Quality control method of sofosbuvir intermediate
Through the isometric elution technology of liquid chromatography, the quality control problem of impurities in sofosbuvir intermediates is solved, efficient separation and quantification of impurities A and impurities B are achieved, and the quality and safety of the product are improved.
Patent Information
- Application Number
- CN202411796905.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-09
- Publication Date
- 2025-03-28
AI Technical Summary
The prior art lacks effective quality control methods for handling specific impurities in sofosbuvir intermediates, resulting in the impact of the quality, safety and effectiveness of the end product.
Using liquid chromatography, an amylose-tris(3-chlorophenylcarbamate) bonded silica gel was used as a filler chromatography column, and was eluted isotropically by a mixed solution of n-hexane and isopropanol to achieve effective elution, separation and quantification of impurities A and impurities B in the sofosbuvir intermediate.
This method avoids the complex process of gradient elution through chromatographic conditions, realizes efficient separation and quantification of specific impurities in sofosbuvir intermediates, and improves the quality, safety and effectiveness of the product.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to a quality control method for a sofosbuvir intermediate, and in particular to a quality control method for impurities in a sofosbuvir intermediate. Background Art
[0002] Sofosbuvir ((S)-isopropyl-2-((S)-(((2R,3R,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl)methoxy)-(phenoxy)phosphoamino)propionate) is a nucleoside polymerase inhibitor against hepatitis C virus. It has a low incidence of adverse reactions and good safety. It is suitable for all hepatitis C virus-infected patients, including those co-infected with AIDS and hepatitis C virus, patients with cirrhosis, hepatocellular carcinoma and patients preparing for liver transplantation.
[0003]
[0004] N-[(S)-(2,3,4,5,6-pentafluorophenoxy)phenoxyphosphoryl]-L-alanine isopropyl ester is an important intermediate for the preparation of sofosbuvir. In the process of synthesizing sofosbuvir, the impurities contained in the intermediate itself will inevitably be introduced into the final product sofosbuvir, thus directly affecting the quality, safety and effectiveness of the API and related preparations.
[0005] The synthetic route of N-[(S)-(2,3,4,5,6-pentafluorophenoxy)phenoxyphosphoryl]-L-alanine isopropyl ester is as follows:
[0006]
[0007] Through impurity analysis, the above synthesis process will produce impurities A and B, where impurity A is a reaction by-product and impurity B is a process impurity. Currently, there is no effective quality control method for specific impurities in the above intermediates. Summary of the invention
[0008] Purpose of the invention: The present invention aims to provide a quality control method that can effectively elute, separate and quantify specific impurities in sofosbuvir intermediates.
[0009] Technical solution: The quality control method of the sofosbuvir intermediate of the present invention,
[0010]
[0011] The following steps are involved:
[0012] (1) Prepare samples;
[0013] (2) Detection: Liquid chromatography was performed using a chromatographic column filled with amylose-tris(3-chlorophenylcarbamate) bonded silica gel and a mixed solution of n-hexane and isopropanol as the mobile phase for isocratic elution;
[0014] (3)Analysis results.
[0015] Preferably, the quality control impurities in the sofosbuvir intermediate are as follows:
[0016]
[0017] Preferably, the mobile phase in step (2) is a mixed solution of n-hexane and isopropanol in a volume ratio of 85:15.
[0018] Preferably, the running time of the isocratic elution in step (2) is 2 to 3 times the retention time of the main peak.
[0019] More preferably, the running time is 40 minutes.
[0020] Preferably, the detection wavelength of the detection in step (2) is 200-220 nm, the column temperature of the chromatographic column is 20-30° C., and the flow rate of the mobile phase is 0.9-1.1 ml / min.
[0021] More preferably, the detection wavelength is 207 nm, the column temperature of the chromatographic column is 25° C., and the flow rate of the mobile phase is 1.0 ml / min.
[0022] More preferably, the chromatographic column has a length of 100 to 250 mm, a diameter of 4.6 mm, and a filler particle size of 3 to 5 μm.
[0023] More preferably, the chromatographic column has a length of 250 mm, a diameter of 4.6 mm, and a filler particle size of 5 μm.
[0024] Still more preferably, the chromatographic column is a CHIRALPAK ID chromatographic column with a column length of 250 mm, a diameter of 4.6 mm, and a filler particle size of 5 μm.
[0025] Preferably, the injection volume for detection in step (2) is 10 μl.
[0026] Preferably, the linear range of detection of the sofosbuvir intermediate is 0.4984 to 5.9816 mg / ml.
[0027] More preferably, the detection linear equation of the sofosbuvir intermediate is y=0.0582x+6.4856.
[0028] Preferably, the detection limit of impurity A is 0.4516 μg / ml, and the quantification limit is 1.5052 μg / ml.
[0029] Preferably, the detection limit of impurity B is 0.4505 μg / ml, and the quantification limit is 1.5018 μg / ml.
[0030] Preferably, the diluent for preparing the sample in step (1) is isopropanol.
[0031] More preferably, the sample prepared in step (1) is a test solution.
[0032] More preferably, the concentration of the sofosbuvir intermediate in the test solution is 5 mg / ml.
[0033] Preferably, in step (3), an area normalization method is used to analyze the results.
[0034] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:
[0035] The quality control method designed by the present invention optimizes the chromatographic conditions and adopts isocratic elution to effectively elute, separate and quantify specific impurities in the sofosbuvir intermediate, thereby avoiding the complex process of gradient elution. The method has strong specificity, wide linear range, high sensitivity, good accuracy, and good solution stability and durability. By effectively controlling the intermediates, it is beneficial to control the quality, safety and effectiveness of the raw materials and related preparations. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Figure 1 It is the HPLC chromatogram of Example 1 of the present invention;
[0037] Figure 2 It is the HPLC chromatogram of Comparative Example 1 of the present invention;
[0038] Figure 3 It is the HPLC chromatogram of Comparative Example 2 of the present invention;
[0039] Figure 4 It is the HPLC chromatogram of Comparative Example 3 of the present invention. DETAILED DESCRIPTION
[0040] The technical solution of the present invention is further described below in conjunction with embodiments.
[0041] Example 1: Isocratic elution
[0042] 1. Experimental methods
[0043] Instrument: Thermo Fisher U-3000;
[0044] Column: CHIRALPAK ID, 4.6 mm × 250 mm, 5 μm;
[0045] Mobile phase: n-hexane-isopropanol (85:15);
[0046] Detection wavelength: 207nm;
[0047] Flow rate: 1.0ml / min;
[0048] Column temperature: 25°C;
[0049] Injection volume: 10 μl;
[0050] Elution procedure: isocratic elution;
[0051] Running time: 40 minutes;
[0052] Dilution solution (blank solution): isopropanol.
[0053] Preparation of control solution: Take appropriate amount of impurity A reference substance and impurity B respectively, weigh accurately, add acetonitrile to dissolve and dilute to prepare a solution with impurity A content of about 5 μg and impurity B content of about 5 μg per 1 ml, as the control solution.
[0054] Preparation of system suitability solution: Take appropriate amounts of sofosbuvir intermediate, impurity A and impurity B, add diluent to dissolve and quantitatively dilute to make a solution containing about 5 mg sofosbuvir intermediate, about 5 μg impurity A and about 1.5 μg impurity B per 1 ml, shake well and set aside.
[0055] The isopropyl alcohol used in the above preparation process is a diluent, and the isopropyl alcohol can be replaced by any other suitable solvent as appropriate.
[0056] Sample determination: Take 10 μl of blank solution, control solution and system suitability solution respectively and inject them into the liquid chromatograph.
[0057] 2. Experimental results
[0058] Depend on Figure 1 It can be seen that RT = 17.933min is the peak of the sofosbuvir intermediate, RT = 15.767min is the peak of impurity A, and RT = 16.960min is the peak of impurity B. The method has good separation between the peak of the sofosbuvir intermediate and the peaks of each known impurity, with a minimum separation of 1.78.
[0059] 3. Methodological validation
[0060] With reference to the "Guidelines for Validation of Analytical Methods", the detection method of Example 1 was methodologically validated.
[0061] Table 1 Methodology validation results
[0062]
[0063]
[0064] It can be seen from the above methodological verification results that the detection method designed by the present invention has strong specificity, wide linear range, high sensitivity, high accuracy, good solution stability and durability, and can achieve effective detection of specific impurities in sofosbuvir intermediates.
[0065] Comparative Example 1: Gradient Elution
[0066] 1. Experimental methods
[0067] Instrument: Thermo Fisher U-3000;
[0068] Column: Waters XBridge Phenyl 4.6×150 mm, 3.5 μm;
[0069] Mobile phase: Phase A: 0.1% phosphoric acid solution, Phase B: acetonitrile;
[0070] Detection wavelength: 207nm;
[0071] Flow rate: 1.0ml / min;
[0072] Column temperature: 25°C;
[0073] Injection volume: 5μl.
[0074] Table 2 Gradient elution program of Comparative Example 1
[0075]
[0076] Dilution solution (blank solution): acetonitrile.
[0077] Preparation of control solution: Take an appropriate amount of impurity A reference substance, weigh it accurately, add acetonitrile to dissolve and dilute it to make a solution with an impurity A content of about 2 μg per 1 ml, which serves as the control solution.
[0078] Preparation of system suitability solution: Take appropriate amount of sofosbuvir intermediate and impurity A, add diluent to dissolve and quantitatively dilute to make a solution containing about 2 mg sofosbuvir intermediate and about 2 μg impurity A per 1 ml, shake well and set aside.
[0079] The acetonitrile used in the above preparation process is a diluent, and acetonitrile can be replaced by any other suitable solvent as appropriate.
[0080] Sample determination: Take 5 μl of blank solution, control solution and system suitability solution respectively and inject them into the liquid chromatograph.
[0081] 2. Experimental results
[0082] Depend on Figure 2It can be seen that RT=18.097min is the peak of the sofosbuvir intermediate, and RT=18.343min is the peak of impurity A. The separation degree of the peak of the sofosbuvir intermediate and impurity A in this method is 0.97 (<1.5), and the separation effect cannot meet the quality control requirements.
[0083] Comparative Example 2: Gradient Elution
[0084] 1. Experimental methods
[0085] Instrument: Thermo Fisher U-3000;
[0086] Column: Phenomenex kinetex PFP 4.6×150 mm, 5.0 μm;
[0087] Mobile phase: Phase A: 0.1% phosphoric acid solution, Phase B: acetonitrile;
[0088] Detection wavelength: 207nm;
[0089] Flow rate: 1.0ml / min;
[0090] Column temperature: 25°C;
[0091] Injection volume: 5μl.
[0092] Table 3 Gradient elution program of Comparative Example 2
[0093]
[0094] Preparation of control solution: Take an appropriate amount of impurity A reference substance, weigh it accurately, add acetonitrile to dissolve and dilute it to make a solution with an impurity A content of about 2 μg per 1 ml, which serves as the control solution.
[0095] Preparation of system suitability solution: Take appropriate amount of sofosbuvir intermediate and impurity A, add diluent to dissolve and quantitatively dilute to make a solution containing about 2 mg sofosbuvir intermediate and about 2 μg impurity A per 1 ml, shake well and set aside.
[0096] The acetonitrile used in the above preparation process is a diluent, and acetonitrile can be replaced by any other suitable solvent as appropriate.
[0097] Sample determination: Take 5 μl of blank solution, control solution and system suitability solution respectively and inject them into the liquid chromatograph.
[0098] 2. Experimental results
[0099] Depend on Figure 3It can be seen that RT=16.657min is the peak of the sofosbuvir intermediate, and RT=16.657min is the peak of impurity A. In this method, the peak of the sofosbuvir intermediate and the peak of impurity A overlap (<1.5), and the separation effect cannot meet the quality control requirements.
[0100] Comparative Example 3: Gradient Elution
[0101] 1. Experimental methods
[0102] Instrument: Thermo Fisher U-3000;
[0103] Column: ACE UltraCore5 Super PhenylHexyl 4.6×250mm, 5.0μm;
[0104] Mobile phase: Phase A: 0.1% phosphoric acid solution, Phase B: acetonitrile;
[0105] Detection wavelength: 207nm;
[0106] Flow rate: 1.0ml / min;
[0107] Column temperature: 25°C;
[0108] Injection volume: 5μl.
[0109] Table 4 Gradient elution program of Comparative Example 3
[0110]
[0111] Dilution solution (blank solution): acetonitrile.
[0112] Preparation of control solution: Take an appropriate amount of impurity A reference substance, weigh it accurately, add acetonitrile to dissolve and dilute it to make a solution with an impurity A content of about 2 μg per 1 ml, which serves as the control solution.
[0113] Preparation of system suitability solution: Take appropriate amount of sofosbuvir intermediate and impurity A, add diluent to dissolve and quantitatively dilute to make a solution containing about 2 mg sofosbuvir intermediate and about 2 μg impurity A per 1 ml, shake well and set aside.
[0114] The acetonitrile used in the above preparation process is a diluent, and acetonitrile can be replaced by any other suitable solvent as appropriate.
[0115] Sample determination: Take 5 μl of blank solution, control solution and system suitability solution respectively and inject them into the liquid chromatograph.
[0116] 2. Experimental results
[0117] Depend on Figure 4It can be seen that RT=20.720min is the peak of the sofosbuvir intermediate, and RT=20.990min is the peak of impurity A. The separation degree of the peak of the sofosbuvir intermediate and impurity A in this method is 1.28 (<1.5), and the separation effect cannot meet the quality control requirements.
Claims
1. A quality control method for a sofosbuvir intermediate, It is characterized in that The following steps are involved: (1) Prepare samples; (2) Detection: Liquid chromatography was performed using a chromatographic column filled with amylose-tris(3-chlorophenylcarbamate) bonded silica gel and a mixed solution of n-hexane and isopropanol as the mobile phase for isocratic elution; (3)Analysis results.
2. The quality control method according to claim 1, characterized in that: The quality control impurities in the sofosbuvir intermediate are as follows:
3. The quality control method according to claim 1, characterized in that: The mobile phase described in step (2) is a mixed solution of n-hexane and isopropanol in a volume ratio of 85:
15.
4. The quality control method according to claim 1, characterized in that: The running time of the isocratic elution in step (2) is 40 min.
5. The quality control method according to claim 1, characterized in that: The detection wavelength of the detection in step (2) is 200-220 nm, the column temperature of the chromatographic column is 20-30° C., and the flow rate of the mobile phase is 0.9-1.1 ml / min.
6. The quality control method according to claim 5, characterized in that: The column length of the chromatographic column is 100-250 mm, the diameter is 4.6 mm, and the filler particle size is 3-5 μm.
7. The quality control method according to claim 6, characterized in that: The chromatographic column has a length of 250 mm, a diameter of 4.6 mm, and a filler particle size of 5 μm.
8. The quality control method according to claim 7, characterized in that: The chromatographic column is CHIRALPAK ID, 4.6 mm×250 mm, 5 μm.
9. The quality control method according to claim 1, characterized in that: The diluent for preparing the sample in step (1) is isopropanol.
10. The quality control method according to claim 1, characterized in that: In step (3), the area normalization method is used to analyze the results.