A method for detecting genotoxic impurities in febuxostat

Abstract

The present invention belongs to the field of medical technology, and specifically provides a method for detecting genotoxic impurities in Febuxostat, wherein the method adopts liquid chromatography. The method for detecting genotoxic impurities in Febuxostat provided by the present invention adopts high performance liquid chromatography to realize rapid and accurate determination of three genotoxic impurities, has high sensitivity and specificity, and is simple to operate. The present invention can be used for the preparation process of Febuxostat and the quality control of the final product.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a liquid phase detection method for genotoxic impurities in febuxostat. Background Art

[0002] Febuxostat (English name: Febuxostat, alias: Febuxostat, Febuxostat), chemical name is 2-[(3-cyano-4-isobutyloxy)phenyl]-4-methyl-5-thiazolecarboxylic acid, molecular formula is C 16 H 16 N2O3S, molecular weight is 316.37, CAS registration number: 144060-53-7. Its structure is shown in formula I.

[0003]

[0004] Febuxostat is a new generation of xanthine oxidase inhibitor developed by Teijin Co., Ltd. of Japan, which is clinically used to treat hyperuricemia (gout). In February 2009, the U.S. Food and Drug Administration (FDA) approved febuxostat for long-term treatment of patients with gout and hyperuricemia.

[0005] Completely different from the xanthine oxidase inhibitor drugs developed 40 years ago, it is a new, highly effective, non-purine xanthine oxidase selective inhibitor. Xanthine oxidase is a key enzyme that promotes uric acid production. Febuxostat can reduce the level of uric acid in the blood of patients with hyperuricemia and gout. Clinical studies have shown that this product is safe and effective. This product is metabolized by the liver and does not rely on kidney excretion. Therefore, there is no need to reduce the dose for patients with moderate to severe liver and kidney dysfunction.

[0006] Febuxostat is usually synthesized from ethyl 2-(3-formyl-4-hydroxyphenyl)-4-methyl-thiazole-5-carboxylate as the starting material through substitution, addition, dehydration, hydrolysis and other reactions. In this synthesis process, there are three genotoxic impurities, including 2-[3-formyl-4-(2-methylpropoxy)phenyl]-4-methyl-5-thiazolecarboxylic acid, ethyl 2-(3-formyl-4-hydroxyphenyl)-4-methyl-thiazole-5-carboxylate, and ethyl 2-(3-formyl-4-isobutoxy-phenyl)-4-methyl-thiazole-5-carboxylate.

[0007] According to the Threshold of Toxicological Concern (TTC) of 1.5 μg / day, the concentration limit of genotoxic impurities in Febuxostat API is calculated to be 18 ppm. If the limit is to be 18 ppm using HPLC, the sample concentration needs to be increased. However, too high a sample concentration will cause the main peak in the chromatographic system to overload and cover the relevant genotoxic impurities, making qualitative and quantitative analysis impossible, and reducing the service life of the chromatographic column.

[0008] According to current research practices, compounds with (in vivo) genotoxicity are likely to cause damage to DNA at any exposure level, and this damage may cause tumors. Therefore, it is particularly urgent for pharmaceutical manufacturers to develop a simple, convenient, and effective detection method for the detection of genotoxic impurities in febuxostat. Summary of the invention

[0009] The object of the present invention is to provide a method for determining three genotoxic impurities in febuxostat by high performance liquid chromatography.

[0010] The genotoxic impurities include:

[0011] 1. Impurity H: 2-[3-Formyl-4-(2-methylpropoxy)phenyl]-4-methyl-5-thiazolecarboxylic acid

[0012] 2. Starting material 1: 2-(3-Formyl-4-hydroxyphenyl)-4-methyl-thiazole-5-carboxylic acid ethyl ester

[0013] 3. Intermediate 1: 2-(3-Formyl-4-isobutoxy-phenyl)-4-methyl-thiazole-5-carboxylic acid ethyl ester

[0014] To solve the above problems, the selection of diluents in the sample processing process of the present invention aims to reduce the dissolution of febuxostat under the condition of dissolving a sufficient amount of genotoxic impurities, so as to reduce the impact of the febuxostat chromatographic peak on the quantification of genotoxic impurities.

[0015] Sample processing method: Dissolve the product with a diluent, extract the impurity H, starting material 1 and intermediate 1 completely by ultrasonication and centrifugation, and then filter the supernatant as the test solution for analysis.

[0016] Chromatographic conditions: The chromatographic column is filled with octadecylsilane bonded silica gel, preferably selected from Kromasil C18, ODS-C18, Thermo Syncronis C18, Thermo Hypersil GOLD C18, Waters symmetry C18, WatersRP C18, Agilent XDB C18, Agilent Eclipse Plus C18,

[0017] Mobile phase A is salt solution; mobile phase B is acetonitrile; gradient elution is performed using salt solution and acetonitrile as mobile phases.

[0018] The detector is one of an ultraviolet detector, a differential detector, an evaporative light scattering detector, and a diode array detector.

[0019] Preferably, the diluent is dimethylformamide, tetrahydrofuran, acetonitrile and aqueous formic acid solution, more preferably a mixed solution of aqueous formic acid solution and acetonitrile.

[0020] Furthermore, the volume of formic acid in the formic acid aqueous solution is 30% to 50% by volume, and more preferably 40% by volume.

[0021] Furthermore, the ratio of the 40% formic acid aqueous solution to acetonitrile is 9:1 to 7:3.

[0022] Preferably, the ultrasonic treatment time is 10 to 20 minutes.

[0023] Preferably, the centrifugal temperature in the centrifugal condition is 20-30° C. and the rotation speed is 8000-13000 r.

[0024] Preferably, the centrifugation time is 10 to 30 minutes.

[0025] Preferably, the pore size of the filter membrane is 0.22 μm to 0.45 μm.

[0026] Preferably, the salt solution is a sodium acetate solution, and the pH of the salt solution is 2.9-3.1.

[0027] Preferably, in the chromatographic conditions, the mobile phase is a salt solution and acetonitrile gradient elution, and during the gradient elution from 0 to 20 min, the volume ratio of the salt solution is 50%, and the volume ratio of the acetonitrile is 50%; during 20 to 30 min, the volume ratio of the salt solution decreases from 50% to 25%, and the volume ratio of the acetonitrile increases from 50% to 75%; during 30 to 50 min, the volume ratio of the salt solution is 25%, and the volume ratio of the acetonitrile is 75%; during 50 to 51 min, the volume ratio of the salt solution increases from 25% to 50%, and the volume ratio of the acetonitrile decreases from 75% to 50%; during 51 to 60 min, the volume ratio of the salt solution is 50%, and the volume ratio of the acetonitrile is 50%.

[0028] More preferably, the chromatographic column is Kromasil C18.

[0029] Preferably, the column temperature is 30-40°C and the flow rate is 0.9-1.1 ml / min.

[0030] Preferably, the detector is a diode array detector.

[0031] Preferably, the detection wavelength of the detector is 300-340 nm.

[0032] The extraction and separation method of the present invention can be achieved according to the following method:

[0033] 1) Preparation of mobile phase A: Dissolve 1.36 g of sodium acetate trihydrate in 1 L of water and adjust the pH to 2.9-3.1 with phosphoric acid;

[0034] 2) Preparation of mobile phase B: acetonitrile;

[0035] 3) Preparation of test solution: accurately weigh 50.0 mg of febuxostat sample into a centrifuge tube, add 2 mL of diluent, sonicate for 10 to 20 minutes, centrifuge for 10 to 30 minutes (centrifugation conditions: 20 to 30°C, 8000 to 13000 r), and filter the supernatant with a 0.22 μm to 0.45 μm filter membrane to obtain the test solution;

[0036] 4) Detection: The sample solution prepared in step 3) is detected using a high performance liquid chromatograph, and the working conditions are as follows:

[0037] Flow rate: 0.9ml / min~1.1ml / min

[0038] Column temperature: 30~40℃

[0039] Injection volume: 10 μL

[0040] Detection wavelength: 300~340nm

[0041] Chromatographic column: Kromasil C18 (5μm, 4.6×250mm)

[0042] Table 1 Column elution program

[0043] Time (min) Mobile phase A (% V / V) Mobile phase B (% V / V) 0 50 50 20 50 50 30 25 75 50 25 75 51 50 50 60 50 50

[0044] The detection method of Febuxostat genotoxic impurities provided by the present invention adopts high performance liquid chromatography to realize the rapid and accurate determination of three genotoxic impurities. The method is simple, has high precision, good stability, good reproducibility, has high sensitivity and specificity, is simple to operate, and the separation degree meets the standard (that is, the separation degree of each adjacent two peaks is greater than 1.50). The present invention can be used for the preparation process of Febuxostat and the quality control of the final product. It also lays a foundation for the research and development and quality detection of such compounds, and has practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 is a liquid chromatogram of the system suitability solution in Example 1;

[0046] Figure 2 is a liquid chromatogram of the system suitability solution in Example 2;

[0047] Figure 3 is a liquid chromatogram of the system suitability solution in Example 3;

[0048] Figure 4 is a liquid chromatogram of the system suitability solution in Example 4;

[0049] Figure 5 is a liquid chromatogram of the system suitability solution in Example 5;

[0050] Figure 6 is the liquid chromatogram of the reference substance solution in Example 5;

[0051] Figure 7 It is the liquid chromatogram of the test solution in Example 5. DETAILED DESCRIPTION

[0052] The above contents are further supplemented by the following examples, but it should not be understood that the present invention is limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0053] Example 1

[0054] 1) Chromatographic conditions

[0055] Flow rate: 0.9ml / min

[0056] Column temperature: 30°C

[0057] Detection wavelength: 320nm

[0058] Chromatographic column: Kromasil C18 (5μm, 4.6×250mm)

[0059] Table 2 Column elution program

[0060] Time (min) Mobile phase A (% V / V) Mobile phase B (% V / V) 0 50 50 20 50 50 30 25 75 50 25 75 51 50 50 60 50 50

[0061] Diluent: 40% formic acid-acetonitrile (7:3)

[0062] 2) Solution preparation

[0063] Reference solution: Accurately weigh appropriate amounts of impurity H, starting material 1 and intermediate 1, dissolve them in a diluent and quantitatively dilute them into a solution containing approximately 0.45 μg of each impurity per 1 ml, as the reference solution.

[0064] System suitability solution: Accurately weigh 50.0 mg of febuxostat sample into a centrifuge tube, add 2 mL of reference solution, sonicate for 20 minutes, centrifuge for 10 minutes (centrifugation conditions: 25°C, 13000r), and filter the supernatant with a 0.45 μm filter membrane to obtain the system suitability solution.

[0065] 3) Detection method

[0066] Accurately measure 10 μL of the system suitability solution and inject it into the HPLC. The chromatogram is as follows: Figure 1 shown.

[0067] 4) Experimental results

[0068] Table 3 Analysis results of impurities in the chromatogram of Example 1

[0069]

[0070]

[0071] As can be seen from Table 3, in this chromatographic system, the retention times of febuxostat impurity H, starting material 1 and intermediate 1 are significantly different, and they can be effectively separated, meeting the requirements, indicating that this method has good specificity.

[0072] Example 2

[0073] 1) Chromatographic conditions

[0074] Flow rate: 1.1ml / min

[0075] Column temperature: 30°C

[0076] Detection wavelength: 320nm

[0077] Chromatographic column: Kromasil C18 (5μm, 4.6×250mm)

[0078] Table 4 Column elution program

[0079] Time (min) Mobile phase A (% V / V) Mobile phase B (% V / V) 0 50 50 20 50 50 30 25 75 50 25 75 51 50 50 60 50 50

[0080] Diluent: 40% formic acid-acetonitrile (7:3)

[0081] 2) Solution preparation

[0082] Reference solution: Accurately weigh appropriate amounts of impurity H, starting material 1 and intermediate 1, dissolve them in a diluent and quantitatively dilute them into a solution containing approximately 0.45 μg of each impurity per 1 ml, as the reference solution.

[0083] System suitability solution: Accurately weigh 50.0 mg of febuxostat sample into a centrifuge tube, add 2 mL of reference solution, sonicate for 10 to 20 minutes, centrifuge for 10 to 30 minutes (centrifugation conditions: 20 to 30°C, 8000 to 13000 r), and filter the supernatant with a 0.45 μm filter membrane to obtain the system suitability solution.

[0084] 3) Detection method

[0085] Accurately measure 10 μL of the system suitability solution and inject it into the HPLC. The chromatogram is as follows: Figure 2 shown.

[0086] 4) Experimental results

[0087] Table 5 Analysis results of impurities in the chromatogram of Example 2

[0088] name Retention time min Separation Febuxostat 17.017 - Impurity H 18.667 2.86 Starting material 1 24.735 10.26 Intermediate 1 42.287 5.57

[0089] As can be seen from Table 5, in this chromatographic system, the retention times of febuxostat impurity H, starting material 1 and intermediate 1 are significantly different, and they can be effectively separated, meeting the requirements, indicating that this method has good specificity.

[0090] Example 3

[0091] 1) Chromatographic conditions

[0092] Flow rate: 1.0ml / min

[0093] Column temperature: 30°C

[0094] Detection wavelength: 320nm

[0095] Chromatographic column: Kromasil C18 (5μm, 4.6×250mm)

[0096] Table 6 Column elution program

[0097] Time (min) Mobile phase A (% V / V) Mobile phase B (% V / V) 0 50 50 20 50 50 30 25 75 50 25 75 51 50 50 60 50 50

[0098] Diluent: 40% formic acid-acetonitrile (7:3)

[0099] 2) Solution preparation

[0100] Reference solution: Accurately weigh appropriate amounts of impurity H, starting material 1 and intermediate 1, dissolve them in a diluent and quantitatively dilute them into a solution containing approximately 0.45 μg of each impurity per 1 ml, as the reference solution.

[0101] System suitability solution: Accurately weigh 50.0 mg of febuxostat sample into a centrifuge tube, add 2 mL of reference solution, sonicate for 20 minutes, centrifuge for 30 minutes (centrifugation conditions: 25°C, 13000r), and filter the supernatant with a 0.45 μm filter membrane to obtain the system suitability solution.

[0102] 3) Detection method

[0103] Accurately measure 10 μL of the system suitability solution and inject it into the HPLC. The chromatogram is as follows: Figure 3 shown.

[0104] 4) Experimental results

[0105] Table 7 Analysis results of impurities in the chromatogram of Example 3

[0106] name Retention time min Separation Febuxostat 18.048 - Impurity H 19.928 2.81 Starting material 1 26.310 9.83 Intermediate 1 44.241 5.93

[0107] As can be seen from Table 7, in this chromatographic system, the retention times of febuxostat impurity H, starting material 1 and intermediate 1 are significantly different, and they can be effectively separated, meeting the requirements, indicating that this method has good specificity.

[0108] Example 4

[0109] 1) Chromatographic conditions

[0110] Flow rate: 1.0ml / min

[0111] Column temperature: 30°C

[0112] Detection wavelength: 320nm

[0113] Chromatographic column: Kromasil C18 (5μm, 4.6×250mm)

[0114] Table 8 Column elution program

[0115]

[0116]

[0117] Diluent: 40% formic acid-acetonitrile (8:2)

[0118] 2) Solution preparation

[0119] Reference solution: Accurately weigh appropriate amounts of impurity H, starting material 1 and intermediate 1, dissolve them in a diluent and quantitatively dilute them into a solution containing approximately 0.45 μg of each impurity per 1 ml, as the reference solution.

[0120] System suitability solution: Accurately weigh 50.0 mg of febuxostat sample into a centrifuge tube, add 2 mL of reference solution, sonicate for 20 minutes, centrifuge for 10 minutes (centrifugation conditions: 25°C, 13000r), and filter the supernatant with a 0.45 μm filter membrane to obtain the system suitability solution.

[0121] 3) Detection method

[0122] Accurately measure 10 μL of the system suitability solution and inject it into the HPLC. The chromatogram is as follows: Figure 4 shown.

[0123] 4) Experimental results

[0124] Table 9 Analysis results of impurities in the chromatogram of Example 4

[0125] name Retention time min Separation Febuxostat 18.056 - Impurity H 19.961 3.27 Starting material 1 26.328 11.36 Intermediate 1 44.264 6.12

[0126] As can be seen from Table 9, in this chromatographic system, the retention times of febuxostat impurity H, starting material 1 and intermediate 1 are significantly different, and they can be effectively separated, meeting the requirements, indicating that this method has good specificity.

[0127] Example 5

[0128] 1) Chromatographic conditions

[0129] Flow rate: 1.0ml / min

[0130] Column temperature: 30°C

[0131] Detection wavelength: 320nm

[0132] Chromatographic column: Kromasil C18 (5μm, 4.6×250mm)

[0133] Table 10 Column elution program

[0134] Time (min) Mobile phase A (% V / V) Mobile phase B (% V / V) 0 50 50 20 50 50 30 25 75 50 25 75 51 50 50 60 50 50

[0135] Diluent: 40% formic acid-acetonitrile (7:3)

[0136] 2) Solution preparation

[0137] Reference solution: Accurately weigh appropriate amounts of impurity H, starting material 1 and intermediate 1, dissolve them in a diluent and quantitatively dilute them into a solution containing approximately 0.45 μg of each impurity per 1 ml, as the reference solution.

[0138] System suitability solution: Accurately weigh 50.0 mg of febuxostat sample into a centrifuge tube, add 2 mL of reference solution, sonicate for 20 minutes, centrifuge for 10 minutes (centrifugation conditions: 25°C, 13000r), and filter the supernatant with a 0.45 μm filter membrane to obtain the system suitability solution.

[0139] Test solution: Accurately weigh 50.0 mg of febuxostat sample into a centrifuge tube, add 2 mL of diluent, sonicate for 20 minutes, centrifuge for 10 minutes (centrifugation conditions: 25°C, 13000r), and filter the supernatant with a 0.45 μm filter membrane to obtain the test solution.

[0140] 3) Detection method

[0141] Accurately measure 10 μL of system suitability solution, reference solution and test solution, and inject them into the HPLC. The chromatogram is as follows: Figures 5 to 7 shown.

[0142] 4) Experimental results

[0143] Table 11 Analysis results of impurities in the chromatogram of Example 5

[0144]

[0145]

[0146] As can be seen from Table 5, in this chromatographic system, the retention times of febuxostat impurity H, starting material 1 and intermediate 1 are significantly different, and they can be effectively separated, meeting the requirements, indicating that this method has good specificity.

[0147] Methodological validation:

[0148] The methodological investigation of the detection method was carried out according to the conventional experimental methods in this field, mainly investigating the specificity, detection limit, quantification limit, linearity, accuracy and repeatability of the detection method. The validation results are summarized in Table 6.

[0149] Table 12 Summary of method validation results

[0150]

[0151] As can be seen from Table 12, this detection method has high sensitivity and specificity and good repeatability.

[0152] Although the embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and preferred embodiments and cannot be understood as limiting the present invention. The sample processing method, mobile phase system and chromatographic conditions of this detection method are optimal under the time, place, environment and operator restrictions of the present invention, but are not limited to this processing method, mobile phase system and chromatographic conditions. Appropriate modification, adjustment and equivalent replacement of the sample processing method, the ratio, concentration and chromatographic conditions of the mobile phase system do not deviate from the technical concept and protection scope of the present invention.

Claims

1. A method for detecting genotoxic impurities in febuxostat, characterized in that: The detection method is high performance liquid chromatography, The chromatographic conditions were as follows: The chromatographic column is an octadecylsilane bonded silica gel chromatographic column; Mobile phase A is sodium acetate solution; mobile phase B is acetonitrile; sodium acetate solution and acetonitrile are used as mobile phases for gradient elution; during gradient elution from 0 to 20 minutes, the volume ratio of the salt solution is 50%, and the volume ratio of the acetonitrile is 50%; during 20 to 30 minutes, the volume ratio of the salt solution is reduced from 50% to 25%, and the volume ratio of the acetonitrile is increased from 50% to 75%; during 30 to 50 minutes, the volume ratio of the salt solution is 25%, and the volume ratio of the acetonitrile is 75%; during 50 to 51 minutes, the volume ratio of the salt solution is increased from 25% to 50%, and the volume ratio of the acetonitrile is reduced from 75% to 50%; during 51 to 60 minutes, the volume ratio of the salt solution is 50%, and the volume ratio of the acetonitrile is 50%; The detector is an ultraviolet detector, a differential detector, an evaporative light scattering detector or a diode array detector; The detection wavelength is 300~340nm; The column temperature is 30-40°C; Flow rate: 0.9-1.1 ml / min; The sample processing step is also included, and the sample processing method is as follows: Take this product and dissolve it with a diluent, sonicate and centrifuge to completely extract the impurity H: 2-[3-formyl-4-(2-methylpropoxy)phenyl]-4-methyl-5-thiazolecarboxylic acid, the starting material 1: 2-(3-formyl-4-hydroxyphenyl)-4-methyl-thiazole-5-carboxylic acid ethyl ester and the intermediate 1: 2-(3-formyl-4-isobutoxy-phenyl)-4-methyl-thiazole-5-carboxylic acid ethyl ester, and then take the supernatant and filter it as the test solution for analysis. The diluent is one or a mixture of dimethylformamide, tetrahydrofuran, acetonitrile or formic acid aqueous solution, the ultrasonic time is 10 to 20 minutes, the centrifugal temperature is 20 to 30°C, the rotation speed is 8000 to 13000r, and the centrifugal time is 10 to 30 minutes; the pore size of the filter membrane is 0.22μm to 0.45μm.

2. The detection method according to claim 1, characterized in that: The diluent is a mixed solution of formic acid aqueous solution and acetonitrile.

3. The detection method according to claim 2, characterized in that: In the formic acid aqueous solution, the volume of formic acid accounts for 30% to 50% by volume.

4. The detection method according to claim 2, characterized in that: In the formic acid aqueous solution, the volume of formic acid accounts for 40% by volume.

5. The detection method according to claim 2, characterized in that: The diluent is a 40% formic acid aqueous solution and acetonitrile in a ratio of 9:1 to 7:

3.

6. The detection method according to claim 1, characterized in that: The pH range of the sodium acetate solution is 2.9-3.

1.

7. The detection method according to claim 1, characterized in that: The chromatographic column is selected from Kromasil C18, ODS-C18, Thermo Syncronis C18, Thermo Hypersil GOLD C18, Waters symmetry C18, WatersRP C18, AgilentXDB C18, and Agilent Eclipse Plus C18.

8. The detection method according to claim 1, characterized in that: The chromatographic column is Kromasil C18.

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