CN111855836A - Method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography - Google Patents
Method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography Download PDFInfo
- Publication number
- CN111855836A CN111855836A CN202010542777.6A CN202010542777A CN111855836A CN 111855836 A CN111855836 A CN 111855836A CN 202010542777 A CN202010542777 A CN 202010542777A CN 111855836 A CN111855836 A CN 111855836A
- Authority
- CN
- China
- Prior art keywords
- rivaroxaban
- ammonia water
- acetonitrile
- aminophenyl
- mobile phase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 63
- 231100000024 genotoxic Toxicity 0.000 title claims abstract description 59
- 230000001738 genotoxic effect Effects 0.000 title claims abstract description 59
- 239000012535 impurity Substances 0.000 title claims abstract description 59
- 229960001148 rivaroxaban Drugs 0.000 title claims abstract description 58
- KGFYHTZWPPHNLQ-AWEZNQCLSA-N rivaroxaban Chemical compound S1C(Cl)=CC=C1C(=O)NC[C@@H]1OC(=O)N(C=2C=CC(=CC=2)N2C(COCC2)=O)C1 KGFYHTZWPPHNLQ-AWEZNQCLSA-N 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 24
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims abstract description 73
- 235000011114 ammonium hydroxide Nutrition 0.000 claims abstract description 73
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 claims abstract description 47
- 239000012488 sample solution Substances 0.000 claims abstract description 47
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 42
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000001514 detection method Methods 0.000 claims abstract description 22
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 12
- 238000010828 elution Methods 0.000 claims abstract description 10
- 239000000523 sample Substances 0.000 claims abstract description 5
- 239000000945 filler Substances 0.000 claims abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 39
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 26
- AKPYISYUMOZZAJ-UHFFFAOYSA-N 4-(2-aminophenyl)morpholin-3-one Chemical compound NC1=CC=CC=C1N1C(=O)COCC1 AKPYISYUMOZZAJ-UHFFFAOYSA-N 0.000 claims description 22
- IABQKUMPNMOSKF-UHFFFAOYSA-N 4-(3-aminophenyl)morpholin-3-one Chemical compound NC1=CC=CC(N2C(COCC2)=O)=C1 IABQKUMPNMOSKF-UHFFFAOYSA-N 0.000 claims description 22
- MHCRLDZZHOVFEE-UHFFFAOYSA-N 4-(4-aminophenyl)morpholin-3-one Chemical group C1=CC(N)=CC=C1N1C(=O)COCC1 MHCRLDZZHOVFEE-UHFFFAOYSA-N 0.000 claims description 22
- 239000000243 solution Substances 0.000 description 55
- 239000002904 solvent Substances 0.000 description 23
- 239000013558 reference substance Substances 0.000 description 17
- 239000012088 reference solution Substances 0.000 description 16
- 239000012085 test solution Substances 0.000 description 14
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 12
- 230000005526 G1 to G0 transition Effects 0.000 description 9
- 238000005303 weighing Methods 0.000 description 7
- 239000007788 liquid Substances 0.000 description 6
- 229910021529 ammonia Inorganic materials 0.000 description 5
- 239000003053 toxin Substances 0.000 description 4
- 231100000765 toxin Toxicity 0.000 description 4
- 108700012359 toxins Proteins 0.000 description 4
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 102000004411 Antithrombin III Human genes 0.000 description 1
- 108090000935 Antithrombin III Proteins 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229960005348 antithrombin iii Drugs 0.000 description 1
- 229960004676 antithrombotic agent Drugs 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000013501 data transformation Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
Images
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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
-
- G01N30/48—
-
- 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
- G01N30/74—Optical detectors
-
- 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
Landscapes
- 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)
- Spectroscopy & Molecular Physics (AREA)
- Engineering & Computer Science (AREA)
- Library & Information Science (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention discloses a method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography, wherein octadecylsilane chemically bonded silica is used as a filler for a chromatographic column; the detection wavelength is 220 nm-240 nm; dissolving a test sample in dimethyl sulfoxide-acetonitrile-0.01% ammonia water to be used as a test sample solution; the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.01-0.1% ammonia water, the mobile phase B is methanol, and the elution mode is gradient elution. The method for determining the potential genotoxic impurities in rivaroxaban by the high performance liquid chromatography provided by the invention can be used for detecting the genotoxic impurities in rivaroxaban, is lower in detection amount, and can better control the quality of rivaroxaban.
Description
Technical Field
The invention belongs to the technical field of substance detection, and particularly relates to a method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography.
Background
Vaxaban tablets are an antithrombotic agent which can directly antagonize free and bound Xa factors without the participation of antithrombin III. Rivaroxaban is a main active ingredient in rivaroxaban tablets, and the quality of the rivaroxaban tablets can be well reflected by measuring potential genotoxic impurities of rivaroxaban. Therefore, a method for determining potential genotoxic impurities in rivaroxaban is established, the quality of rivaroxaban can be better controlled, and the stability of a product can be better controlled.
Disclosure of Invention
The invention aims to establish a method for determining potential genotoxic impurities in rivaroxaban, which can better control the quality of rivaroxaban and better control the stability of a product.
The purpose of the invention is realized by the following technical scheme:
a method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography comprises the following steps:
octadecylsilane chemically bonded silica is used as a filler for the chromatographic column;
the detection wavelength is 220-240 nm;
dissolving a test sample in dimethyl sulfoxide-acetonitrile-0.01% ammonia water as a test sample solution, wherein the volume ratio of the dimethyl sulfoxide to the acetonitrile to the 0.01% ammonia water is (2-3): 3.5-4: 3.5 to 4;
the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.01-0.1% ammonia water, the mobile phase B is methanol, and the elution mode is gradient elution.
Preferably, the conditions of the gradient elution are as follows:
preferably, the conditions of the gradient elution are as follows:
preferably, the temperature of the chromatographic column is 35-45 ℃.
Preferably, the detection wavelength is 230 nm.
Preferably, the volume ratio of the dimethyl sulfoxide to the acetonitrile to 0.01% -0.1% of ammonia water is 2:4: 4.
Preferably, the potentially genotoxic impurity is 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone, or 4- (3-aminophenyl) -3-morpholinone.
Preferably, the sample amount is 10 to 100. mu.l.
Advantageous effects
The method for detecting the potential genotoxic impurities in the rivaroxaban by using the high-number liquid chromatography has the advantages of high separation efficiency, high analysis speed and high detection sensitivity, controls the potential genotoxic impurities in the rivaroxaban to be within 50ppm by detecting the potential genotoxic impurities in the rivaroxaban, can control the quality of the rivaroxaban, and better controls the stability of a product.
Drawings
FIG. 1 HPLC chromatogram of blank solvent of example 2
FIG. 2 HPLC chromatogram of control solution of example 2
FIG. 3 HPLC chromatogram of test solution in example 2
FIG. 4 HPLC chromatogram of sample solution added with standard in example 2
FIG. 5 HPLC chromatogram of control solution of example 4
FIG. 6 HPLC chromatogram of detection limit solution of example 4
FIG. 7 HPLC chromatogram of control solution of example 5
FIG. 8 HPLC chromatogram of quantitative limiting solution of example 5
FIG. 9 HPLC chart of quantitative limiting of standard sample solution in example 5
FIG. 10 HPLC profile of blank solvent of example 6
FIG. 11 HPLC chart of control solution of example 6
FIG. 12 HPLC chart of the sample solution of example 6
FIG. 13 HPLC chart of the sample solution added with the standard in example 6
FIG. 14 HPLC profile of blank solvent of example 7
FIG. 15 HPLC chart of control solution of example 7
FIG. 16 HPLC chart of the sample solution of example 7
FIG. 17 HPLC chart of the sample solution added with the standard in example 7
FIG. 18 HPLC profile of blank solvent of example 8
FIG. 19 HPLC chart of control solution of example 8
FIG. 20 HPLC chart of the sample solution of example 8
FIG. 21 HPLC chart of the sample solution added with the standard in example 8
FIG. 22 HPLC profile of blank solvent of example 9
FIG. 23 HPLC chart of control solution of example 9
FIG. 24 HPLC chart of the sample solution of example 9
FIG. 25 HPLC chart of the sample solution added with the standard in example 9
Detailed Description
The invention is further illustrated by the following examples, which are intended to be illustrative and not limiting. It will be understood by those of ordinary skill in the art that these examples are not intended to limit the present invention in any way and that suitable modifications and data transformations may be made without departing from the spirit and scope of the present invention.
The reagents used in the examples were purchased from the market or simply synthesized, wherein the types of HPLC and some of the reagents used were derived from the following sources:
| device name/model | Manufacturer of the product | Device numbering | Check unit | Checking validity period |
| High performance liquid chromatograph/LC-20 AT | SHIMADZU | JSY-024 | Nanjing measurement supervision and detection institute | 2020.03.26 |
| High performance liquid chromatograph/LC-2030C | SHIMADZU | JSY-049 | Nanjing measurement supervision and detection institute | 2020.08.09 |
| Name (R) | Manufacturer of the product | Batch number | Rank of | Period of validity |
| Acetonitrile | ASTOON | A19T1015 | HPLC | 2021.11.13 |
| Methanol | Honeywell | T1BG2H | HPLC | 2021.11.25 |
| Aqueous ammonia | Shanghai Lingfeng Chemical reagent Limited by | 20180702 | AR | 2021.11.08 |
| Dimethyl sulfoxide | Honeywell | DW672-CN | HPLC | 2021.11.17 |
The columns used in examples 1-8 were Welch C184.6 mm 150mm 3.5um and the columns used in example 9 were Agilent C184.6 mm 150mm 3.5 um.
Example 1
A method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography comprises the following steps:
preparation of a blank solvent: the volume ratio of dimethyl sulfoxide, acetonitrile and 0.01% (V/V) ammonia water is 2:4: 4. The proportions of dimethyl sulfoxide, acetonitrile and 0.01% ammonia referred to in the following and other examples are by volume.
Preparation of control solutions: weighing potential genotoxic impurities 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone and 4- (3-aminophenyl) -3-morpholinone as reference substances, and adding dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) to prepare a solution containing 0.15 mu g of each potential genotoxic impurity per 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4);
Preparation of a test solution: rivaroxaban plus dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) is taken to prepare a solution containing 3.0mg of rivaroxaban per 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4).
Preparing a standard sample solution: taking a proper amount of genotoxic impurities 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone and 4- (3-aminophenyl) -3-morpholinone as reference substances respectively, taking a proper amount of rivaroxaban, and adding dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) to prepare solutions containing 3.0mg of rivaroxaban and 0.15 mu g of each potential genotoxic impurity respectively in each 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4).
And (3) determination: the stationary phase of the HPLC is octadecylsilane chemically bonded silica, the mobile phase A is 0.01% ammonia water solution, the mobile phase B is methanol, the column temperature is 35 ℃, 10 mul of reference solution, sample solution and standard sample solution are respectively absorbed and injected into the HPLC, and data are read. Under the condition, the separation degrees of three peaks of genotoxic impurities, namely 4- (4-aminophenyl) -3-morpholone, 4- (2-aminophenyl) -3-morpholone and 4- (3-aminophenyl) -3-morpholone, in the reference solution meet the requirements; the test solution does not interfere with the detection of 3 potential basic toxins in the reference substance; the sample solution can effectively detect 3 potential base toxins and has good recovery rate.
Example 2
The method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography comprises the following steps
Preparation of a blank solvent: the volume ratio of dimethyl sulfoxide to acetonitrile to 0.01% ammonia water is 2:4: 4.
Preparation of control solutions: weighing potential genotoxic impurities 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone and 4- (3-aminophenyl) -3-morpholinone as reference substances, and adding dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) to prepare a solution containing 0.15 mu g of each potential genotoxic impurity per 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4);
preparation of a test solution: rivaroxaban plus dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) is taken to prepare a solution containing 3.0mg of rivaroxaban per 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4).
Preparing a standard sample solution: taking a proper amount of genotoxic impurities 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone and 4- (3-aminophenyl) -3-morpholinone as reference substances respectively, taking a proper amount of rivaroxaban, and adding dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) to prepare solutions containing 3.0mg of rivaroxaban and 0.15 mu g of each potential genotoxic impurity respectively in each 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4).
And (3) determination: the stationary phase of the HPLC is octadecylsilane chemically bonded silica, the mobile phase A is 0.01% ammonia water solution, the mobile phase B is methanol, the column temperature is 40 ℃, 10 mul of blank solvent, reference solution, sample solution and standard sample solution are respectively absorbed and injected into the HPLC, and data are read. HPLC charts of the blank solvent, the reference solution, the sample solution and the added standard sample solution are respectively shown in fig. 1-4.
Example 3
A method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography comprises the following steps:
preparation of a blank solvent: the volume ratio of dimethyl sulfoxide to acetonitrile to 0.01% ammonia water is 2:4: 4.
Preparation of control solutions: weighing potential genotoxic impurities 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone and 4- (3-aminophenyl) -3-morpholinone as reference substances, and adding dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) to prepare a solution containing 0.15 mu g of each potential genotoxic impurity per 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4);
preparation of a test solution: rivaroxaban plus dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) is taken to prepare a solution containing 3.0mg of rivaroxaban per 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4).
Preparing a standard sample solution: taking a proper amount of genotoxic impurities 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone and 4- (3-aminophenyl) -3-morpholinone as reference substances respectively, taking a proper amount of rivaroxaban, and adding dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) to prepare solutions containing 3.0mg of rivaroxaban and 0.15 mu g of each potential genotoxic impurity respectively in each 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4).
And (3) determination: the stationary phase of the HPLC is octadecylsilane chemically bonded silica, the mobile phase A is 0.01% ammonia water solution, the mobile phase B is methanol, the column temperature is 45 ℃, 10 mul of reference solution, sample solution and standard sample solution are respectively absorbed and injected into the HPLC, and data are read. Under the condition, the separation degrees of three peaks of genotoxic impurities, namely 4- (4-aminophenyl) -3-morpholone, 4- (2-aminophenyl) -3-morpholone and 4- (3-aminophenyl) -3-morpholone, in the reference solution meet the requirements; the test solution does not interfere with the detection of 3 potential basic toxins in the reference substance; the sample solution can effectively detect 3 potential base toxins and has good recovery rate.
Example 4 example of detection limits
Preparation of detection limiting solution: appropriate amounts of genotoxic impurities 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone and 4- (3-aminophenyl) -3-morpholinone as control substances were taken, and dimethylsulfoxide-acetonitrile-0.01% ammonia (2: 4: 4) was added to prepare a solution containing 22ng of each potentially genotoxic impurity per 1ml of dimethylsulfoxide-acetonitrile-0.01% ammonia (2: 4: 4).
And (3) determination: the stationary phase of the HPLC is octadecylsilane chemically bonded silica, the mobile phase A is 0.01% ammonia solution, the mobile phase B is methanol, the column temperature is 40 ℃, 10 mu l of detection limit solution is absorbed and injected into the HPLC, and data is read. The HPLC chart of the control solution is shown in FIG. 5, and that of the detection limit solution is shown in FIG. 6.
EXAMPLE 5 example of quantitative limits
Preparation of a quantitative limiting solution: appropriate amounts of genotoxic impurities 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone and 4- (3-aminophenyl) -3-morpholinone as control substances were taken, and dimethylsulfoxide-acetonitrile-0.01% ammonia (2: 4: 4) was added to prepare a solution containing 75ng of each potentially genotoxic impurity per 1ml of dimethylsulfoxide-acetonitrile-0.01% ammonia (2: 4: 4). That is, 1ml of dimethylsulfoxide-acetonitrile-0.01% aqueous ammonia (2: 4: 4) contained 75ng of 4- (4-aminophenyl) -3-morpholinone, 75ng of 4- (2-aminophenyl) -3-morpholinone, and 75ng of 4- (3-aminophenyl) -3-morpholinone.
Preparing a quantitative limit adding standard test sample solution: taking a proper amount of genotoxic impurities 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone and 4- (3-aminophenyl) -3-morpholinone as reference substances respectively, taking a proper amount of rivaroxaban, and adding dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) to prepare solutions containing 3.0mg of rivaroxaban and 75ng of each potential genotoxic impurity respectively in each 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4).
And (3) determination: the stationary phase of the HPLC is octadecylsilane chemically bonded silica, the mobile phase A is 0.01% ammonia solution, the mobile phase B is methanol, the column temperature is 40 ℃, 10 mul of each of the reference solution, the quantitative limit solution and the quantitative limit standard sample solution is absorbed and injected into the HPLC to read data. The HPLC chart of the control solution is shown in FIG. 7, that of the quantitative limit solution is shown in FIG. 8, and that of the quantitative limit standard test sample solution is shown in FIG. 9.
In the above example, the detector of the HPLC is an ultraviolet detector, and the detection wavelength is 230 nm. Mobile phase a was 0.01% ammonia solution and mobile phase B was methanol.
The mobile phase A and the mobile phase B are carried out according to the program
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0.01 | 90 | 10 |
| 5 | 90 | 10 |
| 25 | 30 | 70 |
| 27 | 30 | 70 |
| 27.1 | 90 | 10 |
。
Example 6
The method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography comprises the following steps
Preparation of a blank solvent: the volume ratio of dimethyl sulfoxide to acetonitrile to 0.01% ammonia water is 2:4: 4.
Preparation of control solutions: weighing potential genotoxic impurities 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone and 4- (3-aminophenyl) -3-morpholinone as reference substances, and adding dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) to prepare a solution containing 0.15 mu g of each potential genotoxic impurity per 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4);
Preparation of a test solution: rivaroxaban plus dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) is taken to prepare a solution containing 3.0mg of rivaroxaban per 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4).
Preparing a standard sample solution: taking a proper amount of genotoxic impurities 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone and 4- (3-aminophenyl) -3-morpholinone as reference substances respectively, taking a proper amount of rivaroxaban, and adding dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) to prepare solutions containing 3.0mg of rivaroxaban and 0.15 mu g of each potential genotoxic impurity respectively in each 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4).
And (3) determination: the stationary phase of the HPLC is octadecylsilane chemically bonded silica, the mobile phase A is 0.01% ammonia water solution, the mobile phase B is methanol, the column temperature is 40 ℃, 10 mul of blank solvent, reference solution, sample solution and standard sample solution are respectively absorbed and injected into the HPLC, and data are read. HPLC charts of the blank solvent, the reference solution, the sample solution and the added standard sample solution are shown in FIGS. 10 to 13, respectively.
Example 7
The method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography comprises the following steps
Preparation of a blank solvent: the volume ratio of dimethyl sulfoxide to acetonitrile to 0.01% ammonia water is 2:4: 4.
Preparation of control solutions: weighing potential genotoxic impurities 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone and 4- (3-aminophenyl) -3-morpholinone as reference substances, and adding dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) to prepare a solution containing 0.15 mu g of each potential genotoxic impurity per 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4);
preparation of a test solution: rivaroxaban plus dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) is taken to prepare a solution containing 3.0mg of rivaroxaban per 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4).
Preparing a standard sample solution: taking a proper amount of genotoxic impurities 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone and 4- (3-aminophenyl) -3-morpholinone as reference substances respectively, taking a proper amount of rivaroxaban, and adding dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) to prepare solutions containing 3.0mg of rivaroxaban and 0.15 mu g of each potential genotoxic impurity respectively in each 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4).
And (3) determination: the stationary phase of the HPLC is octadecylsilane chemically bonded silica, the mobile phase A is 0.01% ammonia water solution, the mobile phase B is methanol, the column temperature is 40 ℃, 10 mul of blank solvent, reference solution, sample solution and standard sample solution are respectively absorbed and injected into the HPLC, and data are read. HPLC charts of the blank solvent, the reference solution, the sample solution and the added standard sample solution are shown in FIGS. 14 to 17, respectively.
EXAMPLE 8 measurement of different wavelengths
A method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography comprises the following steps:
preparation of a blank solvent: the volume ratio of dimethyl sulfoxide to acetonitrile to 0.01% ammonia water is 2:4: 4.
Preparation of control solutions: weighing potential genotoxic impurities 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone and 4- (3-aminophenyl) -3-morpholinone as reference substances, and adding dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) to prepare a solution containing 0.15 mu g of each potential genotoxic impurity per 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4);
preparation of a test solution: rivaroxaban plus dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) is taken to prepare a solution containing 3.0mg of rivaroxaban per 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4).
Preparing a standard sample solution: taking a proper amount of genotoxic impurities 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone and 4- (3-aminophenyl) -3-morpholinone as reference substances respectively, taking a proper amount of rivaroxaban, and adding dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) to prepare solutions containing 3.0mg of rivaroxaban and 0.15 mu g of each potential genotoxic impurity respectively in each 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4).
And (3) determination: the stationary phase of the high performance liquid chromatograph is octadecylsilane chemically bonded silica, the mobile phase A is 0.01% ammonia water solution, the mobile phase B is methanol, the column temperature is 40 ℃, blank solvent, reference solution, sample solution and standard sample solution are respectively absorbed by 10 mu l, the mixture is injected into the high performance liquid chromatograph, detection wavelengths of 225nm, 230nm and 235nm are respectively adopted for detection, and then data are read. The detection spectra of the blank solvent, the reference solution, the sample solution and the added standard sample solution at different wavelengths are shown in FIGS. 18-21.
EXAMPLE 9 selection of different chromatography columns
A method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography comprises the following steps:
preparation of a blank solvent: the volume ratio of dimethyl sulfoxide to acetonitrile to 0.01% ammonia water is 2:4: 4.
Preparation of control solutions: weighing potential genotoxic impurities 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone and 4- (3-aminophenyl) -3-morpholinone as reference substances, and adding dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) to prepare a solution containing 0.15 mu g of each potential genotoxic impurity per 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4);
Preparation of a test solution: rivaroxaban plus dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) is taken to prepare a solution containing 3.0mg of rivaroxaban per 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4).
Preparing a standard sample solution: taking a proper amount of genotoxic impurities 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone and 4- (3-aminophenyl) -3-morpholinone as reference substances respectively, taking a proper amount of rivaroxaban, and adding dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4) to prepare solutions containing 3.0mg of rivaroxaban and 0.15 mu g of each potential genotoxic impurity respectively in each 1ml of dimethyl sulfoxide-acetonitrile-0.01% ammonia water (2: 4: 4).
And (3) determination: the stationary phase of the high performance liquid chromatograph is octadecylsilane chemically bonded silica, the mobile phase A is 0.01% ammonia water solution, the mobile phase B is methanol, the column temperature is 40 ℃, chromatographic columns of different manufacturers and specifications are replaced, Agilent C184.6mm 150mm 3.5um chromatographic columns are adopted, 10 mul of blank solvent, reference solution, test solution and labeled test solution are respectively absorbed, and the blank solvent, the reference solution, the test solution and the labeled test solution are injected into the high performance liquid chromatograph to read data. The detection spectra of the blank solvent, the reference solution, the sample solution and the added standard sample solution at different wavelengths are shown in FIGS. 22-25.
Claims (8)
1. The method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography is characterized by comprising the following steps:
octadecylsilane chemically bonded silica is used as a filler for the chromatographic column;
the detection wavelength is 220-240 nm;
dissolving a test sample in dimethyl sulfoxide-acetonitrile-0.01% ammonia water as a test sample solution, wherein the volume ratio of the dimethyl sulfoxide to the acetonitrile to the 0.01% ammonia water is (2-3): 3.5-4: 3.5 to 4;
the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.01-0.1% ammonia water, the mobile phase B is methanol, and the elution mode is gradient elution.
2. The method of claim 1, wherein the conditions of the gradient elution are:
3. the method of claim 1, wherein the conditions of the gradient elution are:
4. the method of claim 1, wherein the column temperature is 35 ℃ to 45 ℃.
5. The method of claim 1, wherein the detection wavelength is 230 nm.
6. The method of claim 1, wherein the volume ratio of dimethyl sulfoxide, acetonitrile and 0.01% -0.1% ammonia water is 2:4: 4.
7. The method of claim 1, wherein the potentially genotoxic impurity is 4- (4-aminophenyl) -3-morpholinone, 4- (2-aminophenyl) -3-morpholinone, or 4- (3-aminophenyl) -3-morpholinone.
8. The method of claim 1, wherein the sample size is 10 μ l to 100 μ l.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010542777.6A CN111855836A (en) | 2020-06-15 | 2020-06-15 | Method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010542777.6A CN111855836A (en) | 2020-06-15 | 2020-06-15 | Method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111855836A true CN111855836A (en) | 2020-10-30 |
Family
ID=72986647
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010542777.6A Pending CN111855836A (en) | 2020-06-15 | 2020-06-15 | Method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111855836A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114088843A (en) * | 2021-11-26 | 2022-02-25 | 上海皓鸿生物医药科技有限公司 | Method for detecting nitrosamine genotoxic impurities in varenicline intermediate |
| CN114216976A (en) * | 2021-11-30 | 2022-03-22 | 江苏中邦制药有限公司 | Method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography |
-
2020
- 2020-06-15 CN CN202010542777.6A patent/CN111855836A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114088843A (en) * | 2021-11-26 | 2022-02-25 | 上海皓鸿生物医药科技有限公司 | Method for detecting nitrosamine genotoxic impurities in varenicline intermediate |
| CN114088843B (en) * | 2021-11-26 | 2024-01-30 | 上海皓鸿生物医药科技有限公司 | Detection method for nitrosamine genotoxic impurities in valance intermediate |
| CN114216976A (en) * | 2021-11-30 | 2022-03-22 | 江苏中邦制药有限公司 | Method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography |
| CN114216976B (en) * | 2021-11-30 | 2024-04-30 | 江苏中邦制药有限公司 | Method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111855836A (en) | Method for determining potential genotoxic impurities in rivaroxaban by high performance liquid chromatography | |
| CN106855545B (en) | Method for simultaneously detecting fat-soluble vitamins and water-soluble vitamins in feed | |
| CN104122363B (en) | A kind of assay method of methylcobalamin tablet related substance | |
| CN111443151B (en) | Method for detecting content of trace cysteine in compound amino acid injection | |
| Pena-Gallego et al. | Biogenic amine determination in wines using solid-phase extraction: A comparative study | |
| CN105021761B (en) | A kind of method of the content and its true and false of a variety of organic acids in detection grape juice | |
| CN109254087B (en) | HPLC detection method for sodium dodecyl sulfate in ezetimibe tablet sample | |
| CN110441421B (en) | Method for determining dissolution rate of entacapone tablets by high performance liquid chromatography | |
| CN113009028A (en) | Method for detecting related substances of omeprazole sodium for injection | |
| CN104764840B (en) | The separation of palonosetron Hcl and impurity and detection method | |
| CN103063794B (en) | Content detecting and control method of epalrestat tablets | |
| CN108872428B (en) | High performance liquid chromatography analysis and detection method and application of adenine related substances | |
| CN109613163B (en) | Detection method for pimavanserin tartrate and impurities thereof | |
| CN114354801A (en) | Method for analyzing content of three aminopyridine isomers in (R) -3-Boc-aminopiperidine | |
| CN113866318A (en) | Method for detecting (6-aminopyridine-2-yl) (1-methylpiperidine-4-yl) methanone dihydrochloride | |
| CN114166979A (en) | Method for measuring content of 1-tetralone | |
| CN111812253A (en) | Method for detecting potential genotoxic impurities in compound containing benzimidazole structure | |
| CN103175930A (en) | High performance liquid chromatography analysis method for measuring sodium sulfite content | |
| CN107941956B (en) | High performance liquid chromatography analysis and detection method for tenofovir and enantiomer thereof | |
| CN111983108A (en) | Chiral analysis method for main alkaloids in electronic smoke sol | |
| CN111208231B (en) | Method for simultaneously detecting six illegally added compounds in plant beverage | |
| CN111521707A (en) | Method for determining apixaban concentration in blood plasma by LC-MS/MS | |
| CN110824038A (en) | Liquid chromatography analysis method of 2,3,4, 6-tetra-O-trimethylsilyl-D-gluconolactone | |
| CN116338025A (en) | Method for measuring N-nitrosovalinamide by liquid chromatography-mass spectrometer | |
| CN108562674B (en) | Method for measuring mesylate by derivatization HPLC-UV method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20220715 Address after: 210000 No. 36, Shuanggao Road, economic development zone, Gaochun District, Nanjing, Jiangsu Province Applicant after: Nanjing Red Sun Pharmaceutical Research Institute Co.,Ltd. Applicant after: Jiangsu Zhongbang Pharmaceutical Co.,Ltd. Address before: 210000 No. 36, Shuanggao Road, economic development zone, Gaochun District, Nanjing, Jiangsu Province Applicant before: Nanjing Red Sun Pharmaceutical Research Institute Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |