CN111983055B - Method for separating and measuring rivaroxaban intermediate related substances by using HPLC (high performance liquid chromatography) - Google Patents
Method for separating and measuring rivaroxaban intermediate related substances by using HPLC (high performance liquid chromatography) Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—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
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- 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
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- G—PHYSICS
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- 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
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- G—PHYSICS
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- 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
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—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
Abstract
The invention provides a method for separating and measuring related substances of rivaroxaban intermediates by using HPLC (high performance liquid chromatography), which comprises the following steps of: octadecylsilane chemically bonded silica is used as a filling agent, 0.01-0.03 mol/L potassium dihydrogen phosphate solution (the pH value is adjusted to 5.0-8.0 by sodium hydroxide) is used as a mobile phase A, acetonitrile or methanol or acetonitrile-methanol is used as a mobile phase B for gradient elution, the flow rate is 0.8-1.2 ml/min, the column temperature is 25-40 ℃, and related substances of the rivaroxaban intermediate are detected by an ultraviolet detector. The method comprehensively considers the analysis column, the mobile phase, the gradient elution program, the flow rate and the comprehensive influence of the column temperature on the separation detection, optimizes the detection result, has the advantages of rapidness, simplicity, convenience, high sensitivity, accuracy, reliability and wide applicability, and is suitable for separating and determining related substances of rivaroxaban intermediates, thereby effectively controlling the quality of the medicine.
Description
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a method for separating and determining related substances of rivaroxaban intermediates by using HPLC (high performance liquid chromatography).
Background
Rivaroxaban (Rivaroxaban, trade name xarelo), a small molecule oral anticoagulant developed by bayer and qiangsheng, was approved by the U.S. Food and Drug Administration (FDA) on day 01 of year 07/2011 for the prevention of deep venous thrombosis in patients undergoing knee or hip replacement surgery.
(S) -N-glycidylphthalideneThe amine is a rivaroxaban intermediate with a molecular formula: c11H9NO3Molecular weight: 203.19, the chemical formula is shown as the following formula (1):
in the preparation process of the intermediate, a plurality of impurities are generated due to various factors such as starting materials, synthesis process, degradation and the like, wherein the impurities I, II, III and IV are easy to generate in the synthesis process and are used as main inspected impurities in related substance projects, and the limit requirements of the impurities are all not more than 0.50 wt%.
Impurity I is the starting material, molecular formula: c8H5NO2Molecular weight: 147.13, the chemical formula is shown as the following formula (2):
the impurity II is a reaction intermediate in the step 1, and has a molecular formula: c11H10ClNO3Molecular weight: 239.66, the chemical formula is shown as the following formula (3):
impurity III is a hydrolyzed impurity, and the molecular formula is as follows: c11H11NO4Molecular weight: 221.21, the chemical formula is represented by the following formula (4):
impurity IV is a dimer, and the molecular formula is as follows: c 19H14N2O5Molecular weight: 350.32, the chemical formula is shown as the following formula (5):
the analysis and detection of the intermediate have important effects on reaction control and yield improvement, and simultaneously directly influence the quality of a final product, so that the establishment of a stable and effective analysis and detection method with simple operation is very necessary for analyzing and detecting the rivaroxaban intermediate. In the prior art, no analysis method suitable for quickly, simply and accurately analyzing and detecting related substances of rivaroxaban intermediates exists. Therefore, further improvement and optimization needs exist for the determination method of related substances of rivaroxaban intermediates.
Disclosure of Invention
In order to overcome the technical problems in the prior art, the inventor provides a method for separating and determining related substances of rivaroxaban intermediates by using HPLC (high performance liquid chromatography), which has the advantages of rapidness, simplicity, high sensitivity, accuracy and reliability after carrying out a great deal of intensive research.
The technical scheme adopted by the invention is as follows:
a method for separating and determining related substances of rivaroxaban intermediates by using HPLC (high performance liquid chromatography), which comprises the following steps of: performing gradient elution by using octadecylsilane chemically bonded silica as a filling agent and using 0.01-0.03 mol/L potassium dihydrogen phosphate solution as a mobile phase A and acetonitrile or methanol or acetonitrile-methanol as a mobile phase B, wherein the flow rate is 0.8-1.2 ml/min, the column temperature is 25-40 ℃, the pH value of the potassium dihydrogen phosphate solution is adjusted to 5.0-8.0 by using sodium hydroxide, and the volume ratio of the acetonitrile to the methanol is 80: 20-100: 0; and detecting related substances of the rivaroxaban intermediate by adopting an ultraviolet detector, wherein the detection wavelength of the ultraviolet detector is 205-230 nm. The concentration and pH value of the potassium dihydrogen phosphate can affect the column efficiency and the separation degree of the main peak and impurities, and if the concentration of the buffer salt is too low, the column efficiency of the main peak and the impurities can be affected, and if the concentration is too high, the chromatographic column can be damaged.
The invention relates to a method for separating and determining related substances of rivaroxaban intermediates by HPLC (high performance liquid chromatography), wherein the conditions of gradient elution are as follows:
| time, minutes | Mobile phase A, volume% | Mobile phase B, volume% |
| 0 | 70～80 | 20～30 |
| 10 | 70～80 | 20～30 |
| 30 | 45～55 | 45～55 |
| 35 | 45～55 | 45～55 |
| 36 | 70～80 | 20～30 |
| 45 | 70～80 | 20～30 |
An unknown impurity exists between the main peak of the rivaroxaban intermediate and the impurity II, three peaks of the rivaroxaban intermediate are difficult to separate, the separation degree is good by adopting the data, and the separation degree between the three peaks can be reduced by adopting other data.
The invention relates to a method for separating and determining related substances of rivaroxaban intermediates by HPLC (high performance liquid chromatography), wherein the conditions of gradient elution are as follows:
| time in minutes | Mobile phase A, volume% | Mobile phase B, volume% |
| 0 | 75 | 25 |
| 10 | 75 | 25 |
| 30 | 50 | 50 |
| 35 | 50 | 50 |
| 36 | 75 | 25 |
| 45 | 75 | 25 |
The separation obtained is the best and the peak shape is the best.
The invention relates to a method for separating and measuring related substances of rivaroxaban intermediates by HPLC, wherein a mobile phase A is a potassium dihydrogen phosphate solution with the concentration of 0.02mol/L, and the pH value is 7.0; mobile phase B was acetonitrile. Thus, the degree of separation and the number of theoretical plates are both good.
The invention relates to a method for separating and determining related substances of a rivaroxaban intermediate by using HPLC (high performance liquid chromatography), wherein the detection wavelength of an ultraviolet detector is 220 nm. This can further improve the detection sensitivity of impurities.
The invention relates to a method for separating and measuring rivaroxaban intermediate related substances by using HPLC (high performance liquid chromatography), wherein the rivaroxaban intermediate related substances comprise one or more of an impurity I, an impurity II, an impurity III and an impurity IV, and the specific structural formula is as follows:
the method for separating and measuring related substances of the rivaroxaban intermediate by using HPLC (high performance liquid chromatography) is characterized in that the length of a chromatographic column is 150-250 mm, and the particle size of a filling agent is 1.8-5 mu m.
The method for separating and measuring related substances of the rivaroxaban intermediate by using HPLC (high performance liquid chromatography) is characterized in that the length of a chromatographic column is 250 mm. This can further improve the degree of separation.
The invention relates to a method for separating and measuring related substances of a rivaroxaban intermediate by using HPLC (high performance liquid chromatography), wherein the particle size of a filling agent is 5 mu m. This can further improve the degree of separation.
The invention relates to a method for separating and measuring related substances of rivaroxaban intermediates by HPLC, wherein the flow rate is 1.0 ml/min; the column temperature was 30 ℃.
Compared with the prior art, the invention has the following beneficial effects:
the method for separating and determining the rivaroxaban intermediate related substances by using HPLC optimizes the detection result by comprehensively considering the comprehensive influence of an analysis column, a mobile phase, a gradient elution program, flow rate and column temperature on separation detection, can quickly and efficiently separate impurities I, II, III and IV in the rivaroxaban intermediate under the same chromatographic condition, has high sensitivity, strong specificity, strong accuracy, quickness, simplicity and convenience in operation, can effectively control the quality of a medicine, and is suitable for separating and determining the rivaroxaban intermediate related substances.
Drawings
FIG. 1 is a chromatogram of a blank solution tested under the conditions of example 1 in accordance with the present invention;
FIG. 2 is a chromatogram of a system-compatible solution tested under the conditions of example 1 in the present invention;
FIG. 3 is a chromatogram of a test solution tested under the conditions of example 1 in accordance with the present invention;
FIG. 4 is a chromatogram of a limiting quantitation solution detected under the conditions of example 1 in the present invention;
FIG. 5 is a chromatogram of a detection limiting solution detected under the conditions of example 1 in the present invention;
FIG. 6 is a chromatogram of a blank solution tested under the conditions of example 2 in accordance with the present invention;
FIG. 7 is a chromatogram of a system suitability solution tested under the conditions of example 2 in the present invention;
FIG. 8 is a chromatogram of a test solution tested under the conditions of example 2 in accordance with the present invention;
FIG. 9 is a chromatogram of a limiting quantitation solution detected under the conditions of example 2 in the present invention;
FIG. 10 is a chromatogram of a detection limiting solution detected under the conditions of example 2 in the present invention;
FIG. 11 is a chromatogram of a blank solution tested under the conditions of example 3 in accordance with the present invention;
FIG. 12 is a chromatogram of a system suitability solution tested under the conditions of example 3 in the present invention;
FIG. 13 is a chromatogram of a test solution tested under the conditions of example 3 in accordance with the present invention;
FIG. 14 is a chromatogram of a limiting quantitation solution detected under the conditions of example 3 in the present invention;
FIG. 15 is a chromatogram of a detection limiting solution detected under the conditions of example 3 in the present invention.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and the accompanying drawings. The following examples will aid those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any manner. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention. The reagents and instruments used are not indicated by manufacturers, and conventional products can be obtained commercially.
Rivaroxaban intermediates and impurity reference substances used in the embodiment of the invention are prepared by the inventor.
Example 1
The chromatographic conditions were as follows:
a chromatographic column: agilent 5TC-C18250 multiplied by 4.6mm
Mobile phase A: 0.02mol/L potassium dihydrogen phosphate solution (pH adjusted to 6.0 with 10% sodium hydroxide)
Mobile phase B: acetonitrile-methanol (volume ratio 80: 20)
Column temperature: 30 deg.C
Flow rate: 1.0ml/min
Detection wavelength: 220nm
Sample introduction amount: 10 μ L
The gradient elution procedure was:
TABLE 1 gradient elution procedure
| Time, minutes | Mobile phase A,% by volume | Mobile phase B,% by |
| 0 | 75 | 25 |
| 10 | 75 | 25 |
| 30 | 50 | 50 |
| 35 | 50 | 50 |
| 36 | 75 | 25 |
| 45 | 75 | 25 |
Solution preparation:
impurity reference stock solution: accurately weighing about 12.5mg of impurity I reference substance, impurity II reference substance, impurity III reference substance and impurity IV reference substance respectively, placing in a same 50ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking up to obtain the final product; the diluent is as follows: and (3) acetonitrile.
System applicability solution: precisely weighing about 25mg of rivaroxaban intermediate working reference substance, placing the rivaroxaban intermediate working reference substance into a 100ml measuring flask, precisely adding 1ml of impurity reference substance stock solution into the 100ml measuring flask, adding a diluent to dilute to a scale, and shaking up to obtain the rivaroxaban intermediate working reference substance.
Test solution: taking about 25mg of rivaroxaban intermediate, precisely weighing, placing in a 100ml measuring flask, adding a diluent to dissolve and dilute to a scale, and shaking up.
Rivaroxaban intermediate control stock solutions: and (3) precisely weighing about 25mg of rivaroxaban intermediate working reference substance, placing the rivaroxaban intermediate working reference substance into a 100ml measuring flask, adding a diluent to dissolve and dilute the rivaroxaban intermediate working reference substance to a scale, and shaking up the rivaroxaban intermediate working reference substance to obtain the rivaroxaban intermediate.
Quantitative limiting solution: precisely measuring the impurity reference substance storage solution and the rivaroxaban intermediate reference substance storage solution in measuring bottles of 1ml to 100ml respectively, adding a diluent to dilute to a scale, shaking up, precisely measuring 1ml to 100ml, adding the diluent to dilute to the scale, and shaking up to obtain the rivaroxaban intermediate compound.
Detection limiting solution: precisely measuring 5ml of the quantitative limiting solution, putting the quantitative limiting solution into a 10ml measuring flask, adding a diluent to dilute to a scale, and shaking up to obtain the product.
And (3) determination: respectively injecting blank solution (namely diluent), system applicability solution, sample solution, quantitative limiting solution and detection limiting solution into a high performance liquid chromatograph for detection, using octadecylsilane chemically bonded silica as a filler (the particle size is 5 mu m, the inner diameter of a column is 4.6mm, the length of a chromatographic column is 250mm), detecting according to a gradient elution program in Table 1, and recording a chromatogram.
Chromatograms of the blank solution (i.e., diluent), the system applicability solution, the sample solution, the quantification limit solution, and the detection limit solution are shown in fig. 1, 2, 3, 4, and 5, respectively, and it can be seen that fig. 1 shows that the blank does not interfere with the inspection of impurities; FIG. 2 shows that the separation degree between each impurity and rivaroxaban intermediate is good, and the specific applicability profile data of the rivaroxaban intermediate system is shown in Table 2; FIG. 3 shows that no impurity I, no impurity II and no impurity IV are detected in a self-made rivaroxaban intermediate sample, and the detected impurity III is below 0.5 wt%; the other single impurities detected are all below 0.5 wt%; FIG. 4 shows that the quantitation limits of rivaroxaban intermediate and impurity I, impurity II, impurity III and impurity IV are 0.01 wt%, 0.01 wt% and 0.01 wt%, respectively; FIG. 5 shows that the detection limits of the rivaroxaban intermediate and impurity I, impurity II, impurity III and impurity IV are 0.005 wt%, 0.005 wt% and 0.005 wt%, respectively, which are lower than the limit of each impurity by 0.50 wt%; the method has high detection sensitivity.
Table 2 table of applicability profiles of rivaroxaban system in example 1
Example 2
The chromatographic conditions were as follows:
a chromatographic column: agilent 5TC-C18250 multiplied by 4.6mm
Mobile phase A: 0.02mol/L potassium dihydrogen phosphate solution (pH adjusted to 7.0 with 10% sodium hydroxide)
Mobile phase B: acetonitrile
Column temperature: 30 deg.C
Flow rate: 1.0ml/min
Detection wavelength: 220nm
Sample introduction amount: 10 μ l
The gradient elution procedure was:
TABLE 3 gradient elution procedure
| Time in minutes | Mobile phase A, volume% | Mobile phase B, |
| 0 | 75 | 25 |
| 10 | 75 | 25 |
| 30 | 50 | 50 |
| 35 | 50 | 50 |
| 36 | 75 | 25 |
| 45 | 75 | 25 |
Solution preparation:
impurity control stock solution: accurately weighing about 12.5mg of impurity I reference substance, impurity II reference substance, impurity III reference substance and impurity IV reference substance respectively, placing in a same 50ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking up to obtain the final product; the diluent is as follows: and (3) acetonitrile.
System applicability solution: precisely weighing about 25mg of rivaroxaban intermediate working reference substance, placing the rivaroxaban intermediate working reference substance into a 100ml measuring flask, precisely adding 1ml of impurity reference substance stock solution into the 100ml measuring flask, adding a diluent to dilute to a scale, and shaking up to obtain the rivaroxaban intermediate working reference substance.
Test solution: taking about 25mg of rivaroxaban intermediate, precisely weighing, placing in a 100ml measuring flask, adding a diluent to dissolve and dilute to a scale, and shaking up.
Rivaroxaban intermediate control stock solutions: and (3) precisely weighing about 25mg of rivaroxaban intermediate working reference substance, placing the rivaroxaban intermediate working reference substance into a 100ml measuring flask, adding a diluent to dissolve and dilute the rivaroxaban intermediate working reference substance to a scale, and shaking up the rivaroxaban intermediate working reference substance to obtain the rivaroxaban intermediate.
Quantitative limiting solution: precisely measuring the impurity reference substance storage solution and the rivaroxaban intermediate reference substance storage solution in measuring bottles of 1ml to 100ml respectively, adding a diluent to dilute to a scale, shaking up, precisely measuring 1ml to 100ml, adding the diluent to dilute to the scale, and shaking up to obtain the rivaroxaban intermediate compound.
Detection limiting solution: precisely measuring 5ml of the quantitative limiting solution, putting the quantitative limiting solution into a 10ml measuring flask, adding a diluent to dilute to a scale, and shaking up to obtain the product.
And (3) determination: respectively injecting blank solution (namely diluent), system applicability solution, sample solution, quantitative limiting solution and detection limiting solution into a high performance liquid chromatograph for detection, using octadecylsilane chemically bonded silica as a filler (the particle size is 5 mu m, the inner diameter of a column is 4.6mm, the length of a chromatographic column is 250mm), detecting according to a gradient elution program shown in Table 3, and recording a chromatogram.
Chromatograms of the blank solution (i.e., diluent), the system applicability solution, the test sample solution, the quantitation limit solution, and the detection limit solution are shown in fig. 6, 7, 8, 9, and 10, respectively, and it can be seen that fig. 6 shows that the blank does not interfere with the impurity inspection; FIG. 7 shows that the separation degree between each impurity and rivaroxaban intermediate is good, and the applicability map data of the specific rivaroxaban intermediate system is shown in Table 4; FIG. 8 shows that no impurity I, no impurity II and no impurity IV are detected in a self-made rivaroxaban intermediate sample, and the detected impurity III is below 0.5 wt%; the other single impurities detected are all below 0.5 wt%; FIG. 9 shows that the quantitation limits of rivaroxaban intermediate and impurity I, impurity II, impurity III and impurity IV are 0.01 wt%, 0.01 wt% and 0.01 wt%, respectively; FIG. 10 shows that the detection limits of rivaroxaban intermediate and impurity I, impurity II, impurity III and impurity IV are 0.005 wt%, 0.005 wt% and 0.005 wt%, respectively, which are lower than the limit of each impurity by 0.50 wt%; the method has high detection sensitivity.
Table 4 table of applicability profiles of rivaroxaban system in example 2
Example 3
The chromatographic conditions were as follows:
a chromatographic column: agilent 5TC-C18250 multiplied by 4.6mm
Mobile phase A: 0.01mol/L potassium dihydrogen phosphate solution (pH adjusted to 7.5 with 10% sodium hydroxide)
Mobile phase B: acetonitrile
Column temperature: 30 deg.C
Flow rate: 1.0ml/min
Detection wavelength: 220nm
Sample introduction amount: 10 μ l
The gradient elution procedure was:
TABLE 5 gradient elution procedure
| Time in minutes | Mobile phase A, volume% | Mobile phase B, |
| 0 | 75 | 25 |
| 10 | 75 | 25 |
| 30 | 50 | 50 |
| 35 | 50 | 50 |
| 36 | 75 | 25 |
| 45 | 75 | 25 |
Solution preparation:
impurity reference stock solution: accurately weighing about 12.5mg of impurity I reference substance, impurity II reference substance, impurity III reference substance and impurity IV reference substance respectively, placing in a same 50ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking up to obtain the final product; the diluent is as follows: and (3) acetonitrile.
System applicability solution: precisely weighing about 25mg of rivaroxaban intermediate working reference substance, placing the rivaroxaban intermediate working reference substance into a 100ml measuring flask, precisely adding 1ml of impurity reference substance stock solution into the 100ml measuring flask, adding a diluent to dilute to a scale, and shaking up to obtain the rivaroxaban intermediate working reference substance.
Test solution: taking about 25mg of rivaroxaban intermediate, precisely weighing, placing in a 100ml measuring flask, adding a diluent to dissolve and dilute to a scale, and shaking up.
Rivaroxaban intermediate control stock solutions: and (3) precisely weighing about 25mg of rivaroxaban intermediate working reference substance, placing the rivaroxaban intermediate working reference substance into a 100ml measuring flask, adding a diluent to dissolve and dilute the rivaroxaban intermediate working reference substance to a scale, and shaking up the rivaroxaban intermediate working reference substance to obtain the rivaroxaban intermediate.
Quantitative limiting solution: precisely measuring the impurity reference substance storage solution and the rivaroxaban intermediate reference substance storage solution in measuring bottles of 1ml to 100ml respectively, adding a diluent to dilute to a scale, shaking up, precisely measuring 1ml to 100ml, adding the diluent to dilute to the scale, and shaking up to obtain the rivaroxaban intermediate compound.
Detection limiting solution: precisely measuring 5ml of the quantitative limiting solution, putting the quantitative limiting solution into a 10ml measuring flask, adding a diluent to dilute to a scale, and shaking up to obtain the product.
And (3) determination: respectively injecting blank solution (namely diluent), system applicability solution, sample solution, quantitative limiting solution and detection limiting solution into a high performance liquid chromatograph for detection, using octadecylsilane chemically bonded silica as a filler (the particle size is 5 mu m, the inner diameter of a column is 4.6mm, the length of a chromatographic column is 250mm), detecting according to a gradient elution program shown in Table 5, and recording a chromatogram.
Chromatograms of the blank solution (i.e., diluent), the system applicability solution, the sample solution, the quantification limit solution, and the detection limit solution are shown in fig. 11, 12, 13, 14, and 15, respectively, and it can be seen that fig. 11 shows that the blank does not interfere with the impurity inspection; FIG. 12 shows that the separation degree between each impurity and rivaroxaban intermediate is good, and the specific applicability profile data of the rivaroxaban intermediate system is shown in Table 6; FIG. 13 shows that no impurity I, no impurity II and no impurity IV are detected in the home-made rivaroxaban intermediate sample, and the detected impurity III is below 0.5 wt%; the other single impurities detected are all below 0.5 wt%; FIG. 14 shows that the quantitation limits of rivaroxaban intermediate and impurity I, impurity II, impurity III and impurity IV are 0.01 wt%, 0.01 wt% and 0.01 wt%, respectively; FIG. 15 shows that the detection limits of rivaroxaban intermediate and impurity I, impurity II, impurity III and impurity IV are 0.005 wt%, 0.005 wt% and 0.005 wt%, respectively, which are lower than the limit of each impurity by 0.50 wt%; the method has high detection sensitivity.
Table 6 table of applicability profiles of rivaroxaban system in example 3
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention by those skilled in the art should fall within the protection scope defined by the claims of the present invention without departing from the spirit of the present invention.
Claims (7)
1. A method for separating and determining related substances of rivaroxaban intermediates by HPLC (high performance liquid chromatography), which is characterized by comprising the following steps: the method comprises the following steps: performing gradient elution by using octadecylsilane chemically bonded silica as a filler, using 0.01-0.03 mol/L potassium dihydrogen phosphate solution as a mobile phase A and acetonitrile or methanol or acetonitrile-methanol as a mobile phase B, wherein the flow rate is 0.8-1.2 ml/min, the column temperature is 25-40 ℃, the pH value of the potassium dihydrogen phosphate solution is adjusted to 5.0-8.0 by using 10% sodium hydroxide, and the volume ratio of the acetonitrile-methanol is 80: 20-100: 0; detecting related substances of the rivaroxaban intermediate by using an ultraviolet detector, wherein the detection wavelength of the ultraviolet detector is 205-230 nm; the conditions of the gradient elution are as follows:
Related substances of the rivaroxaban intermediate comprise an impurity I, an impurity II, an impurity III and an impurity IV, and the specific structural formula is as follows:
2. the method for separating and determining rivaroxaban intermediate related substances by HPLC as claimed in claim 1, wherein: the mobile phase A is 0.02mol/L potassium dihydrogen phosphate solution, and the pH value is 7.0; mobile phase B was acetonitrile.
3. The method for separating and determining rivaroxaban intermediate related substances by HPLC as claimed in claim 1, wherein: the detection wavelength of the ultraviolet detector is 220 nm.
4. The method for separating and determining related substances of rivaroxaban intermediate by HPLC as claimed in claim 1, wherein: the length of the chromatographic column is 150 mm-250 mm, and the particle size of the filler is 1.8-5 mu m.
5. The method for separating and determining rivaroxaban intermediate related substances by HPLC as claimed in claim 1, wherein: the column length was 250 mm.
6. The method for separating and determining rivaroxaban intermediate related substances by HPLC as claimed in claim 1, wherein: the particle size of the filler is 5 mu m.
7. The method for separating and determining rivaroxaban intermediate related substances by HPLC according to any one of claims 1 to 6, wherein: the flow rate is 1.0 ml/min; the column temperature was 30 ℃.
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