-bromophenylacetic acid and aniline as starting materials, and to produce the pirenzenenaphthalene through condensation, cyanidation and cyclization. Genotoxic impurities are mainly derived from starting materials, intermediates, reagents and reaction byproducts in the raw material synthesis process, and can also be generated by degradation in drug storage.
The international coordination of human drug registration (ICH) mandates guidelines for evaluating and controlling DNA-reactive (mutagenic) impurities in drugs to limit potential carcinogenic risk-ICH M7 (R1), aiming at providing a viable framework for identification, classification, characterization and control of mutagenic impurities for controlling potential carcinogenic risk of impurities. A common method for limiting genotoxic impurities is the toxicological attention threshold method (TTC), which requires a TTC value of 1.5 μg/d for genotoxic impurities in bulk drugs and formulations. In the study on pirenzenenaphthalene, the control limit was calculated as the maximum daily dose, which was 12mg, and thus the control limit for each genotoxic impurity was 0.0125%.
The limit of genotoxic impurities is low, and most detection methods also use LC-MS or GC-MS for detection; because the LC-MS or GC-MS instrument is more expensive, most of the QC departments of pharmaceutical enterprises do not have the detection conditions of LC-MS or GC-MS, so the development of the conventional liquid chromatography method for detecting the genotoxic impurities has more practicability.
Disclosure of Invention
The application provides a detection method for simultaneously detecting 3 aniline genotoxic impurities in pirenzenenaphthalene, which has the advantages of strong specificity, high accuracy, high precision, good durability and the like.
Specifically, the application provides the following technical scheme.
The inventor researches and analyzes the technical process for synthesizing the pirenzenenaphthalene, and finds that the related genotoxic impurities mainly comprise: aniline, o-toluidine and p-toluidine.
Thus, in a first aspect of the present application there is provided the use of one or more of aniline, o-toluidine, p-toluidine as a control in the quality control of a drug substance of pirenzenenaphthalene or a formulation thereof.
In a second aspect of the present application, there is provided a method for simultaneously detecting a plurality of anilines genotoxic impurities in pirenzenenaphthalene, said method employing high performance liquid chromatography comprising:
taking potassium dihydrogen phosphate solution as mobile phase A, acetonitrile as mobile phase B, acetonitrile for dissolving and diluting the pirenzenenaphthalene to prepare a sample solution, acetonitrile-mobile phase A solution for dissolving and diluting the reference substance to prepare a reference substance solution stock solution, and acetonitrile for diluting the reference substance solution stock solution to prepare a reference substance solution;
wherein the plurality of aniline genotoxic impurities are aniline, o-toluidine and p-toluidine.
In some embodiments of the application, the pH of mobile phase a is 4-5.
In the embodiment of the application, the separation effect between aniline and p-toluidine is particularly easy to be influenced by the pH value, wherein the peak of aniline and p-toluidine is difficult to separate when the pH value of the mobile phase A is below 4, and the separation effect is good when the pH value is above 4 although the separation effect is poor when the pH value is 4.
In some embodiments of the application, the mobile phase A is 0.018 to 0.022mol/L potassium dihydrogen phosphate solution, preferably 0.02mol/L potassium dihydrogen phosphate solution. In embodiments of the application, good separation of three impurities can be achieved without adjusting the pH when mobile phase a is within this concentration range, and the degree of separation can be greater than 5.
In some embodiments of the application, the acetonitrile-mobile phase A solution has a volume ratio of acetonitrile to mobile phase A of 20-30:70-80, preferably 20:80.
In some embodiments of the present application, other control solvents, such as 50% acetonitrile-mobile phase A, e.g., 0.05% to 0.2% phosphoric acid solution-acetonitrile (80:20), etc., have also been tried, and as a result, are not ideal, and may have a lower degree of tailing and/or separation between impurities, or even be difficult to separate.
In some embodiments of the application, the method may comprise:
taking a pirenzenenape sample, precisely weighing, adding acetonitrile for dissolving and diluting to a scale, and shaking uniformly to prepare a sample solution;
respectively weighing reference substances of aniline, o-toluidine and p-toluidine, placing into a same measuring flask, dissolving with acetonitrile-mobile phase A solution, diluting to scale, shaking, and collecting as reference substance solution stock solution;
precisely measuring a reference substance solution stock solution, diluting with acetonitrile to a required scale, and shaking to obtain a reference substance solution;
and respectively precisely measuring the sample solution and the reference substance solution, injecting into a high performance liquid chromatograph, and calculating the contents of the aniline, the o-toluidine and the p-toluidine in the sample solution by adopting an external standard method according to peak areas.
In some embodiments of the application, the chromatographic conditions of the method are:
chromatographic column: octadecylsilane chromatographic column;
mobile phase a:0.018 to 0.022mol/L of potassium dihydrogen phosphate solution, preferably 0.02mol/L of potassium dihydrogen phosphate solution;
mobile phase B: acetonitrile;
flow rate: 0.95-1.05 mL/min, preferably 1.0mL/min;
column temperature: 33-37 ℃, preferably 35 ℃;
detection wavelength: 230-234 nm, preferably 232nm;
sample injection amount: 10 mu L.
In some embodiments of the application, the method comprises: linear gradient elution was performed using mobile phase a and mobile phase B, wherein the linear gradient elution procedure was:
the ratio of the mobile phase A to the mobile phase B is kept at 75:25 within 0-25 min;
25-40 min, reducing the proportion of the mobile phase A, and simultaneously increasing the proportion of the mobile phase B to 20:80;
the ratio of the mobile phase A is increased and the ratio of the mobile phase B is reduced to 75:25 in 40-41 min;
and (4) keeping the ratio of the mobile phase A to the mobile phase B at 75:25 in 41-50 min.
In some embodiments of the application, the linear range of aniline is 0.0671-2.2371 mug/mL, the linear range of para-toluidine is 0.1305-2.1755 mug/mL, the linear range of ortho-toluidine is 0.1328-2.2131 mug/mL, all correlation coefficient r values are greater than 0.999, which shows that the method of the application has good linear relation, and the conversion of signal and concentration can be performed by a linear equation in the linear range studied, so as to obtain accurate content or impurity determination result.
In some embodiments of the application, the detection limit of the method is as low as 0.0336 μg/mL, i.e., more than 0.0004% of impurities in pirenzenenaphthalene can be detected, and the quantification limit can be as low as 0.001% (10 ppm).
In some embodiments of the application, the method for detecting and calculating 3 anilide genotoxic impurities (aniline, p-toluidine and o-toluidine) in pirenzenenaphthalene comprises:
1. chromatographic conditions:
instrument: agilent 1260
Chromatographic column: inertisl ODS-3V C18 (250 mm. Times.4.6 mm,5 μm);
mobile phase a:0.02mol/L potassium dihydrogen phosphate solution (natural pH of about 4.63);
mobile phase B: acetonitrile;
flow rate: 1.0mL/min;
column temperature: 35 ℃;
detection wavelength: 232nm;
sample injection amount: 10. Mu.L;
gradient elution, elution procedure is as shown in table 1:
table 1: gradient elution procedure
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 75 | 25 |
| 25 | 75 | 25 |
| 30 | 20 | 80 |
| 40 | 20 | 80 |
| 41 | 75 | 25 |
| 50 | 75 | 25 |
2. Detection method
(1) Preparing test solution
Taking 80mg of a pirenzenenaphthalene sample, precisely weighing, placing into a 10mL measuring flask, adding acetonitrile for dissolution, diluting to a scale, and shaking uniformly to obtain the product;
(2) Preparing reference substance solution
Respectively taking about 25mg of each of aniline, o-toluidine and p-toluidine reference substances, precisely weighing, placing into the same 25mL measuring flask, dissolving with acetonitrile-mobile phase A (20:80), diluting to scale, shaking uniformly, and taking as reference substance solution stock solution; precisely measuring 0.1mL of reference substance solution stock solution, placing into a 100mL measuring flask, diluting to scale with acetonitrile, and shaking to obtain the final product;
(3) Respectively precisely measuring the sample solution and the reference solution, injecting into a liquid chromatograph, recording the chromatograms, and calculating the contents of aniline, o-toluidine and p-toluidine in the sample solution by adopting an external standard method according to peak areas;
wherein, the content calculation formula is:
content (%) =f of each genotoxic impurity in test solution Average of ×A i ×V i /m i ×100％
Wherein: f=m s ×c s /(V s ×A s )；
Wherein: f is a response factor; m is m s Weighing the sample amount of each genotoxic impurity in the reference substance solution;
c s content,%; v (V) s Is used as reference substance in solutionTotal dilution volume of sex impurities; a is that s Peak area for each genotoxic impurity in the control solution; f (f) Average of Is the average value of the response factors; a is that i Peak areas of genotoxic impurities in the sample solution; m is m i Is the sample weighing amount of the test sample; v (V) i Is the dilution volume of the test sample.
The specific features described in the above embodiments of the present application may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
The numerical ranges recited herein include all numbers within the range and include any two values within the range, unless specifically stated otherwise. For example, 4-5, which includes all values between 4-5, and which includes a range of values (4.37-4.63) consisting of any two values (e.g., 4.1, 4.37, 4.63) within the range; the different values of the same index appearing in all embodiments of the application can be combined arbitrarily to form a range value.
Advantages of the present application compared to the prior art include: the method for simultaneously detecting 3 aniline genotoxic impurities in the pirenzenenaphthalene, namely the aniline, the p-toluidine and the o-toluidine can well separate the three impurities and measure the content of the three impurities, has strong specificity, high accuracy and precision and good durability, and is suitable for accurately controlling the 3 aniline genotoxic impurities in the pirenzenenaphthalene.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a chromatogram of a control solution at different mobile phase pH in example 2 (aniline, p-toluidine and o-toluidine in order from left to right).
FIG. 2 is a chromatogram of the control solution in example 3 (elution procedure of Table 2).
FIG. 3 is a chromatogram of the control solution in example 3 (elution procedure of Table 1).
FIG. 4 is a chromatogram of the control solution of example 4.
FIG. 5 is a hollow white solvent chromatogram of example 5.
FIG. 6 is a chromatogram of the sample solution in example 5.
FIG. 7 is a chromatogram of the sample solution of example 5, wherein peak 1 is aniline; peak 2 is p-toluidine; peak 3 is o-toluidine.
FIG. 8 is a standard graph of aniline in example 5.
FIG. 9 is a standard graph of o-toluidine in example 5.
FIG. 10 is a standard graph of p-toluidine in example 5.
Detailed Description
The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or materials used in the present application may be purchased in conventional manners, and unless otherwise indicated, they may be used in conventional manners in the art or according to the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present application. The preferred methods and materials described herein are presented for illustrative purposes only.
The instrument and reagent information used in the following examples of the application are shown in the following table:
example 1
The present embodiment provides a method for simultaneously detecting 3 kinds of aniline genotoxic impurities (aniline, p-toluidine and o-toluidine) in pirenzenenaphthalene, in which 3 batches (220916, 220919, 220920) of pirenzenenaphthalene raw material medicines are weighed respectively, and the content of 3 kinds of aniline genotoxic impurities (aniline, p-toluidine and o-toluidine) in pirenzenenaphthalene is detected and calculated. The method comprises the following steps:
1. chromatographic conditions:
instrument: agilent 1260
Chromatographic column: inertisl ODS-3V C18 (250 mm. Times.4.6 mm,5 μm);
mobile phase a:0.02mol/L potassium dihydrogen phosphate solution (natural pH of about 4.63);
mobile phase B: acetonitrile;
flow rate: 1.0mL/min;
column temperature: 35 ℃;
detection wavelength: 232nm;
sample injection amount: 10 mu L.
Gradient elution, elution procedure is as shown in table 1:
table 1: gradient elution procedure
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 75 | 25 |
| 25 | 75 | 25 |
| 30 | 20 | 80 |
| 40 | 20 | 80 |
| 41 | 75 | 25 |
| 50 | 75 | 25 |
2. Detection method
(1) Preparing test solution
Taking 80mg of a pirenzenenaphthalene sample, precisely weighing, placing into a 10mL measuring flask, adding acetonitrile for dissolution, diluting to a scale, and shaking uniformly to obtain the product;
(2) Preparing reference substance solution
Respectively taking about 25mg of each of aniline, o-toluidine and p-toluidine reference substances, precisely weighing, placing into the same 25mL measuring flask, dissolving with acetonitrile-mobile phase A (20:80), diluting to scale, shaking uniformly, and taking as reference substance solution stock solution; precisely measuring 0.1mL of reference solution stock solution, placing into a 100mL measuring flask, diluting to scale with acetonitrile, and shaking to obtain the final product.
(3) And respectively precisely measuring the sample solution and the reference solution, injecting into a liquid chromatograph, recording the chromatograms, and calculating the contents of the aniline, the o-toluidine and the p-toluidine in the sample solution by adopting an external standard method according to peak areas.
Wherein, the content calculation formula is:
content (%) =f of each genotoxic impurity in test solution Average of ×A i ×V i /m i ×100％
Wherein: f=m s ×c s /(V s ×A s )；
Wherein: f is a response factor; m is m s Weighing the sample amount of each genotoxic impurity in the reference substance solution;
c s content,%; v (V) s The total dilution volume of each genotoxic impurity in the reference substance solution; a is that s Peak area for each genotoxic impurity in the control solution; f (f) Average of Is the average value of the response factors; a is that i Peak areas of genotoxic impurities in the sample solution; m is m i Is the sample weighing amount of the test sample; v (V) i Is the dilution volume of the test sample.
Results: no aniline, o-toluidine and p-toluidine are detected in the 3 batches of pirenzenenaphthalene bulk drugs.
Example 2
The pH of mobile phase A was changed to 3.79, 4.37 and 4 from 0.02mol/L potassium dihydrogen phosphate solution (natural pH of about 4.63) by adjusting the pH to 3.79, 4.37 and 4, respectively, as compared with example 1, and the control solution was again examined in the same manner as in example 1, the examination results being shown in FIG. 1, which shows chromatograms of mobile phase A at pH of 3.79, 4, 4.37 and 4.63, respectively, wherein aniline, p-toluidine and o-toluidine were sequentially listed from left to right in FIG. 1.
As a result, it was found that the separation effect between aniline and p-toluidine was easily affected by pH, and the mobile phase a was difficult to separate at pH of 4 or less, but was able to separate at pH of 4, but was inferior in degree of separation, and was good at pH of more than 4, but the separation effect was more excellent at pH of not adjusting pH alone, that is, the mobile phase a was separated with 0.02mol/L potassium dihydrogen phosphate solution (natural pH of about 4.63).
Example 3
Compared with example 1, the mobile phase A, the solvent and the elution procedure were changed, and the same as example 1, wherein the mobile phase A was a 0.1% ammonium acetate solution (pH was adjusted to 4.0 with glacial acetic acid), the control solvent was 50% acetonitrile-mobile phase A, the gradient elution conditions were as shown in Table 2, and the detection results are shown in FIG. 2.
Table 2: gradient elution procedure
According to the detection result, the baseline fluctuation is large, the signal intensity change is not obvious due to the large baseline fluctuation, the detection limit and the quantitative limit are increased, the analysis sensitivity is low, the integral data is unstable, and the precision and the accuracy of the result are obviously affected.
The elution procedure was adjusted based on the above method, and elution was performed according to the elution procedure shown in table 1, the detection results are shown in fig. 3, and table 3 shows the chromatogram of the control solution. The results show that baseline fluctuations are slightly improved compared to the above method, but there are still larger fluctuations, and the degree of separation of aniline from p-toluidine is lower, both of which are poorly separated.
Example 4
The control solvent was changed to 50% acetonitrile-mobile phase A as compared to example 1, except that example 1 was followed.
The detection results are shown in FIG. 4. The result shows that trailing phenomenon exists in three impurities of the aniline, the p-toluidine and the o-toluidine, the separation degree of the p-toluidine and the o-toluidine is low, and the separation effect is poor.
Example 5Method investigation
The methods described in example 1 were examined for specificity, limit of detection and limit of quantification, linearity and range, accuracy, repeatability, precision, stability and durability of the solution, respectively, and the methods and results were as follows:
1. methodological verification
1.1 specificity
Each impurity localization solution: respectively taking about 25mg of each of aniline, o-toluidine and p-toluidine reference substances, precisely weighing, placing into different 25mL measuring flasks, dissolving with acetonitrile-mobile phase A (20:80, mobile phase A is 0.02mol/L potassium dihydrogen phosphate solution) and diluting to scale, shaking uniformly, and taking as each impurity stock solution; accurately measuring 0.1mL of each impurity stock solution, placing into different 100mL measuring bottles, diluting to scale with acetonitrile, and shaking to obtain the final product.
Test solution: taking about 80mg of the product, placing in a 10mL measuring flask, adding acetonitrile for dissolving and diluting to a scale, and shaking uniformly to obtain the product.
Sample addition test solution: about 80mg of the product is taken, placed in a 10mL measuring flask, 0.1mL of each of aniline, o-toluidine and p-toluidine stock solution is precisely added, and the mixture is dissolved and diluted to a scale by acetonitrile, and is uniformly shaken to obtain the product.
According to the chromatographic conditions of example 1, a blank solution, i.e., acetonitrile-mobile phase A (20:80, mobile phase A is 0.02mol/L potassium dihydrogen phosphate solution), each impurity locating solution, a sample solution and a sample-adding sample solution are respectively injected, chromatograms are recorded, detection results are shown in fig. 5-7 and Table 3, wherein the blank solvent chromatograms are shown in fig. 5, the sample solution chromatograms are shown in fig. 6, the sample-adding sample solution chromatograms are shown in fig. 7, the results show that the separation degree between impurities is greater than 5, and the method of example 1 has good specificity.
Table 3: results of the proprietary test
1.2 detection limit and quantitative limit
Respectively taking appropriate amounts of aniline, o-toluidine and p-toluidine as reference substances, respectively gradually diluting to low concentration, injecting into a liquid chromatograph, and recording chromatogram. The concentration of the signal to noise ratio S/N is more than or equal to 3 is used as the detection limit concentration, and the concentration of the signal to noise ratio S/N is more than or equal to 10 is used as the quantitative limit concentration. The results are shown in Table 4.
Table 4: limit of detection and limit of quantification results
The detection limit and the quantitative limit concentration of each impurity are smaller than the report limit concentration, and both meet the requirements, so that the method of the embodiment 1 is good in sensitivity, the detection limit can be as low as 0.0336 mug/mL, and the impurity higher than 0.0004% in the pirenzenenaphthalene can be detected.
1.3 linearity and Range
Linear stock solution: precisely measuring aniline, o-toluidine and p-toluidine stock solutions respectively by 1mL, placing into the same 10mL measuring flask, diluting to scale with acetonitrile, and shaking uniformly to obtain the final product.
The quantitative limit concentration was taken as a linear 1 solution.
0.5mL, 1.0mL, 1.5mL and 2.0mL of the linear stock solution were precisely measured, placed in 10mL measuring flasks, diluted to the scale with acetonitrile, and shaken well to obtain solutions of linearity 2 (50%), linearity 3 (100%), linearity 4 (150%) and linearity 5 (200%).
According to the chromatographic conditions of example 1, linear regression analysis was performed with the concentration (C) as the abscissa (X-axis) and the peak area (A) as the ordinate (Y-axis), and the r-value of each impurity was greater than 0.999 and approximately 1 in the quantitative limit to 200% limit concentration range, and the Y-axis intercept deviation was within 2%, and the linearity was good. The results are shown in FIGS. 8-10 and Table 5, wherein FIG. 8 is a standard graph of aniline, FIG. 9 is a standard graph of o-toluidine, and FIG. 10 is a standard graph of p-toluidine.
Table 5: results of the Linear test
| Name of the name | Quadratic equation | r | y-axis intercept deviation | Concentration range |
| Aniline | y＝44.5676x-0.6796 | 0.9998 | 1.55％ | 0.0671μg/mL～2.2371μg/mL |
| Para-toluidine | y＝35.5926x-0.4305 | 0.9999 | 1.22％ | 0.1305μg/mL～2.1755μg/mL |
| O-toluidine | y＝36.8111x-0.4334 | 0.9998 | 1.19％ | 0.1328μg/mL～2.2131μg/mL |
1.4 accuracy
A control solution stock was prepared as described in example 1.
50% level test solution: about 80mg of the product is taken, placed in a 10mL measuring flask, 50 mu L of each impurity reference substance solution stock solution is precisely added, acetonitrile is added for dissolution, dilution is carried out to scale, and shaking is carried out uniformly. Triplicate formulations were prepared.
100% level test solution: about 80mg of the product is taken, placed in a 10mL measuring flask, 100 mu L of each impurity reference substance solution stock solution is precisely added, acetonitrile is added for dissolution, dilution is carried out to scale, and shaking is carried out uniformly. Triplicate formulations were prepared.
150% level test solution: about 80mg of the product is taken, placed in a 10mL measuring flask, 150 mu L of each impurity reference substance solution stock solution is precisely added, acetonitrile is added for dissolution, dilution is carried out to scale, and shaking is carried out uniformly. Triplicate formulations were prepared.
Test solution: taking about 80mg of the product, placing in a 10mL measuring flask, adding acetonitrile for dissolving and diluting to a scale, and shaking uniformly to obtain the product. Recovery was calculated according to the external standard method according to the chromatographic conditions of example 1, and the detection results are shown in Table 6.
Table 6: recovery test results
As shown in the table above, the average value of the recovery rate of each impurity at three concentration levels is in the range of 80.0% -120.0%, and the RSD% of the recovery rate results of 9 samples is less than 10%, which proves that the method has good accuracy.
1.5 repeatability
2 portions of control solution were prepared in parallel as described in example 1;
6 parts of test solutions were prepared in parallel according to the method described in example 1;
the detection was carried out according to the chromatographic conditions of example 1, and the content of each impurity was calculated according to the external standard method. The results are shown in Table 7.
Table 7: results of the repeatability test
As shown in the table above, the content RSD% of the aniline, the o-toluidine and the p-toluidine is less than 1.0%, which indicates that the repeatability of the method is good.
1.6 intermediate precision
Taking the same batch of samples, adopting different testers to test by adopting different instruments at different times according to the preparation method of the repeated test sample solution, carrying out intermediate precision test according to the repeated test operation, preparing six samples of the sample solution in parallel, measuring the content of each impurity in the samples, and evaluating the intermediate precision of the method by combining the repeated test results. The results are shown in Table 8.
Table 8: results of intermediate precision test
As shown in the table above, the RSD of each genotoxic impurity content in 12 samples is within 5%, which indicates that the intermediate precision of the method is good.
1.7 solution stability
The control solution was prepared as described in example 1 and the sample solution was prepared as described in example 5, part 1.1, and the sample solution was placed at room temperature and measured at time points of 0,3,6,9, 12, 24, 48, 72h, respectively, according to the chromatographic conditions of example 1. The results are shown in tables 9 and 10.
Table 9: reference substance solution
Table 10: sample adding test solution
| Time (h) | Area of aniline peak | Peak area of p-toluidine | O-toluidine peak area |
| 0 | 49.973 | 35.606 | 38.036 |
| 3 | 49.688 | 35.833 | 38.111 |
| 6 | 49.818 | 35.888 | 38.329 |
| 9 | 50.008 | 35.382 | 37.821 |
| 12 | 50.570 | 36.170 | 38.052 |
| 24 | 50.243 | 35.773 | 38.804 |
| 48 | 50.130 | 36.109 | 38.783 |
| 72 | 50.499 | 36.548 | 38.644 |
| RSD(％) | 0.7 | 1.1 | 1.0 |
The test results show that the peak areas of the aniline, the o-toluidine and the p-toluidine in the control solution and the sample-adding test solution in 72 hours are not obviously changed, and the solution is proved to be stable in 72 hours.
1.8 durability
Under the conditions of changing detection wavelength (230-234 nm), column temperature (33-37 ℃), flow rate (0.95-1.05 mL/min), concentration of monopotassium phosphate solution (0.018-0.022 mol/L), changing different chromatographic columns and the like, the content change condition of each genotoxic impurity in the sample solution and the separation degree condition of each impurity are examined. The detailed results are shown in Table 11.
Table 11: durability test results
The test results show that under the detection conditions, the RSD% of the content change of the aniline, the o-toluidine and the p-toluidine is within 10%, and the method has good durability.
Taken together, the methodology validation results demonstrate that: the method described in example 1 is used for determining the content of 3 aniline genotoxic impurities in the pirenzenenaphthalene, has strong specificity, high accuracy and precision and good durability, and is suitable for accurately controlling the 3 aniline genotoxic impurities in the pirenzenenaphthalene.
The foregoing description is only a preferred embodiment of the present application, and the present application is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present application has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (10)
1. A method for simultaneously detecting a plurality of aniline genotoxic impurities in pirenzenenaphthalene, which is characterized by adopting high performance liquid chromatography and comprising the following steps:
taking potassium dihydrogen phosphate solution as mobile phase A, acetonitrile as mobile phase B, acetonitrile for dissolving and diluting the pirenzenenaphthalene to prepare a sample solution, acetonitrile-mobile phase A solution for dissolving and diluting the reference substance to prepare a reference substance solution stock solution, and acetonitrile for diluting the reference substance solution stock solution to prepare a reference substance solution;
wherein the plurality of aniline genotoxic impurities are aniline, o-toluidine and p-toluidine.
2. The method according to claim 1, wherein the pH of mobile phase a is 4-5.
3. The method of claim 1, wherein mobile phase a is 0.018 to 0.022mol/L potassium dihydrogen phosphate solution.
4. The method according to claim 1, wherein the volume ratio of acetonitrile to mobile phase a in the acetonitrile-mobile phase a solution is 20-30:70-80.
5. The method according to claim 1, wherein the mobile phase A is a 0.02mol/L potassium dihydrogen phosphate solution, and the volume ratio of acetonitrile to mobile phase A in the acetonitrile-mobile phase A solution is 20:80.
6. The method according to claim 1, characterized in that the method comprises:
taking a pirenzenenape sample, precisely weighing, adding acetonitrile for dissolving and diluting to a scale, and shaking uniformly to prepare a sample solution;
respectively weighing reference substances of aniline, o-toluidine and p-toluidine, placing into a same measuring flask, dissolving with acetonitrile-mobile phase A solution, diluting to scale, shaking, and collecting as reference substance solution stock solution;
precisely measuring a reference substance solution stock solution, diluting with acetonitrile to a required scale, and shaking to obtain a reference substance solution;
and respectively precisely measuring the sample solution and the reference substance solution, injecting into a high performance liquid chromatograph, and calculating the contents of the aniline, the o-toluidine and the p-toluidine in the sample solution by adopting an external standard method according to peak areas.
7. The method according to any one of claims 1 to 6, wherein the chromatographic conditions are:
chromatographic column: octadecylsilane chromatographic column
Mobile phase a: 0.018-0.022 mol/L potassium dihydrogen phosphate solution
Mobile phase B: acetonitrile
Flow rate: 0.95-1.05 mL/min
Column temperature: 33-37 DEG C
Detection wavelength: 230-234 nm
Sample injection amount: 10 mu L.
8. The method according to any one of claims 1 to 6, characterized in that the method comprises: linear gradient elution was performed using mobile phase a and mobile phase B, with the linear gradient elution procedure:
the ratio of the mobile phase A to the mobile phase B is kept at 75:25 within 0-25 min;
25-40 min, reducing the proportion of the mobile phase A, and simultaneously increasing the proportion of the mobile phase B to 20:80;
the ratio of the mobile phase A is increased and the ratio of the mobile phase B is reduced to 75:25 in 40-41 min;
and (4) keeping the ratio of the mobile phase A to the mobile phase B at 75:25 in 41-50 min.
9. The method according to any one of claims 1 to 5, wherein in the method, the linear range of aniline is 0.0671 μg/mL to 2.2371 μg/mL, the linear range of para-toluidine is 0.1305 μg/mL to 2.1755 μg/mL, and the linear range of ortho-toluidine is 0.1328 μg/mL to 2.2131 μg/mL.
10. The application of one or more of aniline, o-toluidine and p-toluidine as reference substances in the quality control of pirenzenepamine bulk drug or preparation thereof.
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