Method for separating rivaroxaban intermediate and its impurities and detecting the content of each component

Abstract

The present invention belongs to the field of chemical analysis technology, and in particular to a method for separating rivaroxaban intermediates and their impurities and detecting the content of each component. The method is: a chromatographic column uses a silica gel coated with amylose tris (3,5-diphenylyl carbamate) on the surface as a filler, and a mixed solution of n-hexane and isopropanol is used as a mobile phase for elution, and (S)-(+)-N-(2,3-propylene oxide) phthalimide and its optical isomers, synthetic raw material impurities and hydrolysis impurities are separated; in the mobile phase, the volume ratio of n-hexane and isopropanol is 95-85: 5-15. The self-built high-performance liquid chromatography of the present invention can realize the simultaneous separation and content detection of four components within 30 minutes or more than 30 minutes, and the separation degree between each impurity can meet the requirements. This method is highly sensitive and is not interfered by other impurities and test samples; it is simple, fast, sensitive and effective.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and particularly relates to a method for separating a rivaroxaban intermediate and its impurities and detecting the content of each component. Background Art

[0002] (S)-(+)-N-(2,3-Epoxypropyl)phthalimide, the English chemical name is (S)-(+)-N-(2,3-Epoxypropyl)phthalimide, the structural formula is shown in Formula I, the molecular formula is C 11 H 9 NO 3 , molecular weight is 203.19, CAS number is 161596-47-0.

[0003]

[0004] (S)-(+)-N-(2,3-epoxypropylene)phthalimide is prepared by reacting phthalimide and (S)-epoxyisopropylamine as raw materials, and is a key intermediate in the synthesis of rivaroxaban. (S)-(+)-N-(2,3-epoxypropylene)phthalimide is a key starting material for the synthesis of rivaroxaban, and is the key to the construction of the oxadiazolidine ring of rivaroxaban and the introduction of the chiral center. The subsequent synthesis steps do not involve the introduction of the chiral center and the chiral separation step, and its purity (related substances, optical isomer content) is directly related to the quality of the finished product of rivaroxaban. Therefore, the purpose of detecting related substances of (S)-(+)-N-(2,3-epoxypropylene)phthalimide is to control its purity to ensure the synthesis yield, i.e., the quality, of rivaroxaban; the purpose of detecting optical isomers in (S)-(+)-N-(2,3-epoxypropylene)phthalimide is to control the content of optical isomers in the finished product of rivaroxaban, which is of great significance for providing qualified starting materials for the synthesis of rivaroxaban.

[0005] In the prior art, optical isomers in compounds are usually detected by a separate method using a chiral separation chromatographic column. There are many detection methods, but they have the disadvantages of long detection time and high cost. Summary of the invention

[0006] In view of this, one of the objects of the present invention is to provide a method for separating impurities a, impurity b and impurity c in a rivaroxaban intermediate by HPLC. The method has high sensitivity and provides support for the subsequent simultaneous detection of the contents of the four components in the rivaroxaban intermediate.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for separating impurities a, b and c in a rivaroxaban intermediate by HPLC, wherein the rivaroxaban intermediate is (S)-(+)-N-(2,3-propylene oxide)phthalimide; the components to be separated include (S)-(+)-N-(2,3-propylene oxide)phthalimide, impurity a, impurity b and impurity c; the structural formula of the (S)-(+)-N-(2,3-propylene oxide)phthalimide is shown in Formula I, the structural formula of the impurity a is shown in Formula II, the structural formula of the impurity b is shown in Formula II, The structural formula is shown in Formula III, and the structural formula of the impurity c is shown in Formula IV; in the HPLC method, the chromatographic column uses silica gel coated with amylose-tris (3,5-xylylcarbamate) as a filler, and uses a mixed solution of n-hexane and isopropanol as a mobile phase for elution, and sequentially separates the impurity b, the (S)-(+)-N-(2,3-propylene oxide)phthalimide, the impurity a and the impurity c; in the mobile phase, the volume ratio of n-hexane to isopropanol is 95-85:5-15;

[0009]

[0010] The related impurities of (S)-(+)-N-(2,3-epoxypropylene)phthalimide include impurity a, impurity b and impurity c, among which impurity a is the optical isomer of (S)-(+)-N-(2,3-epoxypropylene)phthalimide; impurity b is the synthetic raw material, phthalimide; impurity c is the hydrolysis impurity of N-(2,3-epoxypropylene)phthalimide, and (S)-(+)-N-(2,3-epoxypropylene)phthalimide is easily hydrolyzed into impurity c. When the conventional reverse phase-liquid phase method is used to detect the content of impurity c and the main component, the solution is extremely unstable and the detection result is unreliable.

[0011] Furthermore, the method separates the impurity b, the (S)-(+)-N-(2,3-propylene oxide)phthalimide, the impurity a and the impurity c within 30 minutes or more.

[0012] Preferably, the elution time is 40 minutes.

[0013] Preferably, in the mobile phase, the volume ratio of n-hexane to isopropanol is 89-91:11-9, more preferably 90:10.

[0014] Furthermore, the flow rate of the mobile phase is 0.8-1.2 ml / min; and the column temperature of the chromatographic column is set at 20-40°C.

[0015] Preferably, the flow rate of the mobile phase is 0.95-1.05 ml / min, more preferably 1 ml / min; the column temperature of the chromatographic column is set at 28°C-32°C, more preferably 30°C.

[0016] Furthermore, the injection solution is prepared using the mobile phase as a diluent, and the injection solution includes a test solution, a control solution and / or a system suitability solution.

[0017] The second object of the present invention is to provide a method for simultaneously detecting the contents of (S)-(+)-N-(2,3-propylene oxide)phthalimide, impurity a, impurity b and impurity c in rivaroxaban intermediates.

[0018] To achieve the above object, the present invention adopts the following technical solutions:

[0019] A method for simultaneously detecting the contents of (S)-(+)-N-(2,3-propylene oxide)phthalimide, impurity a, impurity b and impurity c in a rivaroxaban intermediate, wherein the (S)-(+)-N-(2,3-propylene oxide)phthalimide, impurity a, impurity b and impurity c are separated by the method described in purpose one; after separation, detection is performed using a detector with a wavelength of 220±2nm.

[0020] Preferably, the detection wavelength is 220 nm.

[0021] Furthermore, the retention times of the four components from short to long are: impurity b, (S)-(+)-N-(2,3-propylene oxide)phthalimide, impurity a and impurity c.

[0022] As a preferred method, the following chromatographic conditions are used for elution and a chromatogram is obtained: the volume ratio of n-hexane and isopropanol in the mobile phase is 90:10, the chromatographic column has a specification of 4.6 mm×250 mm×5 μm, a flow rate of 1 ml / min; the column temperature is 30°C; the injection volume is 10 μl; and the detection wavelength is 220 nm.

[0023] Further, the impurity b was found to have a retention time of 8.955±0.5 min, the impurity (S)-(+)-N-(2,3-propylene oxide)phthalimide was found to have a retention time of 12.125±0.5 min, the impurity a was found to have a retention time of 15.221±0.5 min, and the impurity c was found to have a retention time of 23.861±0.5 min.

[0024] Furthermore, standard substances are prepared to obtain a chromatogram of a standard sample; the chromatogram obtained from the test sample is calculated by peak area according to the principal component self-control method with the addition of a correction factor.

[0025] The content of the components of the standard is known, while the content of the components of the test sample is unknown.

[0026] The obtained standard sample chromatogram can be stored in the database as a chromatographic material for quality control in the smart pharmaceutical factory, to help the robot determine whether the products it produces are qualified and to provide quantitative improvement analysis suggestions for unqualified products.

[0027] Further, the impurity b is within the linear range of 0.0131 μg / ml to 0.41 μg / ml, Y=1.9455X+0.0018, r=0.998; the (S)-(+)-N-(2,3-epoxypropylene)phthalimide is within the linear range of 0.0155 μg / ml to 0.8152 μg / ml, Y=2.0207X+0.005, r=0.9991; The impurity a is within the linear range of 0.018 μg / ml to 3.204 μg / ml, Y=1.9658X+0.0346, r=0.9991; the impurity c is within the linear range of 0.0446 μg / ml to 2.124 μg / ml, Y=1.2762X-0.0249, r=0.998; wherein Y is the Y axis, representing the peak area; X is the X axis, representing the concentration; and r represents the correlation coefficient.

[0028] Furthermore, the contents of (S)-(+)-N-(2,3-propylene oxide)phthalimide, impurity a, impurity b and impurity c in the rivaroxaban intermediate were calculated based on the peak area detected by the detector using the principal component self-reference method with correction factor added.

[0029] Furthermore, the correction factor of the impurity a is 1.0, the correction factor of the impurity b is 1.0, and the correction factor of the impurity c is 1.6.

[0030] The beneficial effects of the present invention are

[0031] 1. The high performance liquid chromatography method established by the present invention realizes the simultaneous detection of optical isomers, synthetic raw material impurities, and hydrolysis impurities in (S)-(+)N-(2,3-propylene oxide)phthalimide, the solution is stable, and the results are reliable; the present invention uses a set of methods to comprehensively control the impurities and purity of (S)-(+)N-(2,3-propylene oxide)phthalimide, saving detection time and detection cost.

[0032] 2. This method uses a chiral filler (silica gel coated with amylose-tris (3,5-xylylcarbamate)) chromatographic column and a simple n-hexane-isopropanol elution system. Within 30 minutes, one method is used to simultaneously detect optical isomers, known impurities of related substances (including synthetic raw materials, hydrolysis by-products) and unknown impurities. The separation between each impurity can meet the requirements, and this method has high sensitivity and is not interfered by other impurities and test samples. This method is simple, rapid, sensitive and effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the chromatogram of the mixed solution;

[0034] Figure 2 is the linear relationship diagram of impurity b;

[0035] Figure 3 Linear relationship diagram of the principal components;

[0036] Figure 4 is the linear relationship diagram of impurity a;

[0037] Figure 5 is the linear relationship diagram of impurity c;

[0038] Figure 6 It is the HPLC graph of the quantitative limit solution;

[0039] Figure 7 This is the HPLC graph of the durability-normal condition-mixed solution;

[0040] Figure 8 Durability - column temperature 32°C - HPLC chart of mixed solution;

[0041] Fig. 9 Durability - column temperature 28°C - HPLC chart of mixed solution;

[0042] Fig.10 Durability - flow rate 0.95ml / min - HPLC chart of mixed solution;

[0043] Fig.11 Durability - flow rate 1.05ml / min - HPLC chart of mixed solution;

[0044] Fig.12 The HPLC graph of durability-mobile phase ratio (89:11)-mixed solution;

[0045] Fig.13 The figure shows the HPLC graph of durability-mobile phase ratio (91:9)-mixed solution. DETAILED DESCRIPTION

[0046] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0047] In the embodiment of the present invention, the chromatographic conditions are: using silica gel coated with amylose-tris (3,5-xylylcarbamate) as a filler (Chiralpak AD-H, 4.6mm×250mm, 5μm, or a chromatographic column with equivalent performance); the mobile phase is a mixed solution of n-hexane and isopropanol, wherein the volume ratio of n-hexane to isopropanol is 90:10; the flow rate is 1ml / min; the column temperature is 30°C; the detection wavelength is 220nm; and the injection volume is 10μl.

[0048] In the embodiment of the present invention, the main instrument is a high performance liquid chromatograph.

[0049] In the embodiment of the present invention, the preparation method of the test solution is as follows:

[0050] The diluents were: mobile phase, i.e., n-hexane-isopropanol (90:10);

[0051] Test solution: Take an appropriate amount of this product, dissolve and dilute it with mobile phase to make a solution containing about 0.2 mg per 1 ml, shake well, and use it as the test solution;

[0052] Control solution: Accurately measure 0.2 ml of the test solution and place it in a 100 ml volumetric flask, dilute to the mark with the mobile phase, and shake well;

[0053] System suitability solution: Accurately weigh appropriate amounts of this product, impurity a, impurity b, and impurity c, dissolve and dilute with mobile phase to make a mixed solution containing approximately 0.2 mg of this product and 10 μg of each impurity per 1 ml.

[0054] In the embodiment of the present invention, the content detection method is as follows:

[0055] Accurately measure 10 μl of the system suitability solution, inject it into the liquid chromatograph, and record the chromatogram. The order of peaks is impurity b, main component, impurity a, impurity c, and the separation between the peaks of each component should meet the requirements.

[0056] Accurately measure the test solution and control solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0057] In the chromatogram of the test solution, the contents of impurity a (correction factor 1.0), impurity b (correction factor 1.0), impurity c (correction factor 1.6) and other individual impurities were calculated using the principal component self-reference method with correction factor added.

[0058] Example 1. Specificity

[0059] Prepare the stock solutions and positioning solutions of each impurity: accurately weigh 10 mg of each impurity a, impurity b, and impurity c reference substance, place them in different 50 ml volumetric flasks, add mobile phase to dissolve and dilute to the scale, shake well, and obtain.

[0060] Mixed solution: Weigh 10 mg of the test sample and place it in a 50 ml volumetric flask. Then accurately pipette 10 ml of each impurity stock solution and add it. Dilute to the scale with mobile phase and shake well.

[0061] Take 10 μl of the blank solvent, each positioning solution and the mixed solution, inject them into the liquid chromatograph and record the chromatogram. Figure 1 As shown in Table 1, Figure 1 The integration results are shown in Table 2.

[0062] Table 1. Method specificity test results

[0063]

[0064] Table 2. Figure 1 Points results table

[0065]

[0066]

[0067] Example 2. Limit of Quantitation

[0068] Stock solution: Accurately weigh 10.33 mg of this product, 8.01 mg of impurity a reference substance, 10.25 mg of impurity b reference substance, and 10.62 mg of impurity c reference substance, respectively, and place them in different 50 ml volumetric flasks. Add mobile phase to dissolve and dilute to the scale, shake well, and obtain.

[0069] Quantitative limit solution: Accurately pipette 0.64 ml of impurity b stock solution, 1.1 ml of impurity a stock solution, 0.76 ml of main component stock solution, and 2.1 ml of impurity c stock solution into the same 100 ml volumetric flask, add mobile phase to dilute to the scale, shake well, then accurately measure 1 ml into a 100 ml volumetric flask, add mobile phase to dilute to the scale, shake well, and obtain.

[0070] Accurately measure 10 μl of the quantitative limit solution, inject it 6 times continuously, record the chromatogram, and calculate the signal-to-noise ratio and peak area RSD of each peak. The results are shown in Figure 6 and Table 3, Figure 6 The integration results are shown in Table 4.

[0071] Table 3. Quantitation limit test results

[0072]

[0073]

[0074] Table 4. Figure 6 Points results table

[0075]

[0076] Example 3. Linearity and correction factors

[0077] Impurity stock solution: Accurately weigh 10.33 mg of this product, 8.01 mg of impurity a reference substance, 10.25 mg of impurity b reference substance, and 10.62 mg of impurity c reference substance, respectively, and place them in different 50 ml volumetric flasks. Add mobile phase to dissolve and dilute to the scale, shake well, and obtain.

[0078] Linear stock solution (linear solution 6#): Accurately pipette 0.2 ml of impurity b stock solution, 0.4 ml of main component stock solution, 2 ml of impurity a stock solution, and 1 ml of impurity c stock solution into the same 100 ml volumetric flask, add mobile phase to dilute to the scale, shake well, and obtain.

[0079] Linear solution 1: Accurately pipette 0.64 ml of impurity b stock solution, 1.1 ml of impurity a stock solution, 0.76 ml of main component stock solution, and 2.1 ml of impurity c stock solution into the same 100 ml volumetric flask, add mobile phase to dilute to the scale, shake well, then accurately measure 1 ml into a 100 ml volumetric flask, add mobile phase to dilute to the scale, shake well, and obtain.

[0080] Linear solutions 2#~5#: Accurately pipette 1.0ml, 2.5ml, 4.0ml and 5.0ml of the linear stock solution into 4 different 10ml volumetric flasks, dilute to the scale with mobile phase, shake well, and the solution is ready.

[0081] Take the above linear solutions, inject them in order from low to high concentration, record the chromatogram, use the least squares method to linearly regress the concentration X (μg / ml) against the peak area Y, and calculate the regression equation and correlation coefficient. Figure 2 to Figure 5 And Tables 5 to 8.

[0082] Table 5. Impurity b linearity test data

[0083]

[0084] Table 6. Principal component linearity test data

[0085]

[0086] Table 7. Impurity a linear test data

[0087]

[0088] Table 8. Impurity c linearity test data

[0089]

[0090] The correction factors were calculated using the principal components and the slopes. The results are shown in Table 9.

[0091]

[0092] Table 9. Correction factor test results

[0093] Impurity Name Slope Correction Factor Correction Factor Rounding Impurity b 1.9455 1.04 1.0 principal component 2.0207 - - Impurity a 1.9658 1.03 1.0 Impurity c 1.2762 1.58 1.6

[0094] Example 4. Durability

[0095] Mixed solution: Accurately weigh 10.33 mg of this product, 8.01 mg of impurity a reference substance, 10.25 mg of impurity b reference substance, and 10.62 mg of impurity c reference substance, respectively, and place them in different 50 ml volumetric bottles, add mobile phase to dissolve and dilute to scale, and shake well. Accurately pipette 0.1 ml of impurity b stock solution, 0.2 ml of main component stock solution, 0.5 ml of impurity a stock solution, and 0.5 ml of impurity c stock solution, place them in the same 100 ml volumetric bottle, add mobile phase to dilute to scale, shake well, and you have it.

[0096] Take the mixed solution, fine-tune each chromatographic condition and conduct experiments to examine the separation between the peaks. The test results are shown in Figure 7 to Figure 13 and Table 10, Figure 7 to Figure 13 The integration results are shown in Tables 11 to 17 respectively.

[0097] Table 10. Chromatographic system durability test results

[0098]

[0099] Table 11. Figure 7 Points results table

[0100]

[0101]

[0102] Table 12. Figure 8 Points results table

[0103]

[0104] Table 13. Fig. 9 Points results table

[0105]

[0106] Table 14. Fig.10 Points results table

[0107]

[0108] Table 15. Fig.11 Points results table

[0109]

[0110] Table 16. Fig.12 Points results table

[0111]

[0112]

[0113] Table 17. Fig.13 Points results table

[0114]

[0115] The results showed that when the flow rate, column temperature and mobile phase ratio fluctuated slightly, the peak separation of each component was greater than 1.5; the relative retention time of each impurity varied within the range of ±0.03.

Claims

1. A method for separating impurities a, b and c in a rivaroxaban intermediate by HPLC, characterized in that: The rivaroxaban intermediate is (S)-(+)-N-(2,3-propylene oxide)phthalimide; the components to be separated include (S)-(+)-N-(2,3-propylene oxide)phthalimide, impurity a, impurity b and impurity c; the structural formula of the (S)-(+)-N-(2,3-propylene oxide)phthalimide is shown in Formula I, the structural formula of the impurity a is shown in Formula II, the structural formula of the impurity b is shown in Formula III, the structural formula of the impurity c is shown in Formula The structure is shown in Formula IV; in the HPLC method, the chromatographic column uses silica gel coated with amylose-tris (3,5-xylylcarbamate) as a filler, and uses a mixed solution of n-hexane and isopropanol as a mobile phase for elution, and the impurity b, the (S)-(+)-N-(2,3-propylene oxide)phthalimide, the impurity a and the impurity c are separated in sequence; in the mobile phase, the volume ratio of n-hexane to isopropanol is 95-85:5-15; 2. The method according to claim 1, characterized in that The method separates the impurity b, the (S)-(+)-N-(2,3-propylene oxide)phthalimide, the impurity a and the impurity c within 30 minutes or more.

3. The method according to claim 1, characterized in that The flow rate of the mobile phase is 0.8-1.2 ml / min; the column temperature of the chromatographic column is set at 20-40°C.

4. The method according to claim 1, characterized in that: The injection solution is prepared with the mobile phase as a diluent, and the injection solution includes a test solution, a control solution and / or a system suitability solution.

5. A method for simultaneously detecting the contents of (S)-(+)-N-(2,3-propylene oxide)phthalimide, impurity a, impurity b and impurity c in a rivaroxaban intermediate, characterized in that: The (S)-(+)-N-(2,3-propylene oxide)phthalimide, the impurity a, the impurity b and the impurity c are separated by the method described in any one of claims 1 to 4; after separation, detection is performed using a detector with a wavelength of 220±2 nm.

6. The method according to claim 5, characterized in that The retention times of the four components from short to long are: impurity b, (S)-(+)-N-(2,3-propylene oxide)phthalimide, impurity a and impurity c.

7. The method according to claim 5, characterized in that The following chromatographic conditions were used for elution and to obtain a chromatogram: the volume ratio of n-hexane to isopropanol in the mobile phase was 90:10, the chromatographic column had a specification of 4.6 mm×250 mm×5 μm, a flow rate of 1 ml / min; the column temperature was 30°C; the injection volume was 10 μl; and the detection wavelength was 220 nm.

8. The method according to claim 7, characterized in that The impurity b was found to have a retention time of 8.955±0.5min, the impurity (S)-(+)-N-(2,3-propylene oxide)phthalimide was found to have a retention time of 12.125±0.5min, the impurity (a) was found to have a retention time of 15.221±0.5min, and the impurity (c) was found to have a retention time of 23.861±0.5min.

9. The method according to claim 7, characterized in that: Prepare standard products to obtain the standard sample chromatogram; the chromatogram obtained from the test sample is calculated by peak area using the principal component self-control method with added correction factor.

10. The method according to claim 9, characterized in that The impurity b is within the linear range of 0.0131 μg / ml to 0.41 μg / ml, Y=1.9455X+0.0018, r=0.998; the (S)-(+)-N-(2,3-propylene oxide)phthalimide is within the linear range of 0.0155 μg / ml to 0.8152 μg / ml, Y=2.0207X+0.005, r=0.9991; the impurity b is within the linear range of 0.0131 μg / ml to 0.41 μg / ml, Y=1.9455X+0.0018, r=0.998; the (S)-(+)-N-(2,3-propylene oxide)phthalimide is within the linear range of 0.0155 μg / ml to 0.8152 μg / ml, Y=2.0207X+0.005, r=0.9991; The substance a is in the linear range of 0.018 μg / ml to 3.204 μg / ml, Y=1.9658X+0.0346, r=0.9991; the impurity c is in the linear range of 0.0446 μg / ml to 2.124 μg / ml, Y=1.2762X-0.0249, r=0.998; wherein Y is the Y axis, representing the peak area; X is the X axis, representing the concentration; and r represents the correlation coefficient.

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