CN113552263A

CN113552263A - Apixaban intermediate and separation and detection method of mutagenic impurities thereof

Info

Publication number: CN113552263A
Application number: CN202110864733.XA
Authority: CN
Inventors: 朱巧云; 方晓志; 刘雪芳; 龚彦春; 刘永强
Original assignee: Jiangsu Vcare Pharmatech Co Ltd
Current assignee: Jiangsu Vcare Pharmatech Co Ltd
Priority date: 2021-07-29
Filing date: 2021-07-29
Publication date: 2021-10-26
Anticipated expiration: 2041-07-29
Also published as: CN113552263B

Abstract

The invention relates to an analysis and detection method of aromatic nitro and aromatic amino mutation impurities in 5, 6-dihydro-3- (4-morpholinyl) -1- [4- (2-oxo-1-piperidyl) phenyl ] -2(1H) -pyridone, which comprises the steps of detecting the wavelengths of 242nm and 325nm by a high performance liquid chromatograph, and performing gradient elution by using a mixed solution of ammonium acetate buffer solution and acetonitrile as a mobile phase. The gradient elution adopted by the invention can realize effective separation of impurities, the separation degree of each absorption peak is more than or equal to 1.2, the sample injection precision is good, the RSD is less than or equal to 2%, the durability of the method is good, and the method can be used for quality control in the development and production processes of Apixaban.

Description

Apixaban intermediate and separation and detection method of mutagenic impurities thereof

Technical Field

The invention belongs to the field of drug analysis, and particularly relates to a separation and detection method of 5, 6-dihydro-3- (4-morpholinyl) -1- [4- (2-oxo-1-piperidyl) phenyl ] -2(1H) -pyridone and mutation impurities caused by aromatic nitro and aromatic amino.

Background

The internal quality of the medicine directly influences the clinical curative effect and safety of the medicine, wherein the research on potential mutation-causing impurities is particularly important, and the potential mutation-causing impurities are mainly initial raw materials, intermediates, polymers, side reaction products and the like brought in the production process. Based on the consideration of safety and practical production conditions, potential mutagenic impurities with high toxicity, harm to human health, ineffectiveness or influence on the stability of the medicine have to be researched and strictly controlled.

5, 6-dihydro-3- (4-morpholinyl) -1- [4- (2-oxo-1-piperidinyl) phenyl ] -2(1H) -pyridone (code: 268-7) is an intermediate in the synthesis of apixaban, the potential mutagenic impurity of which is of the structure:

at present, no literature reports 5, 6-dihydro-3- (4-morpholinyl) -1- [4- (2-oxo-1-piperidyl) phenyl ] -2(1H) -pyridone and a separation and detection method of mutation impurities caused by aromatic nitro and aromatic amino.

Disclosure of Invention

The invention provides a method for detecting 5, 6-dihydro-3- (4-morpholinyl) -1- [4- (2-oxo-1-piperidyl) phenyl ] -2(1H) -pyridone and mutation impurities caused by aromatic nitro and aromatic amino by using high performance liquid chromatography, which comprises the following steps:

octadecyl bonded silica gel is used as a filler for the chromatographic column; the detection wavelengths are 242nm and 325 nm;

the column temperature is 35-45 ℃; the sample injection amount is 1-100 mu L; the flow rate is 1.4-1.6 mL/min;

the mobile phase A is a mixed solution of ammonium acetate buffer solution and acetonitrile with the volume ratio of 90: 10;

the mobile phase B is a mixed solution of ammonium acetate buffer solution and acetonitrile with the volume ratio of 5: 95;

elution was performed according to the following gradient:

time (min)	Mobile phase A (v/v)	Mobile phase B (v/v)
			0	90～95	10～5
5	88	12
			28	55	45
28.01	90～95	10～5
			33	90～95	10～5

Wherein the concentration of the ammonium acetate buffer solution is 0.01-0.05 mol/L.

In one embodiment of the present invention, the mutagenic impurity is selected from the group consisting of:

the implementation method of the invention comprises the following steps:

the implementation method of the invention is characterized in that the gradient is as follows:

time (min)	Mobile phase A (v/v)	Mobile phase B (v/v)
			0	92	8
5	88	12
			28	55	45
28.01	92	8
			33	92	8

。

The implementation method of the invention is characterized in that the column temperature is 38-42 ℃, preferably 40-41 ℃.

The implementation method of the invention is characterized in that the sample injection amount is 5-20 mu L.

The implementation method of the invention is characterized in that the flow rate is 1.45-1.55 mL/min.

The method for implementing the invention is implemented, wherein the concentration of the ammonium acetate buffer solution is 0.02-0.04 mol/L, and preferably 0.03 mol/L.

The invention is implemented by the method, wherein the chromatographic column is Inertsil ODS-3C 18.

The separation degree is used for evaluating the separation degree between a substance to be detected and a separated substance, is a key index for measuring the separation efficiency of a chromatographic system and is represented by R, and R is equal to the ratio of the difference of retention time of adjacent chromatographic peaks to the width average value of the two chromatographic peaks. A larger R indicates a better separation of two adjacent components. Generally, when R < 1.0, the peaks overlap partially, the degree of separation can reach 98% when R is 1.0, and 99.7% when R is 1.5.

The detection method of 5, 6-dihydro-3- (4-morpholinyl) -1- [4- (2-oxo-1-piperidyl) phenyl ] -2(1H) -pyridone and aromatic nitro and aromatic amino mutation impurities thereof provided by the invention can realize effective separation of the impurities, the separation degree R of each absorption peak is more than or equal to 1.2, the sampling precision is good, the RSD is less than or equal to 2%, the method has good durability, and the method can be used for quality control in the development and production processes of apixaban.

Drawings

FIG. 1, 268-7 potential mutagenic impurity separation chromatogram of example 1-a;

FIG. 2, 268-7 potential mutagenic impurity separation chromatogram of example 1-b;

FIG. 3, 268-7 potential mutagenic impurity separation chromatogram of example 1-c;

FIG. 4 is a chromatogram of a sample solution using methanol as a solvent;

FIG. 5 is a chromatogram of a sample solution using ethanol as a solvent;

FIG. 6 is a chromatogram of a sample solution using a 20% acetonitrile aqueous solution as a solvent;

FIG. 7 is a chromatogram of a sample solution using an 80% acetonitrile aqueous solution as a solvent;

FIG. 8 is a chromatogram of 268-5 control solution with a sample size of 20. mu.L using acetonitrile as a solvent;

FIG. 9 is a chromatogram of a 268-5 control solution with a sample size of 10. mu.L using acetonitrile as a solvent;

FIG. 10 is a chromatogram of a 268-5 control solution with a sample size of 5. mu.L using acetonitrile as a solvent;

FIG. 11, chromatogram at wavelength 242nm for separation of potential mutagenic impurities from 268-7 of example 1-d;

FIG. 12, chromatogram at 325nm wavelength of separation of the 268-7 potentially mutagenic impurities of example 1-d;

FIG. 13, chromatogram of control 268-5, retention time t 7.653 min;

FIG. 14, chromatogram of control 268-SM, retention time t-9.320 min;

FIG. 15, chromatogram of control 268-2, retention time t 13.327 min;

FIG. 16, chromatogram of control 268-4, retention time t 16.950 min;

FIG. 17 and the chromatogram of control 268-3, with retention time t of 24.413 min;

FIG. 18, chromatogram of control 268-1, retention time t-26.697 min;

FIGS. 19 and 268-5 are chromatograms of labeled test solution.

Detailed Description

Example 1: screening of chromatographic conditions

The chromatographic conditions are determined by screening through chromatographic columns, elution gradients, flow rates, and the like.

Example 1-a

A chromatographic column: phenomenex prodigy ODS 3100A (250X 3.2mm,5 μm);

detection wavelength: 240 nm; column temperature: 40 ℃; sample introduction amount: 10 mu L of the solution; flow rate: 1.00 mL/min;

elution was performed according to the following gradient:

time (min)	Mobile phase A (v/v)	Mobile phase B (v/v)
			0	86	14
2	86	14
			19	55	45
25	55	45
			25.01	86	14
30	86	14

Test results and conclusions: the main peak is seriously tailing, the impurity 268-2 is wrapped, the separation degree of the 268-5 and 268-3 and other impurities is less than 1.0, and the chromatographic column is replaced.

Example 1 b

A chromatographic column: agilent Zorbax GF-250 (250X 4.6mm,5 μm);

elution was performed according to the following gradient:

Test results and conclusions: system suitability gives only one peak in solution and the column is unsuitable.

Examples 1 to c

A chromatographic column: shimadzu Inertsil ODS-3 (150X 4.6mm,5 μm);

detection wavelength: 242 nm; column temperature: 40 ℃; sample introduction amount: 10 mu L of the solution; flow rate: 1.40 mL/min;

elution was performed according to the following gradient:

Test results and conclusions: 268-1 less than 1.0 from adjacent impurities in the sample, and adjusting the mobile phase gradient.

From examples 1-a, 1-b and 1-c, we also found that 5, 6-dihydro-3- (4-morpholinyl) -1- [4- (2-oxo-1-piperidinyl) phenyl ] -2(1H) -pyridone (268-7) is susceptible to the formation of impurity 268-5 in aqueous solution, so we examined five solvents, methanol, ethanol, acetonitrile-water (20: 80), acetonitrile-water (80: 20), respectively, for the detection of impurity 268-5, with the following experimental results: the related map is shown in the attached figures 4-10;

the test results show that: the sample solution is unstable in a solvent containing active hydrogen, and the solvent effect of a high-proportion organic phase (such as methanol, ethanol and 80% acetonitrile aqueous solution) is too large, so acetonitrile is selected as the solvent. The acetonitrile is used as a solvent, the solvent effect still exists, but the influence of the solvent effect can be effectively removed and the sensitivity of impurities can be improved by reducing the sample amount to 5 mu L and increasing the sample concentration to 4 mg/mL.

In addition, the unknown impurities in the sample are more, and the interference on the known mutation-causing impurities is larger, so that the maximum absorption wavelength of different impurities is selected as the detection wavelength, and the interference of unknown single impurities in the sample is avoided.

Examples 1 d

A chromatographic column: shimadzu Inertsil ODS-3 (150X 4.6mm,5 μm);

detection wavelength: 242nm and 325 nm; column temperature: 40 ℃;

sample introduction amount: 5 μ L, 20 μ L; flow rate: 1.45 mL/min;

elution was performed according to the following gradient:

Test results and conclusions: by adjusting the gradient of the mobile phase, different wavelengths are extracted, the separation degree of 5, 6-dihydro-3- (4-morpholinyl) -1- [4- (2-oxo-1-piperidyl) phenyl ] -2(1H) -pyridone and aromatic nitro and aromatic amino mutation-causing impurities thereof achieves a better effect, and the detection of 6 mutation-causing impurities is not interfered, so that the detection requirement can be met.

Example 2: method specificity

Specificity tests were performed according to the method of example 1-d.

Step 1, preparation of a mobile phase:

preparing an ammonium acetate buffer solution (0.03 mol/L): weighing 2.31g of ammonium acetate, and adding water to 1000mL for dissolving to obtain the product.

Mobile phase A: the volume ratio of the ammonium acetate buffer solution (0.03mol/L) to the acetonitrile is 90: 10;

mobile phase B: the volume ratio of the ammonium acetate buffer solution (0.03mol/L) to the acetonitrile is 5: 95.

[ step 2 ] solution preparation:

selecting reference substance 268-7 and its impurities 268-SM, 268-1, 268-2, 268-3, 268-4, 268-5; the specific preparation process of the solution is as follows:

1) for detecting impurities 268-SM, 268-1, 268-2, 268-3, 268-4:

i. blank solvent: respectively weighing 500mL of acetonitrile and water, uniformly mixing, filtering, and standing to room temperature;

test solution: taking 20mg of 268-7 test sample, precisely weighing, placing in a 10mL measuring flask, dissolving with a solvent to a constant volume to obtain a test sample solution;

impurity mixed control solution: 268-SM, 268-1, 268-2, 268-3, 268-4 and 268-5 each impurity reference substance is about 10mg, precisely weighed, placed in a 100mL measuring flask, dissolved by a solvent, subjected to constant volume, and gradually and quantitatively diluted to prepare a solution of 0.3 mu g/mL as a reference solution;

marking the test article solution: taking 20mg of 268-7 test sample, precisely weighing, placing in a 10mL measuring flask, dissolving with a solvent, transferring 2mL of impurity mixed control mother liquor of 3 mug/mL into the measuring flask, and fixing the volume with the solvent;

v. impurity localization solution: taking about 10mg of each impurity reference substance, precisely weighing, placing in a 10mL measuring flask, dissolving with a solvent to a constant volume, and diluting to obtain a solution of 10 mu g/mL as a positioning solution.

2) For the detection impurity 268-5:

i. blank solvent: acetonitrile, and filtering;

test solution: taking 268-7 samples of 40mg, precisely weighing, placing in a 10mL measuring flask, dissolving acetonitrile and fixing the volume to obtain a sample solution;

labeling the test article solution: taking 268-7 sample 40mg, precisely weighing, placing in a 10mL measuring flask, dissolving acetonitrile, transferring 3 microgram/mL impurity mixed reference mother liquor 2mL into the measuring flask, and fixing the volume of acetonitrile;

iv.268-5 localization solution: 268-5 is precisely weighed according to 10mg, and is placed in a 10mL measuring flask, and acetonitrile is dissolved to a constant volume and is diluted into a solution of 10 mu g/mL to be used as a positioning solution.

The chromatographic conditions were as follows:

a chromatographic column: shimadzu Inertsil ODS-3 (150X 4.6mm,5 μm); detection wavelength: 242nm and 325 nm;

column temperature: 40 ℃; sample introduction amount: 5 μ L, 20 μ L; flow rate: 1.45 mL/min.

Elution was performed according to the following gradient:

And (3) respectively sampling the impurity solutions and the mixed solution under the chromatographic conditions, recording a chromatogram, positioning the impurity reference solution as shown in figures 13-18, and marking the sample solution as shown in figure 19, wherein the separation condition of the aromatic nitro and aromatic amino mutation impurities in 268-7 is shown in table 1.

Separation of aromatic Nitro and aromatic amino mutagenic impurities in tables 1, 268-7

Example 3: precision experiment

And taking the reference substance solution, carrying out sample injection for 6 times continuously, recording the chromatogram, and evaluating the result, wherein the result is shown in table 2.

TABLE 2 results of sample injection precision experiment

And the sample injection precision test result shows that: the peak area RSD% of each impurity in the sample injection precision solution is less than or equal to 2%, the method is used for detecting mutation-induced impurities of the aromatic nitro and aromatic amino in 268-7, and the sample injection precision of the instrument is good.

Example 4: durability test

The flow rate, initial ratio of mobile phase, column temperature, etc. under the chromatographic conditions were adjusted to determine the degree of resistance to which the results were not affected, and the results of durability were shown in tables 3 to 5.

TABLE 3 degrees of separation at different flow rates

Durability test results for different flow rates show: the testing method changes between the flow rate of 1.40mL/min and the flow rate of 1.60mL/min, has little influence on the testing result, and shows that the method has good durability.

TABLE 4 degree of separation of the starting proportions of the different mobile phases

The results of the tests for different initial proportions of mobile phase show that: the initial ratio of the mobile phase of the test method is changed between 90: 10 and 95: 5, the test result is not greatly influenced, and the durability of the method is good.

TABLE 5 degrees of separation at different column temperatures

The test results of different column temperatures of durability show that: the test method changes between the column temperature of 35 ℃ and 45 ℃, has little influence on the test result, and shows that the method has good durability.

Claims

1. A method for detecting 5, 6-dihydro-3- (4-morpholinyl) -1- [4- (2-oxo-1-piperidyl) phenyl ] -2(1H) -pyridone and mutation-induced impurities of aromatic nitro and aromatic amino by using high performance liquid chromatography is characterized in that:

elution was performed according to the following gradient:

2. The method of claim 1, wherein: the mutagenic impurity is selected from the following compounds:

3. the method of claim 2, wherein: the detection method of the mutagenic impurities comprises the following steps:

4. the method of claim 2, wherein: the detection method of the mutagenic impurities comprises the following steps:

5. the method according to any one of claims 1 to 4, wherein: elution was performed according to the following gradient:

。

6. The method according to any one of claims 1 to 4, wherein: the column temperature is 38-42 ℃.

7. The method of claim 6, wherein: the column temperature is 40-41 ℃.

8. The method according to any one of claims 1 to 4, wherein: the sample injection amount is 5-20 mu L.

9. The method according to any one of claims 1 to 4, wherein: the flow rate is 1.45-1.55 mL/min.

10. The method according to any one of claims 1 to 4, wherein: the concentration of the ammonium acetate buffer solution is 0.02-0.04 mol/L.

11. The method of claim 10, wherein: the concentration of the ammonium acetate buffer solution is 0.03 mol/L.

12. The method according to any one of claims 1 to 4, wherein: the chromatographic column is Inertsil ODS-3C 18.