CN113552263B

CN113552263B - Apixaban intermediate and separation detection method of mutation-causing impurities thereof

Info

Publication number: CN113552263B
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: 2023-06-06
Anticipated expiration: 2041-07-29
Also published as: CN113552263A

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-piperidinyl) phenyl ] -2 (1H) -pyridone, which comprises the steps of carrying out gradient elution by using a high performance liquid chromatograph and taking a mixed solution of an ammonium acetate buffer solution and acetonitrile as a mobile phase, wherein the detection wavelength of the mixed solution is 242nm and 325 nm. The gradient elution adopted by the invention can realize the effective separation of impurities, the separation degree 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 percent, the method has good durability, and the method can be used for quality control in the development and production processes of apixaban.

Description

Apixaban intermediate and separation detection method of mutation-causing impurities thereof

Technical Field

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

Background

The intrinsic quality of the medicine directly influences the clinical curative effect and safety of the medicine, wherein the research of potential mutagenic impurities is particularly important, and the potential mutagenic impurities mainly comprise initial raw materials, intermediates, polymers, side reaction products and the like which are brought in the production process. Based on safety and practical production considerations, potentially mutagenic impurities that are highly toxic, dangerous to human health, ineffective or affecting drug stability must be studied and tightly 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, with the following structure of potentially mutagenic impurities:

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at present, no method for separating and detecting 5, 6-dihydro-3- (4-morpholinyl) -1- [4- (2-oxo-1-piperidinyl) phenyl ] -2 (1H) -pyridone and aromatic nitro and aromatic amino mutation impurities is reported in the literature.

Disclosure of Invention

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

the chromatographic column takes octadecyl bonded silica gel as a filler; the detection wavelength is 242nm and 325nm;

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

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

the mobile phase B is a mixed solution of an ammonium acetate buffer solution and acetonitrile in a 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.

The method of the invention is practiced wherein the mutagenic impurity is selected from the following compounds:

the implementation method of the invention comprises the following steps of:

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the implementation method of the invention comprises the following steps:

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 process according to the invention is carried out in which the column temperature is 38 to 42℃and preferably 40 to 41 ℃.

The method is implemented, wherein the sample injection amount is 5-20 mu L.

The method is implemented, wherein the flow rate is 1.45-1.55 mL/min.

The method is carried out in which the concentration of the ammonium acetate buffer solution is 0.02 to 0.04mol/L, preferably 0.03mol/L.

The method is implemented by the invention, wherein the chromatographic column is Inertsil ODS-3C18.

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

The detection method of the 5, 6-dihydro-3- (4-morpholinyl) -1- [4- (2-oxo-1-piperidinyl) phenyl ] -2 (1H) -pyridone and the aromatic nitro and aromatic amino mutation impurities 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 durability is good, 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 isolation chromatogram of example 1-a;

FIG. 2, chromatogram for separation of potentially mutagenic impurities at 268-7 of example 1-b;

FIG. 3, chromatogram for separation of potentially mutagenic impurities at 268-7 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 a 268-5 control solution with an injection amount of 20. Mu.L using acetonitrile as a solvent;

FIG. 9 is a chromatogram of a 268-5 control solution with an injection amount of 10. Mu.L using acetonitrile as a solvent;

FIG. 10 is a chromatogram of a 268-5 control solution with an injection amount of 5. Mu.L using acetonitrile as a solvent;

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

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

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

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

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

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

fig. 17, chromatogram of control 268-3, retention time t= 24.413min;

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

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

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

Chromatographic column: phenomenex prodigy ODS 3A (250×3.2mm,5 μm);

detection wavelength: 240nm; column temperature: 40 ℃; sample injection amount: 10. Mu.L; flow rate: 1.00mL/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 tail of the main peak is serious, the impurity 268-2 is wrapped, the separation degree of 268-5 and 268-3 from other impurities is less than 1.0, and the chromatographic column is replaced.

Example 1-b

Chromatographic column: agilent Zorbax GF-250 (250×4.6mm,5 μm);

elution was performed according to the following gradient:

Test results and conclusions: only one peak was seen in the system applicability solution and the chromatographic column was unsuitable.

Example 1-c

Chromatographic column: island body Inertsil ODS-3 (150X 4.6mm,5 μm);

detection wavelength: 242nm; column temperature: 40 ℃; sample injection amount: 10. Mu.L; flow rate: 1.40mL/min;

elution was performed according to the following gradient:

Test results and conclusions: 268-1 and the adjacent impurities in the sample are less than 1.0, and the gradient of the mobile phase is adjusted.

By way of example 1-a, example 1-b and example 1-c, we also found that 5, 6-dihydro-3- (4-morpholino) -1- [4- (2-oxo-1-piperidinyl) phenyl ] -2 (1H) -pyridone (268-7) readily forms impurity 268-5 in aqueous solution, so we examined five solvents, methanol, ethanol, acetonitrile-water (20:80), acetonitrile-water (80:20) for detection of impurity 268-5, respectively, the test results were as follows: the related patterns are shown in figures 4-10;

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

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

Example 1-d

Chromatographic column: island body Inertsil ODS-3 (150X 4.6mm,5 μm);

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

sample injection amount: 5. Mu.L, 20. Mu.L; flow rate: 1.45mL/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-morpholino) -1- [4- (2-oxo-1-piperidinyl) phenyl ] -2 (1H) -pyridone and aromatic nitro and aromatic amino mutation impurities reaches a good effect, and 6 mutation impurity detection is not interfered, so that the detection requirement can be met.

Example 2: method specificity

A specificity test was carried out according to the method of example 1-d.

Preparation of mobile phase:

ammonium acetate buffer solution (0.03 mol/L) was prepared: 2.31g of ammonium acetate is weighed, and water is added to 1000mL for dissolution.

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

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

Solution preparation:

selecting a control material 268-7 and 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 detection of impurities 268-SM, 268-1, 268-2, 268-3, 268-4:

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

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

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

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

v. impurity localization solution: about 10mg of each impurity reference substance is taken, precisely weighed, placed in a 10mL measuring flask, dissolved in a solvent to a certain volume, and diluted into a solution of 10 mug/mL to serve as a positioning solution.

2) For detection of impurity 268-5:

i. blank solvent: acetonitrile, filtering;

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

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

iv.268-5 positioning solution: 268-5 control 10mg was precisely weighed, placed in a 10mL measuring flask, acetonitrile was dissolved to volume, and diluted to 10. Mu.g/mL solution as a positioning solution.

The chromatographic conditions were as follows:

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

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

Elution was performed according to the following gradient:

Taking each impurity solution and mixed solution under the chromatographic condition, respectively injecting samples, recording the chromatograms, positioning the impurity reference substance solutions as shown in figures 13-18, marking the sample solution as shown in figure 19, and separating the impurity caused by mutation of aromatic nitro and aromatic amino in 268-7 by the method as shown in table 1.

Separation of aromatic nitro and aromatic amino-induced mutation impurities in tables 1 and 268-7

Example 3: precision experiments

The control solution was sampled 6 times continuously, the chromatogram was recorded, and the results were evaluated, and the results are shown in Table 2.

TABLE 2 Experimental results of sample injection precision

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 detects the aromatic nitro and aromatic amino mutation impurities in 268-7, and the instrument sample injection precision is good.

Example 4: durability test

The degree of tolerance of the results, which was not affected by the adjustment of the flow rate of the chromatographic conditions, the initial ratio of the mobile phase, the column temperature, etc., was measured, and the durability results are shown in tables 3 to 5.

TABLE 3 degree of separation at different flow rates

The test results for different flow rates of durability showed that: the test method changes between the flow rate of 1.40mL/min and the flow rate of 1.60mL/min, and has little influence on the measurement result, thus indicating that the method has good durability.

TABLE 4 degree of separation of different mobile phase starting ratios

The test results for durability at different initial ratios of mobile phases show that: the initial ratio of the mobile phase of the test method is changed between 90:10 and 95:5, and the test method has little influence on the measurement result, thus indicating that the method has good durability.

TABLE 5 degree 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 ℃ and has little influence on the measurement result, thus showing that the method has good durability.

Claims

1. A method for detecting 5, 6-dihydro-3- (4-morpholino) -1- [4- (2-oxo-1-piperidinyl) phenyl ] -2 (1H) -pyridone and aromatic nitro and aromatic amino mutagenic impurities using high performance liquid chromatography, characterized by:

the chromatographic column is Inertsil ODS-3C18;

the column temperature is 35-45 ℃; the flow rate is 1.4-1.6 mL/min;

elution was performed according to the following gradient:

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

Wherein the concentration of the ammonium acetate buffer solution is 0.03mol/L;

the mutagenic impurities and detection conditions are as follows:

。

2. the method according to claim 1, characterized in that: the column temperature is 38-42 ℃.

3. The method according to claim 2, characterized in that: the column temperature is 40-41 ℃.

4. The method according to claim 1, characterized in that: the flow rate is 1.45-1.55 mL/min.