Method for detecting cis-isomer in vocyclosporine intermediate
Through the combination of high-performance liquid chromatography and porous graphitized carbon chromatography columns, the problem of detection of cis isomer impurities in volcanocyclosporine intermediate OHB is solved, and efficient quality control of volcanocyclosporine intermediates is achieved, ensuring the production of high-purity products.
Patent Information
- Application Number
- CN202311100344.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-29
- Publication Date
- 2025-02-28
AI Technical Summary
The prior art cannot effectively detect cis isomer impurities in volcanosporine intermediate OHB, and the nuclear magnetic method cannot separate the cis isomer from the intermediate OHB.
High performance liquid chromatography was used to use a chromatographic column with porous graphitized carbon as the filler, and the mobile phase was isopropanol-anhydrous ethanol. The separation and quantitative detection of cis isomer impurities in the intermediate OHB were achieved through isometric elution and two chromatographic columns were connected in series.
The efficient separation and quantitative detection of cis isomer impurities in the volcanocyclosporine intermediate OHB is achieved, which improves the quality control level of the product and ensures the high purity production of volcanocyclosporine.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of drug analysis, and relates to a high performance liquid chromatography method for detecting cis isomers in a cyclosporine intermediate. The analytical method of the invention is applied to the quality control of cis isomer impurities in a cyclosporine intermediate. Background Art
[0002] Voclosporin soft capsules were developed by Aurinia Pharmaceutical Inc (Canada). Lupkynis (Voclosporin) was approved by the FDA in January 2021. It is the first oral therapy drug approved by the US FDA for the treatment of lupus nephritis and is not yet available in other countries and regions.
[0003] Vorbis is an analog of cyclosporine A and a potential best-in-class calcineurin inhibitor that blocks IL-2 expression and T cell-mediated immune responses by inhibiting calcineurin, thereby stabilizing renal podocytes. Vorbis has a comparable binding affinity to cyclosporine A for human cyclophilin protein, but the olefinic side chain of Vorbis can induce structural changes in calcineurin upon binding, which may result in enhanced immunosuppressive activity compared to cyclosporine A. The structural formula of Vorbis is shown in the figure below:
[0004]
[0005] In the synthesis process of cyclosporine, because the starting material is a mixture of cis and trans isomers, the intermediate OHB, the intermediate VCS crude product and the product cyclosporine are all mixtures of cis and trans isomers, and the trans isomer is the active ingredient, so the content of the cis isomer produced in the process needs to be controlled. In the process, the cis isomer in the intermediate OHB will react in equal amounts to generate the cis isomer of cyclosporine. The cis isomer cannot be removed in the subsequent reaction and purification process of the cyclosporine product. Therefore, controlling the content of its cis isomer in the intermediate OHB is particularly critical for the production process and quality control of cyclosporine. The structural formula of the intermediate OHB and its cis isomer (impurity BYG) is shown in the figure below:
[0006]
[0007] At present, there is no literature reporting the detection method of the cis isomer in the intermediate OHB of cyclosporine. Referring to the EMA review of Aurinia (the original manufacturer), the cis isomer of cyclosporine can be controlled by the nuclear magnetic resonance method, but when the cis isomer of the intermediate OHB is detected by the nuclear magnetic resonance method, the cis isomer cannot be separated from the intermediate OHB, that is, the nuclear magnetic resonance method is not suitable for detecting the cis isomer impurities of the intermediate OHB. Summary of the invention
[0008] According to the structural difference between the cyclosporine intermediate OHB and its cis isomer (impurity BYG), the present invention develops a high performance liquid chromatography method capable of separating and quantitatively detecting the cis isomer (impurity BYG) in the intermediate OHB, thereby achieving quality control of the cyclosporine intermediate OHB product, and further promoting the use of the intermediate to obtain a high-purity cyclosporine product.
[0009] The present invention provides a method for detecting cis isomers in a cyclosporine intermediate, that is, using high performance liquid chromatography to detect cis isomer impurities in the cyclosporine intermediate OHB, and the chromatographic conditions include:
[0010] Chromatographic column: Use a chromatographic column with porous graphitized carbon as filler;
[0011] Mobile phase: Isopropanol-anhydrous ethanol was used as the mobile phase;
[0012] Elution method: isocratic elution;
[0013] The structural formulas of the cyclosporine intermediate and the cis isomer thereof are respectively:
[0014]
[0015] In some preferred embodiments of the detection method of the present invention, in the high performance liquid chromatography, the preparation process of the test solution is as follows: take the cyclosporine intermediate OHB, first dissolve it in acetonitrile, and then use the mobile phase as a solvent to prepare a test solution, and the concentration of the cyclosporine intermediate OHB in the test solution is 0.4 mg / ml.
[0016] In some preferred embodiments of the detection method of the present invention, in the preparation of the test solution, the volume ratio of acetonitrile to mobile phase is 1:9.
[0017] The detection method of the present invention injects the obtained test sample solution into a high performance liquid chromatograph for detection, thereby completing the detection of the cis-isomer impurities in the cyclosporine intermediate OHB.
[0018] In some preferred embodiments of the detection method of the present invention, the chromatographic columns are two chromatographic columns connected in series.
[0019] In some preferred embodiments of the detection method of the present invention, the porous graphitized carbon chromatographic column has a particle size of 5 μm, a length of 150 mm, and an inner diameter of 4.6 mm.
[0020] In some preferred embodiments of the detection method of the present invention, in the mobile phase, the volume ratio of isopropanol to anhydrous ethanol is 740-760:265-285.
[0021] In some preferred embodiments of the detection method of the present invention, the flow rate of the mobile phase is 1.3 ml / min to 1.6 ml / min, preferably 1.5 ml / min.
[0022] In some preferred embodiments of the detection method of the present invention, the column temperature of the chromatographic column is 75°C to 85°C, preferably 80°C.
[0023] In some preferred embodiments of the detection method of the present invention, the chromatographic conditions further include: a detection wavelength of 225 nm and an injection volume of 20 μl.
[0024] The beneficial effects of the present invention are as follows:
[0025] The method of the invention is simple to operate, rapid and efficient, and has high sensitivity and good reproducibility in detection results.
[0026] The present invention uses porous graphitized carbon as a filler and two chromatographic columns connected in series to control cis-isomer impurities. The detection method is fully verified, proving that the analysis method is effective and feasible.
[0027] The method adopts high performance liquid chromatography and uses a chromatographic column with porous graphitized carbon as a filler, which can better separate the intermediate OHB and its cis-isomer. Since the analysis time is short, the separation effect is good, and the method has good durability, a rapid and effective analysis method is provided for the quality control of the cis-isomer in the intermediate. The analysis method of the invention adopts two chromatographic columns in series, which can effectively avoid the situation that the system applicability does not meet the requirements due to the difference between batches of chromatographic columns, and improves the durability of the method.
[0028] Through the technical solution of the present invention, the separation degree between the intermediate OHB peak and the cis-isomer peak meets the requirements, the system applicability and specificity of the detection method meet the requirements, the linear range, precision and durability of the method also meet the requirements, and the detection method of the present invention can effectively detect the content of cis-isomer impurities in the intermediate OHB. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 This is the blank solvent chromatogram.
[0030] Figure 2 The figure is the chromatogram of the test solution.
[0031] Figure 3 The HPLC spectrum of the test solution in Comparative Example 1 (the fillers of the chromatographic column are different).
[0032] Figure 4 This is the HPLC spectrum of the test solution in Comparative Example 2 (the fillers of the chromatographic column are different).
[0033] Figure 5 This is the HPLC spectrum of the test solution in Comparative Example 3 (different types of mobile phase).
[0034] Figure 6 HPLC spectrum of the test solution under the chromatographic conditions of flow rate = 1.2 ml / min in Comparative Example 4
[0035] Figure 7 The HPLC spectrum of the test solution under the chromatographic conditions of selecting a chromatographic column in Comparative Example 5. DETAILED DESCRIPTION
[0036] The present invention is further described below by multiple groups of specific embodiments. It should be understood that the embodiments of the present invention are only used to illustrate the present invention, rather than to limit the present invention. Technical solutions obtained by simply modifying the present invention or by equivalently replacing the conventional means or active ingredients based on the technical solution of the present invention all belong to the protection scope of the present invention.
[0037] The experimental equipment includes: liquid chromatograph (Waters Company), XS205DU analytical balance (Mettler), chromatographic column (Thermo Fisher Scientific).
[0038] The experimental solvents included: isopropanol purchased from Tianjin Concord Company, anhydrous ethanol purchased from Tianjin Concord Company, and acetonitrile purchased from Zhonghui Chemical Company.
[0039] Example 1 System Applicability
[0040] Preparation of system suitability solution: Accurately weigh about 20 mg of the intermediate reference substance, place it in a 50 ml volumetric flask, add 5 ml of acetonitrile and shake to dissolve, dilute to the scale with mobile phase, and shake well.
[0041] The system suitability solution was injected, and the detection conditions were as follows: using a Waters 2695-2489 high performance liquid chromatograph, the wavelength was set to 225 nm, the chromatographic column was Hypercarb, 4.6 mm × 150 mm, 5 μm, two chromatographic columns were connected in series, the mobile phase was isopropanol-anhydrous ethanol (750:275), the flow rate was 1.5 ml per minute, the column temperature was 80 ° C, and the injection volume was 20 μl.
[0042] Test results: The retention time is 40.645min for the cis isomer impurity BYG. The separation degree between the intermediate OHB peak and the cis isomer impurity BYG peak is 2.4, which meets the requirements. Chromatographic analysis of blank solvent and system suitability solution is shown in Figure 1 , Figure 2 .
[0043] Example 2 Detection Limit and Quantification Limit
[0044] Preparation of cis-isomer impurity stock solution: Accurately weigh 40.02 mg of cis-isomer impurity BYG reference substance, place in a 50 ml volumetric flask, add 5 ml of acetonitrile and shake to dissolve, dilute to scale with mobile phase, shake well,
[0045] Preparation of quantitative limit solution: Accurately measure 1 ml of the stock solution, place it in a 100 ml volumetric flask, dilute to the scale with the mobile phase, and shake well.
[0046] Preparation of detection limit solution: Accurately measure 1 ml of the stock solution, place it in a 200 ml volumetric flask, dilute to the scale with the mobile phase, and shake well.
[0047] The quantitative limit solution and the detection limit solution were injected separately. The detection conditions were as follows: using a waters2695-2489 high performance liquid chromatograph, the wavelength was set to 225nm, the chromatographic column was Hypercarb, 4.6mm×150mm, 5μm, two chromatographic columns were connected in series, the mobile phase was isopropanol-anhydrous ethanol (750:275), the flow rate was 1.5ml per minute, the column temperature was 80℃, and the injection volume was 20μl. The results were recorded as shown in the following table.
[0048] name Limit of Quantitation S / N Limit of quantitation (%) Detection limit S / N Detection limit (%) Cis isomer 14.1 1.03 8.5 0.52
[0049] Test results: The quantitative limit and detection limit of the cis-isomer impurity are equivalent to 1.03% and 0.52% of the test sample concentration, and the impurity limit shall not exceed 10.0%. The results show that the quantitative limit and detection limit of the cis-isomer in the intermediate OHB are far below the limit value when this method is used to separate and detect the cis-isomer, which proves that this method has high sensitivity.
[0050] Example 3 Linear range and correction factor test
[0051] Preparation of cis-isomer impurity linear stock solution: Accurately weigh 40.02 mg of cis-isomer impurity reference substance, place it in a 50 ml volumetric flask, add 5 ml of acetonitrile and shake to dissolve, dilute to the scale with mobile phase, and shake well.
[0052] Preparation of 150% linear, 100% linear, 50% linear and 20% linear of cis-isomer impurity: accurately measure 3ml, 1ml, 1ml and 1ml of cis-isomer impurity linear stock solution and place them in 20ml, 10ml, 20ml and 50ml volumetric flasks respectively, add mobile phase to dilute to the scale and shake well.
[0053] The quantitative limit solution of the cis-isomer impurity is the same as the quantitative limit solution in Example 2.
[0054] 100% linear relationship solution of the intermediate: Accurately weigh 40.03 mg of the intermediate reference substance, place it in a 100 ml volumetric flask, add 10 ml of acetonitrile and shake to dissolve, dilute to the scale with the mobile phase, and shake well.
[0055] Preparation of 50% linear, 30% linear, 20% linear and 10% linear solutions of the intermediate: Accurately measure 5ml, 3ml, 2ml and 1ml of the 100% linear relationship solution of the intermediate, place them in 10ml volumetric flasks respectively, add mobile phase to dilute to the scale, and shake well.
[0056] 200% linear relationship solution of the intermediate: Accurately weigh 40.01 mg of the intermediate reference substance, place it in a 50 ml volumetric flask, add 5 ml of acetonitrile and shake to dissolve, dilute to the scale with the mobile phase, and shake well.
[0057] Take the above linear solution, and the detection conditions are: use Waters 2695-2489 high performance liquid chromatograph, set the wavelength to 225nm, select Hypercarb, 4.6mm×150mm, 5μm as the chromatographic column, connect two chromatographic columns in series, use isopropanol-anhydrous ethanol (750:275) as the mobile phase, the flow rate is 1.5ml per minute, the column temperature is 80℃, and the injection volume is 20μl. Record the results as shown in the following table:
[0058] name Linear range Linear equations Correlation coefficient r Correction Factor Cis isomer impurity 4.1221μg / ml~61.8309μg / m y=8249x-18774 0.9992 1.02 Intermediates 38.4288μg / ml~768.1920μg / ml y=8425x-14113 1.0000 /
[0059] The results showed that the cis isomer had a good linear relationship in the range of 1.03% to 15.46%, the correlation coefficient r was greater than 0.999, the correction factor was 1.02, and the peak area normalization method could be used for calculation.
[0060] Example 4 Precision and Repeatability Test
[0061] Repeatability (precision) Preparation of test solution: Weigh about 20 mg of intermediate OHB sample, place in a 50 ml volumetric flask, add 5 ml of acetonitrile and shake to dissolve, dilute to scale with mobile phase, shake well, and obtain. Prepare 6 portions in the same way.
[0062] The repeatability and precision of the method were investigated on different dates and different instruments. The test conditions were: using a Waters 2695-2489 high performance liquid chromatograph, the wavelength was set to 225nm, the chromatographic column was Hypercarb, 4.6mm×150mm, 5μm, two chromatographic columns were connected in series, the mobile phase was isopropanol-anhydrous ethanol (750:275), the flow rate was 1.5ml per minute, the column temperature was 80℃, and the injection volume was 20μl. The results were recorded as shown in the following table:
[0063]
[0064] The results showed that the repeatability RSD of cis-isomer impurity was 3.6%, and the intermediate precision RSD was 2.8%. The repeatability and precision of this method were very good.
[0065] Example 5 Durability Test
[0066] Preparation of test solution: Accurately weigh about 20 mg of intermediate OHB sample, place it in a 50 ml volumetric flask, add 5 ml of acetonitrile and shake to dissolve, dilute to the scale with mobile phase, and shake well.
[0067] The flow rate, column temperature, mobile phase ratio and chromatographic column were changed, and the system suitability solution was injected and tested. The results are as follows:
[0068] The separation standard requires that the separation should not be less than 1.5. The higher the separation, the better the analytical method.
[0069] The results show that slight changes in the chromatographic conditions within the scope of the present invention have almost no effect on the test results, proving that the method has good durability. In addition, the detection method of the present invention has a separation degree 0.7-1.5 higher than the standard, achieving a good separation effect.
[0070] Comparative Example 1 The chromatographic column filler is amylose-tris (3,5-diphenylcarbamate)
[0071] Preparation of test solution: Accurately weigh about 20 mg of intermediate OHB sample, place it in a 50 ml volumetric flask, add 5 ml of acetonitrile and shake to dissolve, dilute to the scale with mobile phase, and shake well.
[0072] The test solution was injected for detection and the results were recorded. The detection conditions were as follows: using a Waters 2695-2489 high performance liquid chromatograph, the wavelength was set to 225 nm, the chromatographic column was CHIRALPAK AD-H, 4.6 mm × 250 mm, 5 μm, the mobile phase was isopropanol-anhydrous ethanol (750:275), the flow rate was 1.5 ml per minute, the column temperature was 80 ° C, and the injection volume was 20 μl.
[0073] Chromatogram Figure 3 shown.
[0074] The results showed that under the chromatographic conditions, the intermediate OHB peak was not retained and eluted at 2.031 minutes. The intermediate OHB peak and the cis-isomer peak eluted simultaneously and were not separated.
[0075] Comparative Example 2: The chromatographic column filler is octadecylsilane bonded silica gel
[0076] Preparation of test solution: Accurately weigh about 20 mg of intermediate OHB sample, place it in a 50 ml volumetric flask, add 10 ml of acetonitrile and shake to dissolve, dilute to the scale with mobile phase, and shake well.
[0077] The test solution was injected for detection and the results were recorded. The detection conditions were as follows: using a Waters 2695-2489 high performance liquid chromatograph, the wavelength was set to 225 nm, the chromatographic column was Supelco Discovery C18, 4.6 mm × 150 mm, 5 μm, the mobile phase was isopropanol-anhydrous ethanol (750:275), the flow rate was 1.5 ml per minute, the column temperature was 75 ° C, and the injection volume was 20 μl.
[0078] Chromatogram Figure 4 shown.
[0079] The results showed that under the chromatographic conditions, the intermediate OHB peak was not retained and eluted at 1.224 minutes. The intermediate OHB peak and the cis-isomer peak eluted simultaneously and were not separated.
[0080] Comparative Example 3: Changing the type of mobile phase
[0081] Preparation of test solution: Accurately weigh about 20 mg of intermediate OHB sample, place it in a 50 ml volumetric flask, add 5 ml of acetonitrile and shake to dissolve, dilute to the scale with mobile phase, and shake well.
[0082] The test solution was injected for detection and the results were recorded. The detection conditions were as follows: using a Waters 2695-2489 high performance liquid chromatograph, the wavelength was set to 225 nm, the chromatographic column was Hypercarb, 4.6 mm × 150 mm, 5 μm, two chromatographic columns were connected in series, the mobile phase was isopropanol-acetonitrile (750:275), the flow rate was 1.5 ml per minute, the column temperature was 80 ° C, and the injection volume was 20 μl.
[0083] Chromatogram Figure 5 shown.
[0084] The results showed that under the chromatographic conditions, the separation degree between the intermediate OHB peak and the cis-isomer impurity BYG peak was 1.4, and the intermediate OHB peak was not separated from the cis-isomer impurity BYG peak.
[0085] Comparative Example 4: Changing the flow rate
[0086] Preparation of test solution: Accurately weigh about 20 mg of intermediate OHB sample, place it in a 50 ml volumetric flask, add 5 ml of acetonitrile and shake to dissolve, dilute to the scale with mobile phase, and shake well.
[0087] The test solution was injected for testing and the results were recorded. The testing conditions were as follows: using a Waters 2695-2489 high performance liquid chromatograph, the wavelength was set to 225 nm, the chromatographic column was Hypercarb, 4.6 mm × 150 mm, 5 μm, two chromatographic columns were connected in series, the mobile phase was isopropanol-anhydrous ethanol (750:275), the flow rate was 1.2 ml per minute, the column temperature was 80 ° C, and the injection volume was 20 μl.
[0088] Chromatogram Figure 6 shown.
[0089] The results showed that under the chromatographic conditions, the separation degree between the intermediate OHB peak and the cis-isomer impurity BYG peak was 1.3, which did not meet the requirements.
[0090] Comparative Example 5: Select a chromatographic column
[0091] Preparation of test solution: Accurately weigh about 20 mg of intermediate OHB sample, place it in a 50 ml volumetric flask, add 5 ml of acetonitrile and shake to dissolve, dilute to the scale with mobile phase, and shake well.
[0092] The test solution was injected and tested and the results were recorded. The test conditions were: using Waters 2695-2489 high performance liquid chromatograph, the wavelength was set to 225 nm, the chromatographic column was Hypercarb, 4.6 mm × 150 mm, 5 μm, the mobile phase was isopropanol-anhydrous ethanol (750:275), the flow rate was 1.5 ml per minute, the column temperature was 80 ° C, and the injection volume was 20 μl.
[0093] Chromatogram Figure 7 shown.
[0094] The results showed that under the chromatographic conditions, the intermediate OHB peak and the cis-isomer impurity BYG peak were not separated, which did not meet the requirements.
Claims
1. A method for detecting cis isomers in a cyclosporine intermediate, characterized in that: The cis isomer impurity in the cyclosporine intermediate was detected by high performance liquid chromatography, and the chromatographic conditions included: Chromatographic column: Use a chromatographic column with porous graphitized carbon as filler; Mobile phase: Isopropanol-anhydrous ethanol was used as the mobile phase; Elution method: isocratic elution; The structural formulas of the cyclosporine intermediate and the cis isomer thereof are respectively:
2. The method according to claim 1, characterized in that The chromatographic columns are two chromatographic columns connected in series.
3. The method according to claim 1, characterized in that The porous graphitized carbon chromatographic column has a particle size of 5 μm, a length of 150 mm, and an inner diameter of 4.6 mm.
4. The method according to claim 1, characterized in that: In the mobile phase, the volume ratio of isopropanol to anhydrous ethanol is 740-760:265-285, preferably 750:
275.
5. The method according to claim 1, characterized in that The flow rate of the mobile phase is 1.3 ml / min to 1.6 ml / min, preferably 1.5 ml / min.
6. The method according to claim 1, characterized in that The column temperature of the chromatographic column is 75°C to 85°C, preferably 80°C.
7. The method according to claim 1, characterized in that The chromatographic conditions also include: a detection wavelength of 225 nm and an injection volume of 20 μl.