CN114264735A - Method for detecting rivaroxaban isomer content - Google Patents
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Abstract
The application provides a method for detecting rivaroxaban isomer content. The method for detecting the content of the rivaroxaban isomer comprises the following steps: preparing a test solution of rivaroxaban; preparing a control solution of rivaroxaban; and (2) respectively carrying out detection and analysis operations on the test solution and the control solution by adopting a high performance liquid chromatograph, wherein the chromatographic column is a reversed-phase chiral chromatographic column, the mobile phase is acetonitrile, the column temperature is 20-30 ℃, and the flow rate is 0.8-1.2 mL/min. The rivaroxaban isomer content detection method has the advantages of being good in durability and high in accuracy.
Description
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a method for detecting rivaroxaban isomer content.
Background
Anticoagulant drugs play an important role in preventing thrombosis, but traditional anticoagulant drugs such as heparin need to be administered parenterally, and are inconvenient to use outside hospitals. Oral anticoagulants such as warfarin have a narrow therapeutic window, and the prothrombin time needs to be monitored frequently, which brings inconvenience to patients. The novel oral anticoagulant rivaroxaban (rivaroxaban) is a direct factor Xa inhibitor with the advantages of convenient use and no need for monitoring. Rivaroxaban has high selectivity on Xa factor, is used for preventing venous thromboembolism after hip or knee joint replacement, has excellent in-vivo activity and bioavailability, and has good application prospect.
In the rivaroxaban synthesis process, impurity residues affect the purity, yield and quality of rivaroxaban, so that effective control of impurities in the rivaroxaban process is particularly important. At present, the rivaroxaban related substances are mostly measured by adopting a high performance forward liquid chromatography, and the problems of poor durability and low sensitivity of a chromatographic column exist when the forward chromatography is used for detecting rivaroxaban medicines.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the rivaroxaban isomer content detection method with good durability and high accuracy.
The purpose of the invention is realized by the following technical scheme:
a method for detecting the content of rivaroxaban isomer comprises the following steps:
preparing a test solution of rivaroxaban;
preparing a control solution of rivaroxaban;
and (2) respectively detecting and analyzing the test solution and the control solution by adopting a high performance liquid chromatograph, wherein the chromatographic column is a reversed-phase chiral chromatographic column, the mobile phase is acetonitrile water solution or methanol water solution, the column temperature is 20-30 ℃, and the flow rate is 0.8-1.2 mL/min.
In one embodiment, before the step of preparing the sample solution of rivaroxaban, the method for detecting the content of the rivaroxaban isomer further comprises the following steps:
preparing a system applicability test solution of the rivaroxaban.
In one embodiment, the separation degree of rivaroxaban isomer and rivaroxaban in the system suitability test solution is greater than or equal to 1.5.
In one embodiment, after the step of preparing the rivaroxaban control solution and before the step of performing detection and analysis operations on the test solution and the control solution respectively by using a high performance liquid chromatograph, the method for detecting the content of the rivaroxaban isomer further includes the following steps:
preparing a sensitive solution of rivaroxaban.
In one embodiment, the signal-to-noise ratio of the main peak in the sensitivity solution is ≧ 10.
In one embodiment, the reverse phase Chiral chromatography column is Chiral pak ic.250 x 4.6mm, 5 um.
In one embodiment, the sample injection volume in the detection and analysis operation is 15-25 μ L.
In one embodiment, the detector of the high performance liquid chromatograph is an ultraviolet absorption detector, and the detection wavelength is 250 nm.
In one embodiment, the reverse phase Chiral chromatography column is Chiral pak AS-H,250 x 4.6 mm.5um.
In one embodiment, after the step of preparing the sample solution of rivaroxaban and before the step of performing detection and analysis operations on the sample solution and the control solution respectively by using a high performance liquid chromatograph, the method for detecting the content of the rivaroxaban isomer further includes the following steps:
and carrying out ultrasonic dissolution operation on the test solution.
Compared with the prior art, the invention has at least the following advantages:
1. the method for detecting the content of the rivaroxaban isomer adopts a chiral chromatographic column, wherein the chiral chromatographic column is a monomer with optical activity and is fixed on silica gel or other polymers in a chromatogram to form a chiral stationary phase. The chiral environment is introduced, so that the enantiomers of rivaroxaban present physical characteristic difference, the purpose of optical isomer resolution is achieved, and the separation effect is good. Furthermore, the reversed-phase chiral chromatographic column is adopted for carrying out reversed-phase chromatographic determination analysis, so that the separation degree of rivaroxaban and rivaroxaban isomers can be effectively improved, the sensitivity in the rivaroxaban detection process can be improved, the signal-to-noise ratio of chromatographic peaks is further improved, and the accuracy and precision of rivaroxaban determination results are improved.
2. According to the method for detecting the rivaroxaban isomer content, a reversed-phase chiral chromatographic column is used for carrying out reversed-phase chromatographic determination analysis, and due to the fact that a large number of auxiliary materials exist in rivaroxaban drugs, the auxiliary materials have the problem of solubility in organic solvents, water is needed to be involved to dissolve tablet auxiliary materials, but a normal-phase system cannot contain water, and the normal-phase chromatographic column is prone to damage. The method adopts the reversed phase chromatography, can solve the problem of solubility of the rivaroxaban drug, and simultaneously can be compatible with water, thereby improving the durability of the chromatographic column.
3. According to the method for detecting the rivaroxaban isomer content, the column temperature is 20-30 ℃ and the flow rate is 0.8-1.2 mL/min during chromatographic analysis, so that rivaroxaban and rivaroxaban isomers in a sample to be detected are sequentially and fully separated and appear in different retention times, and a better peak shape can be formed, so that the accuracy of a rivaroxaban determination result is improved.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
Fig. 1 is a flow chart of a method for detecting rivaroxaban isomer content according to an embodiment of the present invention.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
It will be understood that when an element is referred to as being "secured to" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present. The terms "vertical," "horizontal," "left," "right," and the like as used herein are for illustrative purposes only and do not represent the only embodiments.
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 to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The application provides a method for detecting rivaroxaban isomer content. The method for detecting the content of the rivaroxaban isomer comprises the following steps: preparing a test solution of rivaroxaban; preparing a control solution of rivaroxaban; and (2) respectively carrying out detection and analysis operations on the test solution and the control solution by adopting a high performance liquid chromatograph, wherein the chromatographic column is a reversed-phase chiral chromatographic column, the mobile phase is acetonitrile, the column temperature is 20-30 ℃, and the flow rate is 0.8-1.2 mL/min.
The method for detecting the content of the rivaroxaban isomer adopts a chiral chromatographic column, wherein the chiral chromatographic column is a monomer with optical activity and is fixed on silica gel or other polymers in a chromatogram to form a chiral stationary phase. The chiral environment is introduced, so that the enantiomers of rivaroxaban present physical characteristic difference, the purpose of optical isomer resolution is achieved, and the separation effect is good. Furthermore, the reversed-phase chiral chromatographic column is adopted for carrying out reversed-phase chromatographic determination analysis, so that the separation degree of rivaroxaban and rivaroxaban isomers can be effectively improved, the sensitivity in the rivaroxaban detection process can be improved, the signal-to-noise ratio of chromatographic peaks is further improved, and the accuracy and precision of rivaroxaban determination results are improved. Because a large amount of auxiliary materials exist in rivaroxaban medicines, the auxiliary materials have the problem of solubility in organic solvents, water is needed to be involved in dissolving the auxiliary materials of the tablets, but a normal phase system cannot be compatible with water, and a normal phase chromatographic column is easily damaged. The method adopts the reversed phase chromatography, so that the problem of solubility of rivaroxaban medicaments can be solved, and the rivaroxaban medicaments can be compatible with water, so that the durability of a chromatographic column is improved; the rivaroxaban isomer content detection method is good in durability and stable in system applicability. Furthermore, the column temperature of the method for detecting the rivaroxaban isomer content is 20-30 ℃ and the flow rate is 0.8-1.2 mL/min during chromatographic analysis, so that rivaroxaban and rivaroxaban isomers in a sample to be detected are fully separated in sequence and appear in different retention times, and a better peak shape can be formed, and the accuracy of a rivaroxaban determination result is improved.
Referring to fig. 1, in order to better understand the method for detecting the content of rivaroxaban isomer in the present application, the following further explains the method for detecting the content of rivaroxaban isomer in the present application, and the method for detecting the content of rivaroxaban isomer in one embodiment includes the following steps:
s100, preparing a test solution of rivaroxaban.
In this embodiment, a preset amount of a sample to be detected is weighed, placed in a measuring flask, and a diluent is added to perform a constant volume operation, so as to obtain a sample solution. Specifically, 640mg of rivaroxaban tablet fine powder, namely 75mg of rivaroxaban, is taken, wherein the rivaroxaban tablet is provided by Guangzhou Hairy pharmaceutical industry Co., Ltd, Yangziang pharmaceutical industry group, precisely weighed and placed in a 50mL measuring flask, a diluent not more than 50mL is added, ultrasonic operation is carried out, then the solution in the volumetric flask is diluted to a scale mark by the diluent, wherein the diluent is acetonitrile aqueous solution, and acetonitrile in the acetonitrile aqueous solution: 50:50 of water; and then shaking up the sample solution in the volumetric flask to finish constant volume operation, thus obtaining a test solution containing 1.5mg of rivaroxaban in every 1 mL.
And S200, preparing a rivaroxaban control solution.
In this embodiment, a predetermined amount of the sample solution is measured, placed in a measuring flask, and a diluent is added to perform a constant volume operation, so as to obtain a control solution. Specifically, 5mL of a sample solution is precisely measured, placed in a 100mL measuring flask, diluted to a scale with a diluent, and shaken up to serve as a reference stock solution; precisely measuring 3mL of the control stock solution, placing the control stock solution into a 100mL measuring flask, and diluting the control stock solution to the scale mark of the measuring flask by using a diluent, wherein the diluent is an acetonitrile aqueous solution, and the acetonitrile in the acetonitrile aqueous solution: 50:50 of water; and then shaking up the solution in the volumetric flask to finish the constant volume operation, thus obtaining a rivaroxaban control solution.
S300, respectively carrying out detection and analysis operations on the sample solution and the control solution by adopting a high performance liquid chromatograph, wherein the chromatographic column is a reversed-phase chiral chromatographic column, the mobile phase is acetonitrile water solution or methanol water solution, the column temperature is 20-30 ℃, and the flow rate is 0.8-1.2 mL/min.
In this embodiment, the reversed-phase chiral chromatographic column is used for reversed-phase chromatographic determination analysis, so that the separation degree of rivaroxaban and rivaroxaban isomers can be effectively improved, the sensitivity in the rivaroxaban detection process can be improved, the signal-to-noise ratio of a chromatographic peak is further improved, and the accuracy and precision of a rivaroxaban determination result are improved. It can be understood that, because a large amount of auxiliary materials exist in rivaroxaban medicine, the auxiliary materials have solubility problem in organic solvent, water is needed to be involved to dissolve tablet auxiliary materials, but a normal phase system cannot be compatible with water, and a normal phase chromatographic column is easy to damage. The method adopts the reversed phase chromatography, can solve the problem of solubility of the rivaroxaban drug, and simultaneously can be compatible with water, thereby improving the durability of the chromatographic column. Further, the column temperature is 20-30 ℃ and the flow rate is 0.8-1.2 mL/min during chromatographic analysis, so that rivaroxaban and rivaroxaban isomers in a sample to be detected are fully separated in sequence and appear in different retention times, and a better peak shape can be formed, thereby improving the accuracy of the rivaroxaban determination result.
In one embodiment, before the step of preparing the sample solution of rivaroxaban, the method for detecting the content of the rivaroxaban isomer further comprises the following steps: a systematic applicability test solution of rivaroxaban was prepared. It can be understood that the system suitability test of the system suitability test solution for chromatographic analysis can determine the effectiveness and suitability of the chromatographic system used for analysis, and a series of system suitability parameters are established through the system suitability test to ensure that the stability of the analysis method can be maintained whenever the system is used, and the system suitability test solution has a greater influence on the system suitability test. In order to improve the accuracy of the system applicability test, in this embodiment, 6mg of a rivaroxaban isomer reference substance is precisely weighed, the weighed rivaroxaban reference substance is placed in a 250mL measuring flask, dissolved and diluted to a scale by a diluent, and shaken up to obtain a rivaroxaban isomer stock solution, wherein the diluent is an acetonitrile aqueous solution, and the acetonitrile in the acetonitrile aqueous solution: 50:50 of water; and additionally, precisely weighing 15mg of rivaroxaban reference substance, placing the weighed rivaroxaban reference substance into a 10mL measuring flask, precisely adding 1mL of rivaroxaban isomer stock solution, dissolving and diluting with a diluent to prepare a mixed solution containing 2.4 mu g of rivaroxaban isomer and 1.5mg of rivaroxaban in each 1mL of the mixed solution as a system applicability solution, so that the accuracy of the applicability test of the rivaroxaban reverse-phase chromatographic analysis system is improved, and the stability of the rivaroxaban reverse-phase chromatographic analysis method is further ensured.
Further, in the system applicability test solution, the separation degree of rivaroxaban isomer and rivaroxaban is more than or equal to 1.5. It is understood that the separation degree is also called resolution, and in order to determine the separation condition of the separated substance in the chromatographic column, the separation degree is often used as an index of the total separation efficiency of the column, and is denoted by R. R is equal to the ratio of the retention time difference of adjacent chromatographic peaks to the width average value of the two chromatographic peaks, and represents the separation degree of the two adjacent peaks, and the larger R represents the better separation of the two adjacent components. In order to determine the accuracy of the rivaroxaban isomer and the content of rivaroxaban, in this embodiment, in the system applicability test solution, the separation degree of the rivaroxaban isomer and the rivaroxaban is not less than 1.5, so that the separation degree of a chromatographic peak of the rivaroxaban isomer and a chromatographic peak of the rivaroxaban in the chromatographic analysis process is improved, and the accuracy of determining the rivaroxaban isomer and the content of rivaroxaban is favorably improved.
In one embodiment, after the step of preparing the rivaroxaban control solution and before the step of performing detection and analysis operations on the test solution and the control solution respectively by using a high performance liquid chromatograph, the method for detecting the content of the rivaroxaban isomer further comprises the following steps: preparing a rivaroxaban sensitivity solution. It can be understood that the liquid chromatograph is beneficial to timely adjusting a rivaroxaban detection and analysis system and improving the signal-to-noise ratio of a main peak of a chromatographic peak by testing and analyzing the sensitive solution, so that the accuracy of rivaroxaban detection is improved, and therefore, the sensitive solution has a great influence on the detection of rivaroxaban. To further improve the accuracy of rivaroxaban detection, in this example, a sensitivity solution of rivaroxaban was prepared and subjected to on-machine assay analysis. Specifically, 1mL of the reference stock solution is precisely measured, the measured reference stock solution is placed in a 100mL measuring flask, diluted to the scale of the volumetric flask by a diluent, and shaken up to obtain the sensitivity solution. Wherein the diluent is acetonitrile aqueous solution, and the content of acetonitrile in the acetonitrile aqueous solution is as follows: 50:50 of water; the sensitivity solution can effectively improve the signal-to-noise ratio of the main peak, so that the accuracy of rivaroxaban detection is improved.
Furthermore, the signal-to-noise ratio of the main peak in the sensitivity solution is more than or equal to 10. It will be appreciated that the signal-to-noise ratio is the ratio between the signal and the noise, for example: a needle sample was then observed for baseline noise values with a noise of 0.1mAu (or 0.1mV) and a peak height of 10mAu could be read. Then this concentration is 100 times the signal-to-noise ratio. In the embodiment, the signal-to-noise ratio of the main peak in the sensitivity solution is not less than 10, so that the influence of baseline noise can be effectively reduced, and the accuracy of rivaroxaban detection is further improved.
In one embodiment, the reverse phase Chiral chromatography column is a Chiral pak ic.250 x 4.6mm, 5 um. It can be understood that the chiral chromatographic column is a chiral stationary phase prepared by fixing optically active monomers on silica gel or other polymers, and enantiomers are subjected to physical characteristic difference by introducing a chiral environment, so that the purpose of optical isomer resolution is achieved. The reverse phase column packing mainly uses silica gel as a matrix, and nonpolar octadecyl functional groups (ODS) are bonded on the surface of the silica gel and are called C18 columns, and other common reverse phase columns comprise C8, C4, C2, phenyl columns and the like. The reversed-phase column has the advantages of stable stationary phase and wide application, and in the embodiment, the reversed-phase Chiral chromatographic column is chiralpak IC.250 x 4.6mm, 5um, Daiiluo medicine Chiral technology Shanghai Co. The Chiral pak IC.250 x 4.6mm and 5um are bonded chromatographic columns, so that the resolution capability of rivaroxaban and rivaroxaban isomers can be effectively improved, namely the separation effect of rivaroxaban and rivaroxaban isomers is improved, the accuracy of rivaroxaban detection is improved, the stability of a stationary phase can be improved, and the stability of rivaroxaban detection is improved. In addition, the chromatographic column Chiral pak IC.250 × 4.6mm, 5um not only can be used for a reverse phase chromatographic system, but also can be used for normal phase chromatography, thereby improving the simplicity of liquid phase switching.
In one embodiment, the sample volume in the assay operation is 15 μ L to 25 μ L. It can be understood that, in the detection and analysis of high performance liquid chromatography, the sample injection volume has a great influence on the retention value, the number of trays or the separation degree of each peak in a chromatogram, for example, as the sample injection amount increases, the spectrum peak is widened, the number of trays/v is reduced, and the separation degree of a sample is deteriorated; if the volume of the sample volume is too small, the peak shape is not obvious or the detection is difficult. In order to ensure the separation degree of each component in rivaroxaban and improve the detection accuracy, in the embodiment, the sample injection volume in the detection and analysis operation is 15 μ L to 25 μ L, so that the stability of the retention value and the separation degree of rivaroxaban in the chromatographic analysis process can be effectively ensured, and the detection accuracy is improved.
In one embodiment, the detector of the high performance liquid chromatograph is an ultraviolet absorption detector, and the detection wavelength is 250 nm. It will be appreciated that the choice of detection wavelength is related to the absorbance and interference of the assay, that rivaroxaban absorbs differently at different detection wavelengths and that the interference of rivaroxaban assays at different wavelengths is also different. In order to further improve the accuracy of rivaroxaban and rivaroxaban isomer detection, in the implementation, the detection wavelength is set to be 250nm, and experiments show that the maximum absorption wavelength of rivaroxaban is 250nm, interference is small at the wavelength of 250nm, and absorption of other impurities is basically avoided. In addition, the derivative of the chromatographic peak at the detection wavelength of 250nm is zero, and some error is introduced because the measurement light is not necessarily strictly monochromatic. In order to make the error of the part small enough, the detection wavelength of 250nm is selected in the embodiment, that is, the position where the derivative of the absorption coefficient to the wavelength is zero is measured, so that the determination error can be effectively reduced, and the accuracy of detecting rivaroxaban and rivaroxaban isomers is further improved.
In one embodiment, the reverse phase Chiral chromatography column is Chiral pak AS-H,250 x 4.6 mm.5um. In the embodiment, the temperature of the chromatographic column is set to be 20-30 ℃, the flow rate is set to be 0.8-1.2 mL/min, and under the chromatographic condition, a reverse-phase Chiral chromatographic column with Chiral pak AS-H and 250 x 4.6mm.5um is selected, so that the separation degree of rivaroxaban and rivaroxaban isomers in a sample to be detected can be further improved, the rivaroxaban and rivaroxaban isomers in the sample to be detected are sequentially and fully separated and appear in different retention time, and a better peak shape can be formed, so that the accuracy of a rivaroxaban determination result is further improved.
In one embodiment, after the step of preparing the sample solution of rivaroxaban and before the step of performing detection and analysis operations on the sample solution and the control solution respectively by using a high performance liquid chromatograph, the method for detecting the content of the rivaroxaban isomer further comprises the following steps: and carrying out ultrasonic dissolution operation on the test solution. In this embodiment, 640mg of rivaroxaban tablet fine powder, which is equivalent to 75mg of rivaroxaban, is taken, wherein the rivaroxaban tablet is provided by Guangzhou Hairy pharmaceutical industry Co., Ltd, Yangzhou pharmaceutical industry group, and is precisely weighed and placed in a 50mL measuring flask, a diluent not exceeding 50mL is added, ultrasonic operation is performed, then the solution in the measuring flask is diluted to a scale mark by the diluent, and the prepared sample solution is subjected to ultrasonic treatment before volume fixing, so that the solubility of the sample can be effectively improved, the accuracy of volume fixing of the sample solution is improved, that is, the accuracy of the concentration of the sample solution is improved, and the accuracy of rivaroxaban content determination is improved.
Further, the ultrasonic time in the ultrasonic dissolving operation is 25 to 35 minutes. It can be understood that if the ultrasonic time is too short, the rivaroxaban tablet fine powder in the solution cannot be sufficiently dissolved, and concentration errors of subsequent samples are easily caused; if the ultrasonic time is too long, the temperature of the test solution in the measuring flask is easily too high, so that the stability of the test solution is damaged, the determination of the rivaroxaban content is influenced, and meanwhile, the efficiency of rivaroxaban detection and analysis is easily influenced due to the too long ultrasonic time. In order to improve the solubility of the rivaroxaban sample to be detected and improve the detection efficiency, in this embodiment, the ultrasonic time in the ultrasonic dissolving operation is 25 to 35 minutes, and after the ultrasonic operation for 25 to 35 minutes, rivaroxaban fine powder can be fully dissolved in the diluent, so that the solubility of the rivaroxaban sample to be detected is effectively improved and the detection efficiency is improved.
In one embodiment, the mobile phase is an aqueous acetonitrile solution further comprising a small amount of phosphoric acid, wherein the ratio of acetonitrile: water: phosphoric acid 85:14.5: 0.5. It will be appreciated that the mobile phase pH will not only have a product impact on the stability of the column, but will also have a crucial effect on the selectivity of the separation and the symmetry of the chromatographic peaks, especially for certain polar acid-base compounds that can be ionized. In the reverse phase chromatography system, the stationary phase is a non-polar compound, the mobile phase is a polar compound, and for a compound containing a polar functional group, the pH affects the existence state of the compound, and further affects the separation selectivity and the symmetry of a chromatographic peak. To improve the chromatographic peak symmetry and retention time of rivaroxaban, in this example, the mobile phase used was acetonitrile: water: phosphoric acid 85:14.5:0.5, and the addition of the small amount of phosphoric acid can inhibit ionization of an acetonitrile aqueous solution, enhance interaction between a mobile phase and a stationary phase of a reversed-phase chiral chromatographic column, and improve the retention time of the rivaroxaban compound. In addition, by changing the pH of the mobile phase, the dissociation of the mobile phase is inhibited, the capacity factor of the polar dissociable compound can be changed, the peak position of the rivaroxaban compound is changed, the separation selectivity is improved, and the symmetry of chromatographic peaks is improved.
Further, the prepared acetonitrile-water-phosphoric acid mobile phase is subjected to an ultrasonic operation before use. It can be understood that in the chromatographic analysis process, the separation degree of related compounds is easily affected by the uneven solubility or the high viscosity of the mobile phase, so that the peak shapes of rivaroxaban and other substances are affected, and the accuracy of the rivaroxaban determination result is reduced. In order to improve the uniformity of the acetonitrile-water-phosphoric acid mobile phase and reduce the viscosity of the acetonitrile-water-phosphoric acid mobile phase, in this embodiment, the solubility of the acetonitrile-water-phosphoric acid mobile phase can be effectively improved, the uniformity of the acetonitrile-water-phosphoric acid mobile phase can be improved, the viscosity of the acetonitrile-water-phosphoric acid mobile phase can be reduced, and the accuracy of the rivaroxaban measurement result can be further improved.
Some specific examples are listed below, and if mentioned%, all are expressed in weight percent. It should be noted that the following examples are not intended to be exhaustive of all possible cases, and that the materials used in the following examples are commercially available without specific recitation.
Example 1
Preparing a test solution of rivaroxaban: taking 640mg of rivaroxaban tablet fine powder, namely equivalent to 75mg of rivaroxaban, wherein the rivaroxaban tablet is provided by Guangzhou Hairy pharmaceutical industry Co., Ltd, Yangzhou pharmaceutical industry group, accurately weighing the rivaroxaban tablet, placing the rivaroxaban tablet in a 50mL measuring flask, adding a diluent not more than 50mL, carrying out ultrasonic operation for 25 minutes, and then diluting the solution in the volumetric flask to a scale mark by using the diluent, wherein the diluent is acetonitrile aqueous solution, and the acetonitrile in the acetonitrile aqueous solution is as follows: 50:50 of water; and then shaking up the sample solution in the volumetric flask to finish constant volume operation, thus obtaining a test solution containing 1.5mg of rivaroxaban in every 1 mL.
Preparing a rivaroxaban control solution: precisely measuring 5mL of a test solution, placing the test solution into a 100mL measuring flask, diluting the test solution to a scale with a diluent, and shaking up to be used as a reference stock solution; precisely measuring 3mL of the control stock solution, placing the control stock solution into a 100mL measuring flask, and diluting the control stock solution to the scale mark of the measuring flask by using a diluent, wherein the diluent is an acetonitrile aqueous solution, and the acetonitrile in the acetonitrile aqueous solution: 50:50 of water; and then shaking up the solution in the volumetric flask to finish the constant volume operation, thus obtaining a rivaroxaban control solution.
And (3) computer determination and analysis: detecting and analyzing the test solution and the control solution by a high performance liquid chromatograph, wherein a Chiral pak IC.250 x 4.6mm chromatographic column with 5um as a chromatographic column and acetonitrile as a mobile phase are adopted, and the sample injection volume is 15 mu L; the detection wavelength is 250nm, the column temperature is 20 ℃, and the flow rate is 0.8 mL/min.
Example 2
Preparing a test solution of rivaroxaban: taking 640mg of rivaroxaban tablet fine powder, namely equivalent to 75mg of rivaroxaban, wherein the rivaroxaban tablet is provided by Guangzhou Hairy pharmaceutical industry Co., Ltd, Yangzhou pharmaceutical industry group, accurately weighing the rivaroxaban tablet, placing the rivaroxaban tablet in a 50mL measuring flask, adding a diluent not more than 50mL, carrying out ultrasonic operation for 25 minutes, and then diluting the solution in the volumetric flask to a scale mark by using the diluent, wherein the diluent is acetonitrile aqueous solution, and the acetonitrile in the acetonitrile aqueous solution is as follows: 50:50 of water; and then shaking up the sample solution in the volumetric flask to finish constant volume operation, thus obtaining a test solution containing 1.5mg of rivaroxaban in every 1 mL.
Preparing a rivaroxaban control solution: precisely measuring 5mL of a test solution, placing the test solution into a 100mL measuring flask, diluting the test solution to a scale with a diluent, and shaking up to be used as a reference stock solution; precisely measuring 3mL of the control stock solution, placing the control stock solution into a 100mL measuring flask, and diluting the control stock solution to the scale mark of the measuring flask by using a diluent, wherein the diluent is an acetonitrile aqueous solution, and the acetonitrile in the acetonitrile aqueous solution: 50:50 of water; and then shaking up the solution in the volumetric flask to finish the constant volume operation, thus obtaining a rivaroxaban control solution.
And (3) computer determination and analysis: detecting and analyzing the test solution and the control solution by a high performance liquid chromatograph, wherein a Chiral pak IC.250 x 4.6mm chromatographic column with 5um as a chromatographic column and acetonitrile as a mobile phase are used, and the sample injection volume is 25 mu L; the detection wavelength was 250nm, the column temperature was 30 ℃ and the flow rate was 1.2 mL/min.
Example 3
Preparing a test solution of rivaroxaban: taking 640mg of rivaroxaban tablet fine powder, namely equivalent to 75mg of rivaroxaban, wherein the rivaroxaban tablet is provided by Guangzhou Hairy pharmaceutical industry Co., Ltd, Yangzhou pharmaceutical industry group, accurately weighing the rivaroxaban tablet, placing the rivaroxaban tablet in a 50mL measuring flask, adding a diluent not more than 50mL, carrying out ultrasonic operation for 25 minutes, and then diluting the solution in the volumetric flask to a scale mark by using the diluent, wherein the diluent is acetonitrile aqueous solution, and the acetonitrile in the acetonitrile aqueous solution is as follows: 50:50 of water; and then shaking up the sample solution in the volumetric flask to finish constant volume operation, thus obtaining a test solution containing 1.5mg of rivaroxaban in every 1 mL.
Preparing a rivaroxaban control solution: precisely measuring 5mL of a test solution, placing the test solution into a 100mL measuring flask, diluting the test solution to a scale with a diluent, and shaking up to be used as a reference stock solution; precisely measuring 3mL of the control stock solution, placing the control stock solution into a 100mL measuring flask, and diluting the control stock solution to the scale mark of the measuring flask by using a diluent, wherein the diluent is an acetonitrile aqueous solution, and the acetonitrile in the acetonitrile aqueous solution: 50:50 of water; and then shaking up the solution in the volumetric flask to finish the constant volume operation, thus obtaining a rivaroxaban control solution.
And (3) computer determination and analysis: detecting and analyzing the test solution and the control solution by a high performance liquid chromatograph, wherein a Chiral pak IC.250 x 4.6mm chromatographic column with 5um as a chromatographic column and acetonitrile as a mobile phase are adopted, and the sample injection volume is 20 mu L; the detection wavelength was 250nm, the column temperature was 25 ℃ and the flow rate was 1.0 mL/min.
Example 4
Preparing a test solution of rivaroxaban: taking 640mg of rivaroxaban tablet fine powder, namely equivalent to 75mg of rivaroxaban, wherein the rivaroxaban tablet is provided by Guangzhou Hairy pharmaceutical industry Co., Ltd, Yangzhou pharmaceutical industry group, accurately weighing the rivaroxaban tablet, placing the rivaroxaban tablet in a 50mL measuring flask, adding a diluent not more than 50mL, carrying out ultrasonic operation for 25 minutes, and then diluting the solution in the volumetric flask to a scale mark by using the diluent, wherein the diluent is acetonitrile aqueous solution, and the acetonitrile in the acetonitrile aqueous solution is as follows: 50:50 of water; and then shaking up the sample solution in the volumetric flask to finish constant volume operation, thus obtaining a test solution containing 1.5mg of rivaroxaban in every 1 mL.
Preparing a rivaroxaban control solution: precisely measuring 5mL of a test solution, placing the test solution into a 100mL measuring flask, diluting the test solution to a scale with a diluent, and shaking up to be used as a reference stock solution; precisely measuring 3mL of the control stock solution, placing the control stock solution into a 100mL measuring flask, and diluting the control stock solution to the scale mark of the measuring flask by using a diluent, wherein the diluent is an acetonitrile aqueous solution, and the acetonitrile in the acetonitrile aqueous solution: 50:50 of water; and then shaking up the solution in the volumetric flask to finish the constant volume operation, thus obtaining a rivaroxaban control solution.
And (3) computer determination and analysis: respectively carrying out detection analysis operation on the test solution and the control solution by adopting a high performance liquid chromatograph, taking Chiral pak AS-H,250 x 4.6mm.5um AS a chromatographic column, taking acetonitrile AS a mobile phase, and taking the sample injection volume of 20 mu L; the detection wavelength was 250nm, the column temperature was 25 ℃ and the flow rate was 1.0 mL/min.
1. Accuracy of detection method for determining rivaroxaban isomer content
Weighing rivaroxaban isomers according to three concentration levels of 50%, 100% and 120%, adding the rivaroxaban isomers into a test solution, and adopting the method for detection and calculating the sample adding recovery rate, wherein the results are shown in table 1:
TABLE 1
From the results in table 1, the average recovery rate is 101.2% and the RSD is 0.6% (n ═ 9) at the three concentration levels of 50%, 100% and 120%, and the results meet the requirements, which indicates that the method for detecting the content of rivaroxaban isomers has better accuracy.
2. Repeatability of detection method for determining rivaroxaban isomer content
The same batch is taken, 6 parts of samples are prepared and subjected to sample injection detection respectively, and the results show that the determination results of the 6 parts of samples are all between 0.032% and 0.035%, and the RSD is 3.2% (n is 6), which indicates that the repeatability of the rivaroxaban isomer content detection method is good.
3. Solution stability in the detection method for determining rivaroxaban isomer content of the present application
Taking 1 part of test solution, injecting samples for 4h, 8h, 12h, 16h, 20h and 24h after sample preparation, and counting the isomer content, wherein the results are shown in Table 2:
TABLE 2
From the results in table 2, it can be seen that the sample solutions in the present application have better stability within 24 h.
Compared with the prior art, the invention has at least the following advantages:
1. the method for detecting the content of the rivaroxaban isomer adopts a chiral chromatographic column, wherein the chiral chromatographic column is a monomer with optical activity and is fixed on silica gel or other polymers in a chromatogram to form a chiral stationary phase. The chiral environment is introduced, so that the enantiomers of rivaroxaban present physical characteristic difference, the purpose of optical isomer resolution is achieved, and the separation effect is good. Furthermore, the reversed-phase chiral chromatographic column is adopted for carrying out reversed-phase chromatographic determination analysis, so that the separation degree of rivaroxaban and rivaroxaban isomers can be effectively improved, the sensitivity in the rivaroxaban detection process can be improved, the signal-to-noise ratio of chromatographic peaks is further improved, and the accuracy and precision of rivaroxaban determination results are improved.
2. According to the method for detecting the rivaroxaban isomer content, a reversed-phase chiral chromatographic column is used for carrying out reversed-phase chromatographic determination analysis, and due to the fact that a large number of auxiliary materials exist in rivaroxaban drugs, the auxiliary materials have the problem of solubility in organic solvents, water is needed to be involved to dissolve tablet auxiliary materials, but a normal-phase system cannot contain water, and the normal-phase chromatographic column is prone to damage. The method adopts the reversed phase chromatography, can solve the problem of solubility of the rivaroxaban drug, and simultaneously can be compatible with water, thereby improving the durability of the chromatographic column.
3. According to the method for detecting the rivaroxaban isomer content, the column temperature is 20-30 ℃ and the flow rate is 0.8-1.2 mL/min during chromatographic analysis, so that rivaroxaban and rivaroxaban isomers in a sample to be detected are sequentially and fully separated and appear in different retention times, and a better peak shape can be formed, so that the accuracy of a rivaroxaban determination result is improved.
The above examples only show some embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A method for detecting the content of rivaroxaban isomer is characterized by comprising the following steps:
preparing a test solution of rivaroxaban;
preparing a control solution of rivaroxaban;
and (2) respectively detecting and analyzing the test solution and the control solution by adopting a high performance liquid chromatograph, wherein the chromatographic column is a reversed-phase chiral chromatographic column, the mobile phase is acetonitrile aqueous solution or methanol aqueous solution, the column temperature is 20-30 ℃, and the flow rate is 0.8-1.2 mL/min.
2. The method for detecting the content of rivaroxaban isomer according to claim 1, wherein before the step of preparing the sample solution of rivaroxaban, the method for detecting the content of rivaroxaban isomer further comprises the following steps:
preparing a system applicability test solution of the rivaroxaban.
3. The method for detecting the content of rivaroxaban isomers according to claim 2, wherein the separation degree of rivaroxaban isomers from rivaroxaban in the system suitability test solution is not less than 1.5.
4. The method for detecting the content of rivaroxaban isomer according to claim 1, wherein after the step of preparing the control solution of rivaroxaban and before the step of performing detection and analysis operations on the test solution and the control solution respectively by using a high performance liquid chromatograph, the method for detecting the content of rivaroxaban isomer further comprises the following steps:
preparing a sensitive solution of rivaroxaban.
5. The method for detecting the content of rivaroxaban isomers according to claim 4, wherein the signal-to-noise ratio of a main peak in the sensitive solution is not less than 10.
6. The method for detecting the content of rivaroxaban isomers according to claim 1, wherein the reversed-phase Chiral chromatographic column is Chiral pak IC.250 x 4.6mm, 5 um.
7. The method for detecting the content of rivaroxaban isomers according to claim 1, wherein the injection volume in the detection analysis operation is 15 μ L to 25 μ L.
8. The method for detecting the rivaroxaban isomer content according to claim 1, wherein the detector of the high performance liquid chromatograph is an ultraviolet absorption detector, and the detection wavelength is 250 nm.
9. The method for detecting rivaroxaban isomer content according to claim 1, wherein the reversed-phase Chiral chromatographic column is chiralpak AS-H,250 x 4.6mm.5 um.
10. The method for detecting the content of the rivaroxaban isomer according to claim 1, wherein after the step of preparing the sample solution of rivaroxaban and before the step of performing detection and analysis operations on the sample solution and the control solution respectively by using a high performance liquid chromatograph, the method for detecting the content of the rivaroxaban isomer further comprises the following steps:
and carrying out ultrasonic dissolution operation on the test solution.
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