Method for detecting related substances in a regadenoson starting material
The detection of impurities in the starting material of Reganoxen by high performance liquid chromatography overcomes the shortcomings of existing detection methods, achieves highly sensitive and specific impurity detection, and ensures drug quality.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-26
- Publication Date
- 2026-06-26
AI Technical Summary
There is a lack of effective methods in the current technology to detect related substances in the starting materials of Reganoxane, which affects the quality control of the starting materials and the purity of the final drug.
High-performance liquid chromatography (HPLC) was used with an Ultimate XB-C18 column, gradient elution, a detection wavelength of 240 nm, a flow rate of 1.0 mL/min, and a mobile phase of a mixture of acetic acid aqueous solution and methanol. Impurities A-12, A-13, A-14, A-15 and diastereomeric A-16 in the starting material of Reganoxen were detected. The resolution between the main peak and the impurity peak was greater than 1.5, and the sensitivity solution signal-to-noise ratio was greater than 10.
It achieves highly sensitive and specific detection of impurities in Reganoxane starting materials, effectively controls impurity content, ensures drug quality, and is suitable for quantitative analysis of Reganoxane starting materials.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical analysis, specifically relating to a method for detecting related substances in starting materials of Reganoxane. Background Technology
[0002] Regadenoson, chemically named 1-(6-amino-9-((2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofurancyclo-yl)-9H-purine-2-yl)-N-methyl-1H-pyrazole-4-carboxamide monohydrate, has the structural formula shown below.
[0003]
[0004] Regadenoson (trade name Lexiscan) is a selective adenosine receptor agonist developed by CV Therapeutics that causes coronary artery dilation. This drug is used for radionuclide myocardial perfusion imaging in patients who cannot undergo exercise stress testing.
[0005] Myocardial perfusion imaging drug stress testing is an examination conducted for patients who cannot tolerate exercise testing during clinical examinations. Studies have shown that the adenosine receptor agonist retardon can effectively avoid the adverse reactions of traditional myocardial ischemia stimulants, such as nausea, vomiting, bronchospasm, dyspnea, chest pain, and headache. It has advantages such as rapid onset of action, long duration of action, stable efficacy, and few side effects. Moreover, the procedure is simpler to perform and has been widely recognized in the international market, greatly assisting in myocardial perfusion imaging examinations.
[0006] Reganoxane starting materials are one of the most important starting materials for Reganoxane. Related substances are an important test item for the quality control of starting materials, which directly affects the impurity types and control limits of Reganoxane intermediates and finished products, as well as the quality of Reganoxane raw materials. Currently, no relevant reports have been found on the detection methods for related substances in Reganoxane starting materials. Therefore, developing a method for detecting related substances in Reganoxane starting materials is of great significance in the process of drug quality control. Summary of the Invention
[0007] This invention addresses the shortcomings of existing technologies by providing a highly specific and sensitive method for detecting related substances in Reganorogen starting materials, thereby solving the problem that existing methods cannot detect related substances in Reganorogen starting materials.
[0008] The present invention discloses a method for detecting related substances in Reganoxane starting materials, the method comprising the following steps: (1) Preparation of test solution: Take the starting material of Reganoxen, accurately weigh it, dissolve it with diluent and quantitatively dilute it to prepare a solution containing 0.1~5.0 mg of Reganoxen starting material per 1 mL; The starting material for the aforementioned Reganoxon is 2-chloroadenosine; (2) Preparation of control solution: Accurately measure 0.5 mL of the test solution, place it in a volumetric flask, dilute it to 100 mL with diluent, and shake well; (3) Preparation of sensitivity solution: Accurately measure 1 mL of the reference solution, place it in a volumetric flask, dilute it to 10 mL with diluent, and shake well; (4) Take the above-mentioned test solution, control solution and sensitivity solution respectively, and inject them into the high-performance liquid chromatograph for determination. The chromatographic conditions are as follows: Column: The packing material is octadecylsilane-bonded silica gel; Detector: UV detector Detection wavelength: 210nm~315nm Column temperature: 20℃~40℃ Flow rate: 0.5 mL / min ~ 1.2 mL / min Injection volume: 10-20µL Mobile phase: Under the chromatographic conditions described, mobile phase A is aqueous acetic acid solution; mobile phase B is methanol.
[0009] Elution method: gradient elution.
[0010] Furthermore, the chromatographic column is an Ultimate XB-C18 column.
[0011] Furthermore, the chromatographic column is 200mm to 350mm long, has an inner diameter of 3mm to 10mm, and the packing material has a particle size of 3μm to 10μm.
[0012] Furthermore, the detection wavelength is 240 nm.
[0013] Furthermore, the column temperature is 30°C.
[0014] Furthermore, the flow rate is 1.0 mL / min.
[0015] Furthermore, the diluent is a 10% methanol solution.
[0016] Furthermore, the suitability requirements for the high performance liquid chromatography system are: the signal-to-noise ratio of the main peak in the chromatogram of the sensitive solution is greater than 10; and the resolution between the main peak and the impurity peak in the chromatogram of the test sample solution is greater than or equal to 1.5.
[0017] Furthermore, based on the starting material, the concentration of the test solution is 0.5 mg / mL; the concentration of the control solution is 2.5 μg / mL; and the concentration of the sensitivity solution is 0.25 μg / mL.
[0018] Furthermore, the impurities with a relatively large content are impurity A-12, impurity A-13, impurity A-14, and impurity A-15, and the diastereomer is impurity A-16.
[0019] The impurity A-12 is: (2R,3R,4S,5R)-2-(6-amino-2-(((2R,3R,4R,5R)-2-(6-amino-2-chloro-9H-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol Impurity A-13: (2R,3R,4S,5R)-2-(6-amino-2-(((2R,3S,4R,5R)-5-(6-amino-2-chloro-9H-purin-9-yl)-4-hydroxy-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol Impurity A-14: (2S,3R,4S,5R)-2-(2-chloro-6-((9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-methoxy-9H-purin-2-yl)amino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol Impurity A-15: (2R,3R,4S,5R)-2-(6-amino-2-((2-chloro-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)amino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol The diastereomer impurities are: The impurity A-16 is 2-chloro-9-α-D-rifuranosyl-9H-adenine.
[0020] Beneficial effects of this invention: This invention considers not only the required separation degree between each impurity and the main peak, but also the separation between the impurities themselves. Through research, this invention has developed a method for detecting related substances in 2-chloroadenosine, a starting material for Reganoxon, which boasts numerous advantages: it can quantitatively control the content of related substances, exhibits high specificity, high sensitivity, and precision, and can effectively control the limits of impurities in the starting material 2-chloroadenosine. It is applicable to the determination of related substances in 2-chloroadenosine, a starting material for Reganoxon. Using the method of this invention, complete separation of specific impurities in the starting material 2-chloroadenosine can be achieved, and quantitative analysis can be performed. Attached Figure Description
[0021] Figure 1 : Blank solution Figure 2-3 Impurity Mixture Solution Detailed Implementation
[0022] The following examples are only for further illustration of the present invention and do not limit the scope of the present invention in any way.
[0023] Example This embodiment is a verification of the method for determining related substances in the starting material 2-chloroadenosine of Reganoxon.
[0024] The starting material 2-chloroadenosine, impurity A-12, impurity A-13, impurity A-14, impurity A-15, and impurity A-16 used in this embodiment were all purchased externally.
[0025] The instruments used in this embodiment are: Thermo Vanquish high performance liquid chromatograph and XS205 analytical electronic balance (METTLER TOLEDO, Switzerland).
[0026] Column: Ultimate XB-C18 column; Mobile phases: Mobile phase A is an aqueous solution of acetic acid; Mobile phase B is methanol; Detection wavelength: 240nm; Flow rate: 1.0 mL / min; Injection volume: 20 μL; Column temperature: 30℃; Elution method: gradient elution.
[0027] The elution gradient is: .
[0028] 1.1 Specificity Test Blank solvent: 10% methanol solution.
[0029] Test solution: Accurately weigh 2-chloroadenosine, the starting material of Reganoxen, dissolve it with diluent and dilute quantitatively to prepare a solution containing 0.5 mg of 2-chloroadenosine, the starting material of Reganoxen, per 1 mL.
[0030] Mixed solution of main component and impurities: Take appropriate amounts of 2-chloroadenosine reference standard, impurity A-12 reference standard, impurity A-13 reference standard, impurity A-14 reference standard, impurity A-15 reference standard, and impurity A-16 reference standard, dissolve them in an appropriate amount of methanol, and dilute with 10% methanol-water to prepare a mixed solution containing approximately 1.0 mg of 2-chloroadenosine and approximately 50 µg each of impurities A-12, A-13, A-14, A-15, and A-16 per ml. Inject 20 μl each of the blank solution, the test solution, and the mixed solution of main component and impurities into the liquid chromatograph and record the chromatograms.
[0031] The results showed that the blank solvent did not interfere with the determination of related substances in this product. The resolution between the starting material 2-chloroadenosine and its adjacent impurities was >1.5, and the resolution between each impurity was >1.5. (See attached table). Figure 1-3 .
[0032] 1.2 Linearity and Range Test Accurately weigh appropriate amounts of 2-chloroadenosine and each impurity reference standard, place them in volumetric flasks, and dissolve and dilute them with blank solvent to prepare a series of mixed standard solutions of varying concentrations. Accurately measure 20 μL of each solution and inject them for analysis under the chromatographic conditions described above. The lowest concentration was defined as 0.05%–0.1% of the relative concentration of each component compared to the test solution, and the highest concentration was defined as 2% of the relative concentration of the test solution. Linear regression was performed with concentration on the x-axis and peak area on the y-axis. The results are shown in Table 1. The results show that the correlation coefficients (R) of the regression lines for the standard curves of 2-chloroadenosine and each impurity are all > 0.990, which meets the requirements.
[0033] Table 1 Results of Linear Range Examination .
[0035] 1.3 Limit of Detection and Limit of Quantification Tests Take appropriate amounts of 2-chloroadenosine and each impurity reference standard, quantitatively dilute them stepwise with blank solvent, and then analyze them under the above chromatographic conditions. The corresponding concentration when S / N≥10:1 is taken as the limit of quantitation, and the corresponding concentration when S / N≥3:1 is taken as the limit of detection. The results of the limit of quantitation are shown in Table 2, and the results of the limit of detection are shown in Table 3.
[0036] Table 2 Results of the Limit of Quantitation Study
[0037] See Table 2
[0038] Table 3 Results of the detection limit study
[0039] 1.4 Solution stability test Accurately measured amounts of the test solution, control solution, and sensitivity solution were placed at room temperature for 0 h, 18 h, 24 h, 36 h, and 70 h, respectively. 20 μL of each solution was then precisely injected into the liquid chromatograph, and the chromatograms were recorded. Changes in impurity content in the test solution, changes in the peak area of the control and sensitivity solutions, and the signal-to-noise ratio of the peak in the sensitivity solution were investigated. The results showed that the sensitivity solution, control solution, and test solution were stable within 70 h at room temperature.
[0040] 1.5 Accuracy Test Take appropriate amounts of each impurity reference standard, dissolve them in methanol using ultrasound, and dilute with 10% methanol solution to prepare a mixed solution containing approximately 50 µg each of impurities A-12, A-13, A-14, A-15, and A-16 per 1 ml, as the reference standard stock solution. Accurately measure 1 ml of the reference standard stock solution into a 10 ml volumetric flask, dilute to the mark with 10% methanol solution, and shake well to prepare the reference standard solution. Accurately weigh approximately 10 mg of the test sample into 10 portions, place them in 20 ml volumetric flasks, and accurately add 0 mL, 1.0 mL, 1.0 mL, 1.0 mL, 2.0 mL, 2.0 mL, 2.0 mL, 3.0 mL, 3.0 mL, and 3.0 mL of the reference standard stock solution, respectively. Dilute to the mark with blank solvent, shake well, and prepare the test sample solution. Accurately measure 20 μL each of the reference solution and the test solution, and inject them for analysis under the chromatographic conditions described above. Calculate the recovery rate of each impurity in the test solution at low, medium, and high impurity concentration levels. The results show that the average recovery rates of each impurity at the low, medium, and high concentration levels are all between 80.0% and 120.0%, and the RSD (n=9) is less than 10.0%. The results indicate that this method has good accuracy.
[0041] 1.6 Precision Test Two researchers, on different dates, accurately weighed approximately 10 mg of the test sample, placed it in a 20 mL volumetric flask, dissolved it in 10% methanol, diluted it to the mark, and mixed it thoroughly. Six parallel aliquots of the test sample solution were prepared for each researcher. Using different instruments, 20 μL of each solution was accurately measured and injected for analysis under the chromatographic conditions described above. The results showed that the range (n=12) of the results obtained by the two researchers for each impurity did not exceed 0.05%. The results indicate that this method has good precision.
[0042] 1.7 Durability Test The analysis was conducted under varying detection wavelengths (±2 nm), column temperatures (±2 °C), flow rates (±0.1 ml / min), and with different batches of the same type of column. Blank solutions, mixed solutions of main components and impurities, test solutions, self-control solutions, and sensitivity solutions were injected for analysis.
[0043] The results showed that under all conditions, the blank solvent did not interfere with the determination of related substances in this product; in the mixed solution of the main component and impurities, the resolution between the main component peak and adjacent known impurities was greater than 1.5, and the resolution between adjacent known impurities was greater than 1.5; the signal-to-noise ratio of the sensitivity solution was greater than 10; compared with the original conditions, the absolute values of the differences in the measured impurities in the test solution were as follows: for impurity measured values ≤0.1%, the absolute values of the differences were all <0.05%; for impurity measured values ≤0.5%, the absolute values of the differences were all ≤0.1%; for impurity measured values >0.5%, the absolute values of the differences were all ≤0.2%. The results indicate that this method has good robustness.
Claims
1. A method for detecting related substances in Reganoxant starting materials, wherein the method is high-performance liquid chromatography, characterized in that, The starting material for Reganoxon is 2-chloroadenosine, and its chromatographic conditions are as follows: Column: the packing material is octadecylsilane-bonded silica gel; Detector: UV detector; detection wavelength: 210nm~315nm; column temperature: 20℃~40℃; flow rate: 0.5mL / min~1.2mL / min; injection volume: 10-20µL; mobile phase: as described in the chromatographic conditions; mobile phase A: acetic acid aqueous solution; mobile phase B: methanol; elution method: gradient elution.
2. The method for detecting related substances in Reganoxant starting materials according to claim 1, characterized in that, The detection method includes the following steps: (1) Preparation of test solution: Take the starting material of Reganoxen, accurately weigh it, dissolve it with diluent and quantitatively dilute it to prepare a solution containing 0.1~5.0 mg of Reganoxen starting material per 1 mL; (2) Preparation of control solution: Accurately measure 0.5 mL of the test solution, place it in a volumetric flask, dilute it to 100 mL with diluent, and shake well; (3) Preparation of sensitivity solution: Accurately measure 1 mL of the reference solution, place it in a volumetric flask, dilute it to 10 mL with diluent, and shake well; (4) Take the above test solution, control solution and sensitivity solution respectively and inject them into the high performance liquid chromatograph for determination.
3. The method for detecting related substances in Reganoxant starting materials according to claim 1, characterized in that, The detection method uses liquid chromatography to detect the content of 2-chloroadenosine and impurities.
4. The method for detecting related substances in Reganoxant starting materials according to claim 1, characterized in that, The gradient elution is as follows: 。 5. The method for detecting related substances in Reganoxant starting materials according to claim 1, characterized in that, The diluent for the liquid chromatography is a 10% methanol solution. The chromatographic column is an Ultimate XB-C18 column with a length of 200 mm to 350 mm, an inner diameter of 3 mm to 10 mm, a packing particle size of 3 µm to 10 µm, a detection wavelength of 240 nm, a column temperature of 30 °C, a flow rate of 1.0 mL / min, and an injection volume of 20 µL.
6. A method for detecting related substances in Reganoxant starting materials according to any one of claims 1 or 2, characterized in that... The suitability requirements for high performance liquid chromatography (HPLC) systems are: the signal-to-noise ratio of the main peak in the chromatogram of the sensitive solution is greater than 10; and the resolution between the main peak and the impurity peak in the chromatogram of the test sample solution is greater than or equal to 1.
5.
7. The method for detecting related substances in Reganoxant starting materials according to claim 2, characterized in that, Based on the starting material 2-chloroadenosine, the concentration of the test solution is 0.5 mg / mL; the concentration of the control solution is 2.5 μg / mL; and the concentration of the sensitivity solution is 0.25 μg / mL.
8. A method for detecting related substances in Reganoxant starting materials according to any one of claims 1-7, characterized in that, It can detect residual impurities in the starting material 2-chloroadenosine, impurities in process byproducts, and diastereomers.
9. The method for detecting related substances in Reganoxant starting materials according to claim 8, characterized in that, The process impurities with a relatively high content are: Impurity A-12: (2 R ,3 R 4 S 5 R )-2-(6-amino-2-(((2) R ,3 R 4 R 5 R )-2-(6-amino-2-chloro-9 H -purine-9-yl)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)-9H-purine-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol; Impurity A-13: (2 R ,3 R 4 S 5 R )-2-(6-amino-2-(((2) R ,3 S 4 R 5 R )-5-(6-amino-2-chloro-9 H -purine-9-yl)-4-hydroxy-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)-9 H -Purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol; Impurity A-14: (2S,3R,4S,5R)-2-(2-chloro-6-((9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-methoxy-9H-purin-2-yl)amino)-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol; Impurity A-15: (2 R ,3 R 4 S 5 R )-2-(6-amino-2-((2-chloro-9-((2) R ,3 R 4 S 5 R )-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9 H -purine-6-yl)amino)-9 H -Purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol; The diastereomer impurity is: Impurity A-16: 2-chloro-9- α -D-ribofuranosyl-9 H -Adenine.