CN112557543B

CN112557543B - Method for determining rivaroxaban and related substances thereof

Info

Publication number: CN112557543B
Application number: CN202011427144.7A
Authority: CN
Inventors: 张晓飞; 黄乐群
Original assignee: Jiangsu Sinobiopharma Co ltd
Current assignee: Jiangsu Sinobiopharma Co ltd
Priority date: 2020-12-09
Filing date: 2020-12-09
Publication date: 2022-11-22
Anticipated expiration: 2040-12-09
Also published as: CN112557543A

Abstract

The invention provides a method for measuring rivaroxaban and related substances thereof, which comprises the steps of adopting a high performance liquid chromatography to measure, dissolving rivaroxaban in a diluent to obtain a sample solution, injecting the sample solution into a high performance liquid chromatograph with a core-shell chromatographic column as a stationary phase, adopting a flow without an ion pair reagent to carry out gradient elution relative to the sample solution, separating to obtain an eluent, and injecting the eluent into a detector to measure. The invention adopts the core-shell chromatographic column, has good column efficiency and can bring good peak shape and separation; meanwhile, the mobile phase does not contain an ion pair reagent, so that various ghost peaks generated in the analysis process are avoided, the interference of a base line is effectively reduced, the sensitivity of impurity detection is greatly improved, various processes and degradation impurities in rivaroxaban can be simultaneously separated, and the method has important significance for impurity control and product safety procedures in rivaroxaban bulk drugs.

Description

Method for determining rivaroxaban and related substances thereof

Technical Field

The invention relates to a method for determining rivaroxaban and related substances thereof, and belongs to the technical field of medical chemical analysis.

Background

Rivaroxaban is an oral anticoagulant with high selectivity for directly inhibiting blood coagulation factor xa, and has a molecular formula of C19H18ClN3O5S and a chemical name of: 5-chloro-N- ({ (5S) -2-oxo-3- [4- (3-oxo-4-morpholinyl) -phenyl ] -1, 3-oxazolidin-5-yl } -methyl) -2-thiophene-carboxamide, having the formula:

in the chemical synthesis process of rivaroxaban, various intermediates, side reaction impurities and degradation products are inevitably contained in the raw material medicine due to the residue of the starting raw material intermediate and the occurrence of chemical reaction side reactions. In order to ensure the quality of rivaroxaban bulk drug, a scientific analysis method is needed to detect and control various impurities in the rivaroxaban bulk drug.

Related quality standards for rivaroxaban impurity control are not yet collected by United states Pharmacopeia, european Pharmacopeia, chinese Pharmacopeia and other right Wei Pharmacopeia, and an authoritative method which can be referred to in the current industry is a related substance method collected in the imported registration standard of rivaroxaban preparations, because the preparation related substance method mainly detects degradation impurities of the preparations as targets, the method has weak separation capability on process impurities, intermediate impurities and the like existing in bulk drugs.

The invention patent CN107941936B discloses a method for separating and measuring rivaroxaban and impurities thereof and application thereof, wherein an impurity in front of a main peak cannot be completely separated from the main peak in the method, and the separation degree of the main peak and the impurities cannot be ensured to meet requirements under the condition similar to shoulder seams.

The invention patent CN105004802B discloses a method for separating and measuring rivaroxaban and related impurities by using a liquid chromatography, wherein an ion pair is added into a mobile phase A, the balancing time is long, the ion pair reagent is unstable, a base line has a plurality of unknown small peaks, the measurement of related substances is greatly interfered, and ghost peak small columns cannot be added for removal; and ions can cause irreversible damage to the chromatographic column to affect the active sites on the stationary phase of the chromatographic column, thereby not only affecting the reproducibility and durability of the method, but also greatly shortening the service life of the chromatographic column.

The invention patent CN2019104208181A discloses a method for detecting related substances of rivaroxaban and a preparation thereof, which has the advantages that although a plurality of impurities are well separated, the analysis time of the analysis method is long, the total operation time reaches 70 minutes, the peak-off time of the final impurities is 58 minutes, the analysis time is prolonged to improve the separation degree, the column efficiency of partial chromatographic peaks is reduced, the analysis efficiency is low, the cost control of the production angle is very unfavorable, and the waste liquid generated by the long-time operation brings the cost of waste liquid treatment.

Based on the above reasons, in the prior art, methods used for separation and detection of rivaroxaban and related substances thereof have the problems of interference peak influence, poor durability, poor reproducibility, low detection sensitivity, manual blank solvent deduction in the calculation process and the like, and have great influence on calculation and judgment of detection results.

Disclosure of Invention

The invention aims to provide a method for measuring rivaroxaban and related substances thereof, which does not contain ion pairs, uses a core-shell chromatographic column, can separate more impurities and solves the problem of poor separation of impurity peaks close to a main peak and the main peak.

The method has the advantages of simple steps, high sensitivity, good durability, good reproducibility, high accuracy of the measurement result and high separation speed,

the technical scheme of the invention is as follows: a method for measuring rivaroxaban and related substances thereof specifically comprises the following steps:

1) Dissolving rivaroxaban in a diluent to obtain a sample solution;

2) Preparing a mobile phase A and a mobile phase B;

3) Performing determination by adopting a high performance liquid chromatography, injecting the sample solution prepared in the step 1) into a high performance liquid chromatograph, performing gradient elution on the sample solution by adopting the mobile phase A and the mobile phase B prepared in the step 2), and separating to obtain an eluent;

4) The eluent is injected into a detector for measurement.

The diluent in the step 1) is acetonitrile-water solution, wherein the acetonitrile: the volume ratio of water is 60.

The flow rate of the mobile phase in the step 3) is 0.8-1.5 ml/min, and preferably 1.2 ml/min.

The volume ratio of the mobile phase A to the mobile phase B in the step 3) is (5-95) to (95-5) (the sum of the volume ratios of the mobile phase A to the mobile phase B is 100), and the specific process of the gradient elution is shown in the following table 1:

TABLE 1

The high performance liquid chromatography comprises the following conditions:

(1) the stationary phase is a core-shell chromatographic column of octadecylsilane chemically bonded silica;

further, the chromatographic column is a core-shell type C18 chromatographic column, preferably, the chromatographic column is an Agilent poroshell EC-C18 column which adopts a special core-shell type structure and provides different chromatographic selectivity from a conventional end-capped chromatographic column.

Further, the temperature of the column box of the chromatographic column is 40-60 ℃, preferably 50 ℃. High column temperatures can provide better column efficiency and separation within the tolerable range of the chromatography column.

(2) The mobile phase A is a mixed solvent of a buffered saline solution and methanol and does not contain an ion-pair reagent. The mass ratio of the buffered saline solution to the methanol in the mixed solvent is (100).

Further, the buffer salt in the buffer saline solution is an alkali metal phosphate salt including a sodium phosphate salt or a potassium phosphate salt, preferably one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate, and more preferably potassium dihydrogen phosphate or sodium dihydrogen phosphate. The concentration of the buffer salt in the buffer salt aqueous solution is 1 to 50mmol/L, preferably 5 to 20mmol/L.

Further, 100 to 500. Mu.l of phosphoric acid, preferably 150 to 250. Mu.l of phosphoric acid is added to the buffered saline solution.

(3) The mobile phase B is a ternary mixed solvent of water, acetonitrile and methanol; acetonitrile: methanol: the volume ratio of water is 10.

(4) The detector is an ultraviolet detector, and the wavelength of ultraviolet rays is 240-260 nm, preferably 250nm.

The related substances comprise process impurities and degradation impurities, and specifically comprise one or more of impurities A, B, C, D, E, F, G, H, I, J, K, M, N, O and P, and the structural formula of each impurity is as follows:

compared with the prior art, the invention has the advantages that:

1) The core-shell chromatographic column is adopted, so that the column effect is good, and good peak pattern and separation can be brought;

2) The mobile phase does not contain ion pair reagents, so that various ghost peaks generated in the analysis process are avoided, ghost peak trapping small columns can be applied, the interference of a base line is effectively reduced, and the sensitivity of impurity detection is greatly improved;

3) 15 known processes and degradation impurities in rivaroxaban can be separated simultaneously, two unknown impurities (wrapped in the main peak and incapable of being separated) with similar polarities to the main peak before and after the main peak can be separated, and the separation quantity and the detection precision are greatly improved compared with the prior art;

4) When more impurities are separated, the analysis time is short, the peak emergence time of the impurities with the minimum polarity is 35min, and the time efficiency is improved higher compared with the peak emergence time near 58min in other patents.

5) The method is simple, convenient and feasible, high in sensitivity, high in accuracy and good in reproducibility, can be used for quickly and effectively separating and determining the content of each related substance in rivaroxaban API, and has important significance on impurity control and product safety procedures in rivaroxaban bulk drugs.

Drawings

FIG. 1 is a high performance liquid chromatogram of the blank diluent of example 1.

Figure 2 is a high performance liquid chromatogram of a rivaroxaban mixed impurities solution of example 1.

FIG. 3 is a high performance liquid chromatogram of a 0.1% rivaroxaban self-control solution of example 1.

FIG. 4 is a high performance liquid chromatogram of a test solution of rivaroxaban as in example 1.

FIG. 5 is a high performance liquid chromatogram of the blank diluent of example 2.

FIG. 6 is a high performance liquid chromatogram of a rivaroxaban mixed impurities solution of example 2.

FIG. 7 is a high performance liquid chromatogram of the blank diluent of example 3.

FIG. 8 is a high performance liquid chromatogram of a rivaroxaban mixed impurities solution of example 3.

FIG. 9 is a high performance liquid chromatogram of the blank diluent of example 4.

FIG. 10 is a high performance liquid chromatogram of a rivaroxaban mixed impurities solution of example 4.

FIG. 11 is a high performance liquid chromatogram of the blank diluent of example 5.

FIG. 12 is a high performance liquid chromatogram of a rivaroxaban mixed impurities solution of example 5.

FIG. 13 is a high performance liquid chromatogram of a blank solvent of example 6.

Figure 14 is a high performance liquid chromatogram of a sample solution of rivaroxaban of example 6.

Detailed Description

The technical scheme of the invention is further illustrated by the following examples. The technical solutions and related parameter settings given in the embodiments are exemplary and are intended to be used for explaining the present invention, and should not be construed as limiting the present invention.

Example 1

The method for separating and measuring rivaroxaban and impurities thereof specifically comprises the following steps:

1) Apparatus and conditions

The instrument comprises the following steps: a high performance liquid chromatograph; a chromatographic column: a core-shell chromatographic column using octadecylsilane chemically bonded silica as a filler, wherein the type of the chromatographic column is Agilent Poroshell 120EC-C18, 150 multiplied by 4.6 mm, 2.7 mu m or a chromatographic column with equivalent performance; the detector detects the wavelength: 250nm; flow rate of mobile phase: 1.2 ml/min; mobile phase A: mobile phase a was prepared by using 10mmol/mL potassium dihydrogen phosphate buffer solution (1.36 g potassium dihydrogen phosphate with 1000mL water, 200 μ l phosphoric acid) -methanol (95; mobile phase B: acetonitrile-methanol-water (50; diluent agent: acetonitrile-water (70; the column temperature of the chromatographic column is 50 ℃; the elution gradient is shown in table 2 below:

TABLE 2

Sample injection amount: 5 μ l.

2) Experimental procedure

(1) Preparing a mixed impurity solution: taking a proper amount of each of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M, impurity N, impurity O and rivaroxaban, and diluting with a solvent to prepare a mixed impurity solution containing 0.4 mg of rivaroxaban and 0.4 mu G of each impurity in each 1 ml;

(2) Preparing a test solution: dissolving the product with solvent [ acetonitrile-water (70) 30) ] by ultrasonic treatment, cooling to room temperature, and diluting with solvent to obtain solution containing 0.4 mg per 1ml as test solution;

(3) Preparing a control solution: precisely measuring 1ml of a test solution, placing the test solution in a 100ml measuring flask, diluting the test solution to a scale by using a solvent [ acetonitrile-water (70) ], and shaking up; precisely measuring 1ml of the solution, placing the solution in a 10ml measuring flask, diluting the solution to a scale with a mobile phase, and shaking up to obtain a control solution;

(4) Injecting 5 μ l of each of the above diluent, system suitability test solution, sample solution and control solution into high performance liquid chromatograph, measuring according to the above chromatographic conditions, performing linear gradient elution according to data shown in Table 1, and recording chromatogram, wherein the results are shown in FIGS. 1-5.

3) The results of the experiment are shown in table 3 below:

TABLE 3

As a result: the components can be separated from each other at a base line, the separation requirement of pharmacopeia is met, the blank solvent does not interfere the detection of each component, the signal-to-noise ratio of 0.1 percent of the self-contrast peak is good, and the detection sensitivity is high. (as shown in FIGS. 1-4 and Table 3).

Example 2

1) The flow rate was adjusted to 1.0 ml/min based on example 1

2) The results of the experiment are shown in table 4 below:

TABLE 4

As a result: baseline separation between each component was achieved, pharmacopeia separation requirements were met, and blank solvent did not interfere with the detection of each component (as shown in figures 5-6 and table 4).

Example 3

1) The flow rate was adjusted to 1.4 ml/min based on example 1

2) The results of the experiment are shown in table 5 below:

TABLE 5

As a result: the components can be separated from each other at a baseline, the separation requirement of pharmacopeia is met, and the blank solvent does not interfere the detection of the components. (as shown in figures 7-8 and table 5).

Example 4

1) The column temperature was adjusted to 55 ℃ based on example 1

2) The results of the experiment are shown in table 6 below:

TABLE 6

As a result: the components except the impurities G and H can be separated from each other at a baseline, the separation requirement of pharmacopeia is met, and the blank solvent does not interfere the detection of each component. (as shown in FIGS. 9-10 and Table 6).

Example 5

1) The column temperature was adjusted to 45 ℃ based on example 1

2) The results of the experiment are shown in table 7 below:

TABLE 7

As a result: all components can be separated from each other at a baseline, the separation requirement of pharmacopeia is met, and the blank solvent does not interfere the detection of all components. (as shown in FIGS. 11-12 and Table 7).

Example 6

The method for separating and determining rivaroxaban and impurities thereof specifically comprises the following steps:

1) Apparatus and conditions

A high performance liquid chromatograph: shimadzu LC-2010C; a chromatographic column: purospher Star RP-18 (55X 4mm, 3 μm); the detection wavelength of the detector is as follows: 250nm; sample introduction amount: 5 mul; diluent (b): diluent (b): acetonitrile-0.01 mol/L phosphoric acid solution (11.5 g or 6.7 ml of 85% phosphoric acid, diluted to 10.0L with water) = (3);

2) Experimental procedure

(1) Preparing a mixed impurity solution: taking an appropriate amount of each of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M, impurity N, impurity O and rivaroxaban, and diluting with a solvent to prepare a mixed impurity solution containing 0.4 mg of rivaroxaban and 0.4 mu G of each impurity per 1 ml;

(2) Preparing a mobile phase A: taking 1000ml of water, adding 0.1% phosphoric acid, and mixing uniformly; the mobile phase B is acetonitrile;

(3) And (2) injecting 10 mu l of diluent and the sample solution obtained in the step (1) into a high performance liquid chromatograph, wherein the model of a chromatographic column is Purospher Star RP-18, the flow rate of a mobile phase is set to be 1.0 ml/min, the detection wavelength is 250nm, and the temperature of a chromatographic column box is 45 ℃. The elution gradient is shown in table 8 below:

TABLE 8

And (5) carrying out linear gradient elution according to the data shown in the table 8 to complete the separation and determination of related substances of rivaroxaban. Recording chromatogram, high performance liquid chromatogram is shown in FIGS. 6-7. Under the condition, compared with example 1, rivaroxaban and impurity peaks only have 12 peaks, and a plurality of impurity peaks cannot be separated and overlapped or have poor separation degree.

Compared with the method in the embodiment 2 (the existing method), the method in the embodiment 1 has the advantages that the amount of separable impurities is large, and the separation degree is good, so that the contents of various related substances in rivaroxaban API can be effectively separated and measured, the quality of rivaroxaban bulk drug and preparation thereof is controllable, and the safety and effectiveness of the product are finally determined.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention by equivalent replacement or change according to the technical solution and the inventive concept of the present invention within the technical scope of the present invention.

Claims

1. A method for measuring rivaroxaban and related substances thereof specifically comprises the following steps:

1) Dissolving rivaroxaban in a diluent to obtain a sample solution, wherein the diluent is an acetonitrile-water solution;

2) Preparing a mobile phase A and a mobile phase B;

4) Injecting the eluent obtained in the step 3) into a detector for measurement;

the method is characterized in that: the stationary phase in the high performance liquid chromatograph is a core-shell chromatographic column of octadecylsilane chemically bonded silica; the mobile phase A is a mixed solvent of a buffered saline solution and methanol, the buffered saline solution does not contain an ion-pairing reagent, and a buffer salt in the buffered saline solution is an alkali metal phosphate;

the mass ratio of the buffer saline solution to the methanol in the mobile phase A is 100-85;

the mobile phase B is a ternary mixed solvent of water, acetonitrile and methanol; wherein acetonitrile: methanol: the volume ratio of water is 10;

the flow rate of the mobile phase in the step 3) is 0.8-1.5 ml/min;

the specific process of the gradient elution is as follows:

；

the related substances comprise one or more of impurities A, B, C, D, E, F, G, H, I, J, K, M, N, O and P,

the structural formula of each impurity is specifically as follows:

。

2. the method for determining rivaroxaban and related substances according to claim 1, wherein: the temperature of the column box of the chromatographic column is 40-60 ℃.

3. The method for determining rivaroxaban and related substances according to claim 1, wherein: the buffer salt in the buffer saline solution is sodium phosphate or potassium phosphate.

4. The method for determining rivaroxaban and related substances thereof according to claim 3, characterized in that: the buffer salt in the buffer saline solution is one or more of dipotassium phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate and sodium dihydrogen phosphate.

5. The method for determining rivaroxaban and related substances according to claim 3, wherein: the concentration of the buffer salt in the buffer salt water solution is 1-50 mmol/L.

6. The method for determining rivaroxaban and related substances according to claim 3, wherein: the volume of the phosphoric acid is 100-500 mul, and the phosphoric acid is also added into the buffer saline solution.

7. The method for determining rivaroxaban and related substances according to claim 1, wherein: the detector is an ultraviolet detector, and the ultraviolet wavelength of the ultraviolet detector is 240-260 nm.

8. The method for determining rivaroxaban and related substances according to claim 1, wherein: the diluent in the step 1) is acetonitrile-water solution, wherein the acetonitrile: the volume ratio of water is 60.