A HPLC detection method for related substances of edosapan intermediate 2

Abstract

The invention discloses an HPLC detection method for related substances of edosapan intermediate 2. Purified water is used as a diluent for dissolution, and a system applicability solution containing edosapan intermediate 2, known impurities M07, M08, and pyridine is prepared by quantitative dilution. A test solution and a quantitative limit solution containing edosapan intermediate 2 are prepared by quantitative dilution. The system applicability solution, the test solution, and the quantitative limit solution are respectively injected into a liquid chromatograph, a chromatogram is recorded, and the area percentage of related substances of the test sample is detected. The method adopts a high performance liquid chromatography method, and purified water is used as a diluent to determine the related substances of edosapan intermediate 2. The separation degree between edosapan intermediate 2 and its related substances and adjacent peaks meets the requirements, the method has strong specificity, good linearity, good repeatability, and convenient preparation of test samples.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, relates to a method for analyzing and detecting drugs, and particularly relates to a method for detecting edosapan intermediate 2 and related substances thereof by using HPLC. Background Art

[0002] Edoxaban, also known as edoxaban, is a small molecule oral anticoagulant developed by Daiichi Sankyo Co., Ltd. of Japan. It is a coagulation factor X (FXa) inhibitor. During the coagulation process, activated coagulation factor X (FXa) activates prothrombin (FII) into thrombin (FIIa), which promotes the formation of fibrin and thus forms a thrombus. Therefore, FXa has become the main target for the development of a new generation of anticoagulant drugs.

[0003] Edoxaban intermediate 2, also known as edoxaban impurity A (monooxalate), edoxaban intermediate 2, English name is Tert-Butyl [(1R,2S,5S)-2-amino-5-[(dimethylamino)carbonyl]cyclohexyl]carbamate oxalate, chemical name is [(1R,2S,5S)-2-amino-5-[(dimethylamino)carbonyl]cyclohexyl]carbamate oxalate, molecular formula is C 16 H 29 N3O7, molecular weight is 375.42, CAS number is 1210348-34-7, chemical structure is as follows:

[0004] .

[0005] According to the concept of source control and process control, effective detection methods should be established to implement strict control for the preparation of edosapan and edosapan intermediate 2. After searching, no relevant patents or public literature reports on the detection methods of related substances of edosapan intermediate 2 have been found. Summary of the invention

[0006] The object of the present invention is to provide a method for detecting related substances of edosapan intermediate 2, which has strong specificity, good linearity and good repeatability, and can be applied to the impurity control of edosapan intermediate 2.

[0007] The technical solution adopted by the present invention to solve the technical problem is: an HPLC detection method for related substances of edosapan intermediate 2, the chemical name of the edosapan intermediate 2 is [(1R, 2S, 5S)-2-amino-5-[(dimethylamino)carbonyl]cyclohexyl]carbamic acid tert-butyl ester oxalate, the molecular formula is C 16 H 29 N3O7, the structural formula is

[0008] ,

[0009] The structural formulas of the controlled known impurities M07, M08 and pyridine (Py) are

[0010] , , ;

[0011] Reverse phase high performance liquid chromatography was used with an ultraviolet detector. The HPLC detection conditions were as follows: chromatographic column: ZefexSupfex AQ-C18, 150*4.6mm, 5μm, hydrophilic C18 chromatographic column, column temperature was 40°C, conventional temperature, suitable for most instruments; mobile phase: aqueous phase: 20mM sodium perchlorate buffer; organic phase: acetonitrile; flow rate was 1.0 ml / min; elution program: gradient elution; detection wavelength: 210nm; including the following steps:

[0012] (1) Dissolve edosapan intermediate 2, known impurities M07, M08, and pyridine (Py) in purified water as a diluent, and quantitatively dilute to prepare a system suitability solution containing 2.0 mg of edosapan intermediate 2, 0.02 mg of known impurity M07, 0.02 mg of known impurity M08, and 0.02 mg of known impurity pyridine (Py) per 1 ml of diluent;

[0013] (2) Dissolve edosapan intermediate 2 in purified water as a diluent, and quantitatively dilute to prepare a test solution containing 2.0 mg of edosapan intermediate 2 per 1 ml of diluent;

[0014] (3) Dissolve the edosapan intermediate 2 in purified water as a diluent, and quantitatively dilute to produce a quantitative limit solution containing 0.001 mg of edosapan intermediate 2 per 1 ml of diluent;

[0015] (4) Take 5 μl of the system suitability solution, the test solution, and the quantitation limit solution and inject them into the liquid chromatograph. Record the chromatogram to complete the detection of related substances of edosapan intermediate 2.

[0016] Furthermore, the gradient elution is first eluted with 10% acetonitrile for 2 min, then the acetonitrile gradient is increased to 70% for 13 min, then washed with 70% acetonitrile for 2 min, and finally adjusted back to the initial proportion of 10% acetonitrile for 0.1 min and equilibrated for 5 min.

[0017] The beneficial effects of the present invention are as follows: the present invention adopts an AQ-C18 hydrophilic chromatographic column, which is a conventional C18 column and is easily available on the market; the mobile phase aqueous phase is a 20 mM sodium perchlorate buffer, which is an ion pair reagent but can solve the problems of poor separation and poor peak shape of the edosapan intermediate 2 and its related substances; the column temperature of the chromatographic column of the present invention is 40° C., which is a conventional temperature and is suitable for most instruments, thus avoiding the problem that most factories cannot detect due to insufficient instrument preparation; the detection time of the present invention is 22 minutes, which greatly improves the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a specific overlay diagram for the implementation case of the present invention;

[0019] Figure 2 The quantitative limit spectrum of the implementation case of the present invention;

[0020] Figure 3 This is a screen overlay of the sample solution of Comparative Example 1 of the present invention on an Agilent EC-C18 / CS-C18 / PFP / Bonus-RP chromatographic column;

[0021] Figure 4 This is an overlay of the screening of sample solutions in different mobile phase systems in Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0023] The present invention screens the chromatographic column and the mobile phase, and finally the chromatographic conditions of this implementation case can effectively separate it from related substances. The established detection method controls the analysis time within 22 minutes, and the separation degrees of the main component and the related substances and their adjacent peaks are all ≥2.5. The method has good specificity, high sensitivity, good linearity, good repeatability, and the method is accurate and reliable, achieving the above-mentioned purpose of the invention.

[0024] The specific HPLC detection chromatographic conditions are as follows:

[0025] Chromatographic column: Zefex Supfex AQ-C18, 150*4.6mm, 5μm, hydrophilic C18 column, column temperature is 40℃.

[0026] Mobile phase: aqueous phase: 20 mM sodium perchlorate buffer, organic phase: acetonitrile; flow rate: 1.0 ml / min.

[0027] Elution procedure: gradient elution, first eluting with 10% acetonitrile for 2 min, then increasing the acetonitrile gradient to 70% for 13 min, then washing with 70% acetonitrile for 2 min, finally adjusting back to the initial proportion of 10% acetonitrile in 0.1 min, and balancing for 5 min.

[0028] Detection wavelength: 210nm.

[0029] The following steps are involved:

[0030] (1) Dissolve edosapan intermediate 2, known impurities M07, M08, and pyridine (Py) in purified water as a diluent, and quantitatively dilute to prepare a system suitability solution containing 2.0 mg of edosapan intermediate 2, 0.02 mg of known impurities M07, 0.02 mg of M08, and 0.02 mg of pyridine (Py) per 1 ml of diluent.

[0031] (2) Dissolve edosapan intermediate 2 in purified water as a diluent and quantitatively dilute to prepare a test solution containing 2.0 mg of edosapan intermediate 2 per 1 ml of diluent.

[0032] (3) Dissolve the edosapan intermediate 2 in purified water as a diluent and quantitatively dilute it to produce a quantitative limit solution containing 0.001 mg of edosapan intermediate 2 per 1 ml of diluent.

[0033] (4) Take 5 μl of the system suitability solution, the test solution, and the quantitation limit solution and inject them into the liquid chromatograph. Record the chromatogram to complete the detection of related substances of edosapan intermediate 2.

[0034] The pre-verification investigation was carried out according to the above experimental conditions, and the specific implementation is as follows:

[0035] (1) Specificity investigation: Take 5 μl of the diluent and the system suitability solution in step 1 above, inject them into the liquid chromatograph, record the chromatogram, and complete the specificity investigation.

[0036] The results are as follows Figure 1 As shown: Peak 1 in the figure is the oxalate peak released from the sample of edosapan intermediate 2, which may not be integrated in the actual detection process; Peak 2 is M08, Peak 3 is Py, Peak 4 is edosapan intermediate 2, and Peak 5 is M07; edosapan intermediate 2 and known impurities M07, M08, and Py are completely baseline separated from their respective adjacent peaks, with a separation degree ≥ 2.5, and the blank has no interference with edosapan intermediate 2 and known impurities M07, M08, and Py. The spectral purity of edosapan intermediate 2 is 1000, and the specificity is good; wherein M08 has a poor peak shape, but can be quantified at a sample concentration level of 0.1. In view of the fact that the method of the present invention is a method for controlling the quality of the intermediate, M08 can be quantified, and although the peak shape is poor, it does not affect the quality control of edosapan intermediate 2, and has no significant effect on the quality control of the final product edosapan.

[0037] (2) Sensitivity study: Take the known impurities M07, M08 and Py and dissolve them in purified water as diluent. Dilute them quantitatively to make quantitative limit solutions containing 0.001 mg M07, 0.002 mg M08 and 0.002 mg Py per 1 ml of diluent. Take 5 μl of each quantitative limit solution and inject it into the liquid chromatograph. Record the chromatogram to complete the sensitivity study.

[0038] The results are as follows Figure 2 As shown: The S / N of the three known impurities is ≥ 15, which meets the acceptance criteria (S / N ≥ 10).

[0039] (3) Linearity test: Dissolve the edosapan intermediate 2 in purified water as a diluent and quantitatively dilute it to prepare a series of linear solutions at the sample concentration level of 0.05% to 120%. Take 5 μl and inject it into the liquid chromatograph, record the chromatogram, and complete the linearity test.

[0040] Results: The linear equation is Y=67.0962X-0.6122, correlation coefficient=0.9999; the ratio of intercept b to Y at 100X=0.4%, and the linear relationship is good.

[0041] (4) Repeatability: Dissolve edosapan intermediate 2 in purified water as diluent and quantitatively dilute to prepare a test solution containing 2.0 mg of edosapan intermediate 2 per 1 ml of diluent; prepare 3 replicates in parallel.

[0042] Results: The RSD of 2%Area of ​​polysaiban intermediate in three sample solutions was 0.0%, and the maximum absolute difference of %Area of ​​impurities (including known impurities and unknown impurities detected) was ADmax=0.03%, with good repeatability.

[0043] (5) Solution stability: Dissolve the edosapan intermediate 2 in purified water as a diluent and quantitatively dilute it to prepare a test solution containing 2.0 mg of edosapan intermediate 2 per 1 ml of diluent. After standing at room temperature for 16.2 h, re-inject the sample.

[0044] Results: After the sample solution was placed at room temperature for 16.2 hours, the absolute difference of the known impurity M08%Area was 0.21%, which was significantly increased. It is recommended that the sample solution be freshly prepared before use.

[0045] The above data show that the method of the present invention is accurate and reliable and can be used for the detection of related substances of edosapan intermediate 2.

[0046] Comparative Example 1

[0047] Chromatographic column screening, HPLC detection chromatographic conditions are as follows:

[0048] Chromatographic columns: Agilent Poroshell 120 EC-C18 / CS-C18 / PFP / Bonus-RP, the four columns are all 100*4.6mm, 2.7μm was screened, and the column temperature was 30℃.

[0049] Mobile phase: aqueous phase: 0.1% formic acid aqueous solution, organic phase: acetonitrile; flow rate is 1.0 ml / min.

[0050] Elution procedure: gradient elution, first eluting with 10% acetonitrile for 1 min, then increasing the acetonitrile gradient to 90% for 7 min, then washing with 90% acetonitrile for 2 min, finally adjusting back to the initial proportion of 10% acetonitrile in 0.1 min, and balancing for 5 min.

[0051] Detection wavelength: 220nm, 254nm.

[0052] The following steps are involved:

[0053] (1) Take a sample of edosapan intermediate 2 and dissolve it in a 50% acetonitrile aqueous solution as a diluent, and quantitatively dilute it to prepare a sample solution containing 1.0 mg of the sample per 1 ml of the diluent.

[0054] (2) Take 5 μl of the sample solution and inject it into the liquid chromatograph, record the chromatogram, and complete the development of the method for related substances of edosapan intermediate 2.

[0055] The results are as follows Figure 3 As shown: poor peak shape and response, EC-C18 has a slight response.

[0056] Comparative Example 2

[0057] The mobile phase system was optimized and the HPLC detection chromatographic conditions were as follows:

[0058] Chromatographic column: Agilent Poroshell 120 EC-C18 100*4.6mm, 2.7μm, column temperature 30℃.

[0059] Mobile phase 1: aqueous phase: 0.1% formic acid aqueous solution, organic phase: acetonitrile; flow rate is 1.0 ml / min.

[0060] Mobile phase 2: aqueous phase: 0.1% trifluoroacetic acid aqueous solution, organic phase: acetonitrile; flow rate is 1.0 ml / min.

[0061] Mobile phase 3: aqueous phase: 20 mM K2HPO4 buffer, organic phase: acetonitrile; flow rate is 1.0 ml / min.

[0062] Elution procedure (mobile phase 1, mobile phase 2): gradient elution, first eluting with 10% acetonitrile for 1 min, then increasing the acetonitrile gradient to 90% for 7 min, then washing with 90% acetonitrile for 2 min, and finally adjusting back to the initial proportion of 10% acetonitrile in 0.1 min and balancing for 5 min.

[0063] Elution program (mobile phase 3): gradient elution, first eluting with 10% acetonitrile for 1 min, then increasing the acetonitrile gradient to 70% for 7 min, then washing with 70% acetonitrile for 2 min, and finally adjusting back to the initial proportion of 10% acetonitrile in 0.1 min and balancing for 5 min.

[0064] Detection wavelength: 220nm.

[0065] The following steps are involved:

[0066] (1) Take a sample of edosapan intermediate 2 and dissolve it in a 50% acetonitrile aqueous solution as a diluent, and quantitatively dilute it to prepare a sample solution containing 1.0 mg of the sample per 1 ml of the diluent.

[0067] (2) Take 5 μl of the sample solution and inject it into the liquid chromatograph, record the chromatogram, and complete the development of the method for related substances of edosapan intermediate 2.

[0068] The results are as follows Figure 4 As shown: 20mM K2HPO4 buffer has the best response, but the peak is delayed and still needs further optimization.

[0069] Comparative Example 3

[0070] The mobile phase system was optimized and the HPLC detection chromatographic conditions were as follows:

[0071] Chromatographic column: Agilent Poroshell 120 EC-C18 100*4.6mm, 2.7μm, column temperature 40℃.

[0072] Mobile phase: aqueous phase: 20 mM K2HPO4 buffer, organic phase: acetonitrile; flow rate is 1.0 ml / min.

[0073] Elution procedure: gradient elution, first eluting with 5% acetonitrile for 1 min, then increasing the acetonitrile gradient to 70% for 19 min, then washing with 70% acetonitrile for 5 min, finally adjusting back to the initial proportion of 5% acetonitrile in 0.1 min, and balancing for 5 min.

[0074] Detection wavelength: 210nm.

[0075] The following steps are involved:

[0076] (1) Dissolve the edosapan intermediate 2 sample, M07, M08, and Py impurity reference substance in 50% acetonitrile aqueous solution as a diluent, and quantitatively dilute to prepare a sample solution containing 2.0 mg of sample, 2.0 mg of M07, 2.0 mg of M08, and 2.0 mg of Py per 1 ml of diluent, and a M07, M08, and Py positioning solution.

[0077] (2) Take 5 μl of sample solution, M07, M08, and Py positioning solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0078] Results: The retention time of M08 was 2.17 min, which was basically unretained. Combined with the chemical structure of M08, it was judged that M08 was alkaline. In the 20 mM K2HPO4 buffer system, it should be in a molecular state, but it was still unretained. Therefore, an ion pair reagent was used to enhance retention.

[0079] The above embodiments are merely illustrative of the principles and effects of the present invention, as well as some embodiments of its application. It is understandable to those skilled in the art that various changes, modifications, substitutions and variations may be made without departing from the principles and concepts of the present invention, and all of these fall within the scope of protection of the present invention.

Claims

1. A HPLC detection method for related substances of edosapan intermediate 2, wherein the chemical name of the edosapan intermediate 2 is [(1R, 2S, 5S)-2-amino-5-[(dimethylamino)carbonyl]cyclohexyl]carbamic acid tert-butyl ester oxalate, and the molecular formula is C 16 H 29 N3O7, the structural formula is ， Features: Reverse phase high performance liquid chromatography, UV detector, HPLC detection conditions are as follows Chromatographic column: Zefex Supfex AQ-C18, 150*4.6mm, 5μm, hydrophilic C18 column, column temperature 40℃; Mobile phase: aqueous phase: 20 mM sodium perchlorate buffer; organic phase: acetonitrile; flow rate: 1.0 ml / min; Elution procedure: first elute with 10% acetonitrile for 2 min, then increase the acetonitrile gradient to 70% for 13 min, then rinse with 70% acetonitrile for 2 min, finally adjust back to the starting ratio of 10% acetonitrile for 0.1 min, and balance for 5 min; Detection wavelength: 210nm; The following steps are involved: (1) Take edosapan intermediate 2, dissolve known impurities pyridine, M07 and M08 in purified water as diluent, and quantitatively dilute to prepare a system suitability solution containing 2.0 mg edosapan intermediate 2, 0.02 mg M07, 0.02 mg M08 and 0.02 mg pyridine per 1 ml of diluent; the structural formulas of M07 and M08 are and ; (2) Dissolve edosapan intermediate 2 in purified water as a diluent, and quantitatively dilute to prepare a test solution containing 2.0 mg of edosapan intermediate 2 per 1 ml of diluent; (3) Dissolve the edosapan intermediate 2 in purified water as a diluent, and quantitatively dilute to produce a quantitative limit solution containing 0.001 mg of edosapan intermediate 2 per 1 ml of diluent; (4) Take 5 μl of the system suitability solution, the test solution, and the quantitation limit solution and inject them into the liquid chromatograph. Record the chromatogram to complete the detection of the related substances of edosapan intermediate 2.

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