A method for detecting the enantiomer of edosapan intermediate 2

Abstract

The invention discloses a method for detecting the enantiomers of edosapan intermediate 2. The method comprises the following steps: using ethanol as a diluent to dissolve, quantitatively diluting to prepare a racemic solution containing 1.2 mg of the racemic form of edosapan intermediate 2 per 1 ml of the diluent, quantitatively diluting to prepare a test solution containing 2.0 mg of edosapan intermediate 2 per 1 ml of the diluent, taking 5 μl of the racemic solution and the test solution, injecting them into a liquid chromatograph respectively, recording a chromatogram, and detecting the enantiomer area percentage of the test sample. The method adopts a high performance liquid chromatography method and uses ethanol as a diluent to determine the enantiomers of edosapan intermediate 2. The method has the following requirements for the separation between edosapan intermediate 2 and its enantiomers and adjacent peaks, has strong specificity, good linearity, good repeatability, is convenient for preparing the test sample, and has good stability of the test sample solution.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and relates to a method for analyzing and detecting a drug, and in particular to a method for determining an edosapan intermediate 2 and related substances thereof. 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]

[0004] 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:

[0005] .

[0006] According to the concept of source control and process control, the preparation of edosapan and edosapan intermediate 2 also need to establish an effective detection method for strict control. After searching, there are currently no relevant patents or public literature reports on the enantiomer detection method of edosapan intermediate 2. Summary of the invention

[0007] The object of the present invention is to provide an enantiomer detection method for edosapan intermediate 2, which has strong specificity, good linearity, good repeatability, good stability of the test solution, and can be applied to the impurity control of edosapan intermediate 2.

[0008] The technical solution adopted by the present invention to solve the technical problem is: a method for detecting the enantiomers of edoxaban intermediate 2, wherein the chemical name of the edoxaban 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 H29 N3O7, the structural formula is

[0009] ,

[0010] Normal phase high performance liquid chromatography was used, and an ultraviolet detector was used. The HPLC detection conditions were as follows: chromatographic column: CHIRALPAK@OX-H, 250*4.6mm, 5μm, a polysaccharide derivative normal phase coated chiral chromatographic column, the silica gel surface was coated with cellulose-tris(4-chloro-3-methylphenylcarbamate), the column temperature was 35°C, a conventional temperature, suitable for most instruments; mobile phase: a mixed solution of n-hexane-ethanol-diethylamine; the flow rate was 0.7ml / min; elution procedure: isocratic elution for 10min; detection wavelength: 220nm is the maximum absorption wavelength; the following steps were included:

[0011] (1) dissolving the edosapan intermediate 2 racemate in ethanol as a diluent, and quantitatively diluting the solution to prepare a racemate solution containing 1.2 mg of the edosapan intermediate 2 racemate per 1 ml of the diluent;

[0012] (2) Dissolve edosapan intermediate 2 in ethanol as a diluent, and quantitatively dilute to prepare a test solution containing 2.0 mg of edosapan intermediate 2 per 1 ml of diluent;

[0013] (3) Take 5 μl of the racemic solution and the test solution and inject them into the liquid chromatograph, record the chromatogram, and complete the detection of the enantiomers of the edosapan intermediate 2.

[0014] Furthermore, the mobile phase is n-hexane:ethanol:diethylamine=500:500:1 (v / v / v).

[0015] The invention has the following beneficial effects: the invention adopts CHIRALPAK@OX-H, a polysaccharide derivative normal phase coated chiral chromatographic column, the surface of the silica gel is coated with cellulose-tris(4-chloro-3-methylphenylcarbamate), which is an uncommon chiral column, and solves the problem of difficulty in separating the edosapan intermediate 2 and its enantiomer; the column temperature of the chromatographic column of the invention is 35°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 configuration; the detection time of the invention is 10 minutes, which greatly improves the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is an overlay of the racemic solution and the sample of the embodiment of the present invention;

[0017] Figure 2 This is a screening overlay of the mixed solution of comparative example 2 of the present invention in AD / OJ in methanol:ethanol:diethylamine=40:60:0.3 (v / v / v);

[0018] Figure 3 This is a screen overlay of the mixed solution of Comparative Example 1 of the present invention on chromatographic columns AD / OD / OJ / AS;

[0019] Figure 4 This is a specific overlay diagram for the implementation case of the present invention. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0021] The present invention screens the chromatographic column and the mobile phase, and finally the chromatographic conditions of the implementation case can effectively separate it from the enantiomers, and the established detection method controls the analysis time within 10 minutes, and the separation degrees of the main component and the enantiomer and their adjacent peaks are all ≥3.9. The method has good specificity, high sensitivity, good linearity, good repeatability, certain solution stability, and the method is accurate and reliable, achieving the above-mentioned invention purpose.

[0022] The specific HPLC detection chromatographic conditions are as follows:

[0023] Chromatographic column: CHIRALPAK OX-H, 250*4.6 mm, 5 μm, polysaccharide derivative normal phase coated chiral chromatographic column, the silica gel surface is coated with cellulose-tris (4-chloro-3-methylphenylcarbamate), the column temperature is 35°C.

[0024] Mobile phase: n-hexane: ethanol: diethylamine = 500: 500: 1 (v / v / v); flow rate is 0.7 ml / min.

[0025] Elution program: isocratic elution, 10 min.

[0026] Detection wavelength: 220nm.

[0027] The following steps are involved:

[0028] (1) dissolving the edosapan intermediate 2 racemate in ethanol as a diluent, and quantitatively diluting to prepare a racemate solution containing 1.2 mg of the edosapan intermediate 2 racemate per 1 ml of the diluent;

[0029] (2) Dissolve edosapan intermediate 2 in ethanol as a diluent, and quantitatively dilute to prepare a test solution containing 2.0 mg of edosapan intermediate 2 per 1 ml of diluent;

[0030] (3) Dissolve the edosapan intermediate 2 in ethanol as a diluent, and quantitatively dilute it to prepare a sensitivity solution containing 0.004 mg of the edosapan intermediate per 1 ml of diluent;

[0031] (4) Take 5 μl of the racemic solution and the test solution and inject them into the liquid chromatograph, record the chromatogram, and complete the detection of the enantiomers of the edosapan intermediate 2. Figure 1 shown.

[0032] The pre-verification investigation was carried out according to the above experimental conditions, and the specific implementation is as follows:

[0033] (1) Specificity investigation: Based on the solution preparation in the above steps, other known impurity solutions (M06, M07, M08, Py) were prepared separately. Other known impurities were dissolved in ethanol as a diluent, and quantitatively diluted to prepare a mixed localization solution with 2.0 mg of each known impurity per 1 ml of diluent. The results are shown in Figure 4 As shown: the edosapan intermediate 2 and its enantiomers are completely baseline separated from the adjacent peaks, and the blank and other known impurities have no interference on the two chromatographic peaks.

[0034] (2) Sensitivity test: Take the sensitivity solution in step 3 above, take 5 μl and inject it into the liquid chromatograph, record the chromatogram, and complete the sensitivity test. Result: The main component S / N = 12, which meets the acceptance standard (S / N> 10).

[0035] (3) Linearity test: Take edosapan intermediate 2 and dissolve it in ethanol as diluent, and quantitatively dilute it to make a series of linear solutions at the sample concentration level of 0.2% to 120%. Take 5μl and inject it into the liquid chromatograph, record the chromatogram, and complete the linearity test. Results: The linear equation is Y=54.2038X-0.6768, the correlation coefficient is 0.9999; the ratio of intercept b to Y at 100X is 0.5%, and the linear relationship is good.

[0036] (4) Repeatability: Dissolve the edosapan intermediate 2 in ethanol as a diluent and quantitatively dilute it to make a test solution containing 2.0 mg of edosapan intermediate per 1 ml of diluent; prepare 3 copies in parallel. Result: The RSD of the isomer %Area of ​​the 3 sample solutions was 0.0%, which showed good repeatability.

[0037] (5) Solution stability: Dissolve the edosapan intermediate 2 in ethanol as a diluent and quantitatively dilute it to make a test solution containing 2.0 mg of edosapan intermediate per 1 ml of diluent. After standing at room temperature for 12.8 hours, re-inject the sample. Result: The sample solution is stable within 12.8 hours at room temperature.

[0038] The above data show that the method of the present invention is accurate and reliable. Figure 1 , where peak 1 is the enantiomer peak and peak 2 is the chromatographic peak of edosapan intermediate 2.

[0039] Comparative Example 1 Chromatographic column screening, HPLC detection chromatographic conditions are as follows:

[0040] Chromatographic columns: CHIRALPAK AD-H, OD-H, OJ-H, and AS-H were screened, with dimensions of 250*4.6 mm, 5 μm, and a column temperature of 30 °C.

[0041] Mobile phase: n-hexane: isopropanol = 60:40 (V / V); flow rate is 0.5 ml / min.

[0042] Elution program: isocratic elution, 30 min.

[0043] Detection wavelength: 220nm, 254nm.

[0044] The following steps are involved:

[0045] (1) Take a sample of the racemate of edosapan intermediate 2 and dissolve it in methanol as a diluent, and quantitatively dilute it to prepare a development solution containing 1.2 mg of the racemate per 1 ml of diluent.

[0046] (2) Take 5 μl of the development solution and inject it into the liquid chromatograph, record the chromatogram, and complete the development of the enantiomer method of edosapan intermediate 2.

[0047] The results are as follows Figure 3 Shown: Poor separation.

[0048] Comparative Example 2 The mobile phase system was optimized, and the HPLC detection chromatographic conditions were as follows:

[0049] Chromatographic columns: CHIRALPAK OJ-H, AD-H, both with dimensions of 250*4.6 mm, 5 μm, and column temperature of 30 °C.

[0050] Mobile phase: methanol: ethanol: diethylamine = 40: 60: 0.3 (v / v / v); flow rate is 0.5 ml / min.

[0051] Elution program: isocratic elution, 60 min.

[0052] Detection wavelength: 220nm, 254nm.

[0053] The following steps are involved:

[0054] (1) Take a sample of the racemate of edosapan intermediate 2 and dissolve it in methanol as a diluent, and quantitatively dilute it to prepare a development solution containing 1.2 mg of the racemate per 1 ml of diluent.

[0055] (2) Take 20 μl of the development solution and inject it into the liquid chromatograph, record the chromatogram, and complete the development of the enantiomer method of edosapan intermediate 2.

[0056] The results are as follows Figure 2 Shown: Poor separation.

[0057] 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 method for detecting the enantiomers of an edoxaban intermediate 2, wherein the chemical name of the edoxaban 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: Normal phase high performance liquid chromatography with UV detector was used. The HPLC detection conditions were as follows: Chromatographic column: CHIRALPAK OX-H, 250*4.6mm, 5μm, polysaccharide derivative normal phase coated chiral chromatographic column, the silica surface is coated with cellulose-tris (4-chloro-3-methylphenylcarbamate), the column temperature is 35℃; Mobile phase: a mixed solution of n-hexane, ethanol and diethylamine with a volume ratio of 500:500:1; flow rate is 0.7 ml / min; Elution procedure: isocratic elution for 10 min; Detection wavelength: 220nm; The following steps are involved: (1) dissolving the edosapan intermediate 2 racemate in ethanol as a diluent, and quantitatively diluting the solution to obtain a racemate solution containing 1.2 mg of the edosapan intermediate 2 racemate per 1 ml of the diluent; (2) Dissolve edosapan intermediate 2 in ethanol 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) Take 5 μl of the racemic solution and the test solution and inject them into the liquid chromatograph, record the chromatogram, and complete the detection of the enantiomers of the edosapan intermediate 2.

Images