Quality control method for related isomer impurities in novel antiviral drug intermediate

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a quality control method for related isomer impurities in a new antiviral drug intermediate, and the new antiviral drug intermediate comprises three related isomer impurities, namely an impurity I, an impurity II and an impurity III. The related isomer impurities are detected by adopting a gradient elution procedure through high performance liquid chromatography, and the result shows that the quality control method provided by the invention can effectively separate the related isomer impurities, can accurately determine the content of the isomer impurities, effectively solves the problem that the isomer impurities are difficult to separate and detect, and is suitable for industrial production. The quality control method has the advantages of strong specificity, high sensitivity and good accuracy, can accurately and quantitatively detect each isomer impurity in a sample, formulates a reasonable control limit, and provides a powerful guarantee for the quality control of the final product of the novel antiviral drug.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a quality control method for related isomer impurities in a new antiviral drug intermediate. Background Art

[0002] The intermediate of the new antiviral drug is a small molecule RNA polymerase PA subunit inhibitor of a class of chemical drugs. It is currently in the phase III clinical trial stage and is indicated for the treatment of influenza A and B. WXSH0208-SM1 refers to the intermediate of the new antiviral drug, and its chemical structure is as follows: WXSH0208-SM1 includes three related isomeric impurities, as shown in the following table: In order to ensure the quality of WXSH0208-SM1, it is necessary to conduct qualitative and quantitative analysis of its related isomeric impurities. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the object of the present invention is to provide a quality control method for related isomeric impurities in an intermediate of a new antiviral drug.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is: A quality control method for related isomeric impurities in a new antiviral drug intermediate, the structural formula of the new antiviral drug intermediate is as follows: ; Related isomeric impurities include one or more of impurities I, II and III; Among them, the structural formula of impurity I is as follows: ; The structural formula of impurity II is as follows: ; The structural formula of impurity III is as follows: ; The related isomeric impurities were detected by high performance liquid chromatography using a gradient elution procedure, and the chromatographic conditions were as follows: Chromatographic column: chiral column; The mobile phase includes mobile phase A and mobile phase B, the initial volume proportion of mobile phase A is 72% to 65%, and the initial volume proportion of mobile phase B is 28% to 35%; Wherein, mobile phase A is selected from an aqueous solution of diammonium phosphate, and mobile phase B is selected from acetonitrile; The initial flow rate of gradient elution was 0.36 ml / min to 0.44 ml / min.

[0005] Furthermore, in the gradient elution process, the volume ratios of mobile phase A and mobile phase B are as follows: constant from 0 min to 25 min, with mobile phase A maintained at 65% and mobile phase B maintained at 35%; then changing at a constant speed to the 45th min, with mobile phase A at 55% and mobile phase B at 45%; then changing at a constant speed to the 46th min, with mobile phase A at 65% and mobile phase B at 35%; constant from 46th min to 60 min, with mobile phase A maintained at 65% and mobile phase B maintained at 35%.

[0006] Furthermore, in the gradient elution process, the volume ratios of mobile phase A and mobile phase B are as follows: constant from 0 min to 25 min, with mobile phase A maintained at 70% and mobile phase B maintained at 30%; then changing uniformly to the 65th min, with mobile phase A at 55% and mobile phase B at 45%; then changing uniformly to the 66th min, with mobile phase A at 70% and mobile phase B at 30%; constant from 66 min to 80 min, with mobile phase A maintained at 70% and mobile phase B maintained at 30%.

[0007] Furthermore, in the gradient elution process, the volume proportions of mobile phase A and mobile phase B are as follows: constant from 0 min to 5 min, with mobile phase A maintained at 70% and mobile phase B maintained at 30%; then changing at a constant speed to the 45th min, with mobile phase A at 55% and mobile phase B at 45%; then changing at a constant speed to the 46th min, with mobile phase A at 70% and mobile phase B at 30%; constant from 46th min to 60th min, with mobile phase A maintained at 70% and mobile phase B maintained at 30%.

[0008] Furthermore, the specifications of the chromatographic column are 4.6×150 mm, 3 μm.

[0009] Furthermore, the column temperature during the detection process was 23°C to 27°C.

[0010] Furthermore, during the detection process, the new antiviral drug intermediate is prepared into an injection solution using methanol as a solvent.

[0011] Furthermore, the pH of the diammonium hydrogen phosphate aqueous solution is adjusted to 8.5 to 9.0 using aqueous ammonia.

[0012] Furthermore, the limit of quantification of impurity I is not higher than 0.50µg / ml, the limit of quantification of impurity II is not higher than 0.40µg / ml, and the limit of quantification of impurity III is not higher than 0.43µg / ml.

[0013] Furthermore, the detection limit of impurity I is not higher than 0.15µg / ml, the quantification limit of impurity II is not higher than 0.12µg / ml, and the quantification limit of impurity III is not higher than 0.13µg / ml.

[0014] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: The quality control method of related isomeric impurities in the intermediate of the new antiviral drug provided by the present invention can effectively separate the related isomeric impurities in WXSH0208-SM1, accurately determine the content of each isomeric impurity, effectively solve the problem of separation and detection difficulties of isomeric impurities in WXSH0208-SM1, thereby ensuring the quality control of WXSH0208-SM1. The quality control method has strong specificity, high sensitivity, and good accuracy, can accurately and quantitatively detect each isomeric impurity in the sample, formulate reasonable control limits, and provide a strong guarantee for the quality control of the final product antiviral new drug.

[0015] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the HLPC chromatogram of the mixed solution of WXSH0208-SM1 and three impurities provided in Example 1 of the present invention.

[0017] Figure 2 It is the HPLC chromatogram of the mixed solution of WXSH0208-SM1 and three impurities provided in Example 2 of the present invention.

[0018] Figure 3 It is the HPLC chromatogram of the mixed solution of WXSH0208-SM1 and three impurities provided in Example 3 of the present invention.

[0019] Figure 4 It is the HPLC chromatogram of the detection limit solution in Example 4 of the present invention.

[0020] Figure 5 It is the HPLC chromatogram of the quantitative limit solution in Example 4 of the present invention. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0022] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used are all commercially available unless otherwise specified.

[0023] In the following examples, the drugs, reagents and instruments used are as follows: Instruments and equipment: High performance liquid chromatography (HPLC) instrument (Vanquish-DAD, Thermo Fisher Scientific (China) Co., Ltd.); Chromatographic column: OX-3R type, chromatographic column (4.6×150mm, 3μm), Daicel Pharmaceutical Chiral Technology (Shanghai) Co., Ltd.; Reagents: methanol (chromatographic grade, batch number 240506, Tianjin Concord Technology Co., Ltd.); acetonitrile (chromatographic grade, batch number 240619, Tianjin Concord Technology Co., Ltd.); diammonium hydrogen phosphate (analytical grade, batch number 20221026, Sinopharm Group); water (ultrapure water, homemade); Reference Material Information: Impurity I (abbreviated as Z-1): batch number E040639-048-02, Duchuang (Chongqing) Pharmaceutical Technology Co., Ltd.; Impurity II (abbreviated as Z-2): batch number WS-ES18228-279-P1, Shanghai WuXi AppTec Pharmaceutical Co., Ltd.; Impurity III (abbreviated as Z-3): Batch number E040639-048-01, Duchuang (Chongqing) Pharmaceutical Technology Co., Ltd.

[0024] Example 1 Chromatographic column: OX-3R chromatographic column, specification: 4.6×150mm, 3μm; Mobile phase: 0.02 mol / L diammonium hydrogen phosphate aqueous solution (pH adjusted to 8.8 with ammonia water) as mobile phase A, acetonitrile as mobile phase B; The gradient elution program is shown in the following table: The flow rate is 0.4 ml / min; The column temperature was 25 °C; The detection wavelength is 290nm; The injection volume was 5 μl; Diluent: 50% acetonitrile aqueous solution; Operation steps: weigh about 10 mg of WXSH0208-SM1 sample into a 10 ml volumetric flask, add 1 ml of methanol to dissolve it, add Z-1 and Z-2 at 1% of the mass concentration of the test sample, add Z-3 at 5% of the mass concentration of the test sample, and then add diluent to dilute to the scale, mix well, and obtain a mixed solution of the test sample and related isomer impurities. The test results of the mixed solution are as follows: Figure 1 As shown, the results show that the separation degree of Z-1 and Z-2 detected by this method is 1.65.

[0025] Example 2 Chromatographic column: OX-3R chromatographic column, specification: 4.6×150mm, 3μm; Mobile phase: 0.02 mol / L diammonium hydrogen phosphate aqueous solution (pH adjusted to 8.8 with ammonia water) as mobile phase A, acetonitrile as mobile phase B; The gradient elution program is shown in the following table: The flow rate is 0.4 ml / min; The column temperature was 25°C; The detection wavelength is 290nm; The injection volume was 5 μl; Diluent: 50% acetonitrile aqueous solution; Operation steps: weigh about 10 mg of WXSH0208-SM1 sample into a 10 ml volumetric flask, add 1 ml of methanol to dissolve it, add Z-1 and Z-2 at 1% of the mass concentration of the test sample, add Z-3 at 5% of the mass concentration of the test sample, and then add diluent to dilute to the scale, mix well, and obtain a mixed solution of the test sample and related isomer impurities. The test results of the mixed solution are as follows: Figure 2 As shown, the results show that the separation degree of Z-1 and Z-2 detected by this method is 1.88, and Z-3 and adjacent peaks are separated by baseline. However, this method takes too long and the detection cost is high, so the method time needs to be shortened.

[0026] Example 3 1. Chromatographic conditions and operation process.

[0027] Chromatographic column: OX-3R chromatographic column, specification: 4.6×150mm, 3μm; Mobile phase: 0.02 mol / L diammonium hydrogen phosphate aqueous solution (pH adjusted to 8.8 with ammonia water) as mobile phase A, acetonitrile as mobile phase B; The gradient elution program is shown in the following table: The flow rate is 0.4 ml / min; The column temperature was 25°C; The detection wavelength is 290nm; The injection volume was 5 μl; Diluent: 50% acetonitrile aqueous solution; Operation process: weigh about 20 mg of WXSH0208-SM1 sample into a 10 ml volumetric flask, add 2 ml of methanol to dissolve it, add Z-1 and Z-2 at 1% of the mass concentration of the test sample, add Z-3 at 5% of the mass concentration of the test sample, and then add diluent to dilute to the scale, mix well, and obtain a mixed solution of the test sample and related isomer impurities. The test results of the mixed solution are as follows: Figure 3 As shown, the results show that the separation degree of Z-1 and Z-2 detected by this method is 1.87, Z-3 and adjacent peaks also achieve baseline separation, and the detection time using this method only takes about 60 minutes.

[0028] 2. Investigation on the durability range of method parameters.

[0029] As shown in the following table, the durability range of this method is investigated from the aspects of initial proportion of mobile phase B, column temperature and flow rate: Initial volume proportion of mobile phase B: 28% to 32%; When the initial volume proportion of mobile phase B was 28%, 30%, and 32%, respectively, the higher the initial volume proportion of mobile phase B, the earlier the peak elution time. However, the separation between Z-1, Z-2, and Z-3 under the three conditions could meet the detection requirements.

[0030] Column temperature: 23℃~27℃; The column temperatures of 23°C, 25°C and 27°C were examined in turn. Under the three column temperature conditions, the separation between Z-1, Z-2 and Z-3 could meet the detection requirements.

[0031] Flow rate: range 0.36ml / min~0.44ml / min; The flow rate conditions of 0.36ml / min, 0.40ml / min and 0.44ml / min were examined in turn. Under the same conditions, the higher the flow rate, the earlier the peak time. Under the three flow rate conditions, the separation degrees between Z-1, Z-2 and Z-3 can meet the requirements.

[0032] Example 4 1. Chromatographic conditions and operation process.

[0033] Chromatographic column: OX-3R chromatographic column, specification: 4.6×150mm, 3μm; Mobile phase: 0.02 mol / L diammonium hydrogen phosphate aqueous solution (pH adjusted to 8.8 with ammonia water) as mobile phase A, acetonitrile as mobile phase B; The gradient elution program is shown in the following table: The flow rate is 0.4 ml / min; The column temperature was 25°C; The detection wavelength is 290nm; The injection volume was 5 μl; Diluent: 50% acetonitrile aqueous solution; 2. Investigation of limit of quantification and limit of detection.

[0034] Operation steps: Accurately weigh appropriate amounts of Z-1, Z-2 and Z-3 impurity reference substances, dissolve them in methanol and dilute to the scale to obtain each impurity reference substance solution; gradually dilute to prepare each impurity quantitative limit and detection limit solution, inject into the liquid chromatograph, record the chromatogram, and the test results are as follows: Figure 4 and Figure 5 The quantitative limits and detection limits of each impurity are shown in the following table: From the data provided in the above table, it can be seen that based on the test solution concentration of 2 mg / ml, the detection limits are all less than 0.010%, with high sensitivity, which meets the detection requirements.

[0035] 3. Investigation of recovery rate.

[0036] Chromatographic conditions: the same as those in Example 4.

[0037] Purpose: To investigate the recovery rates at the 50%, 100% and 150% levels.

[0038] Reference substance solution: Accurately weigh appropriate amounts of Z-1, Z-2 and Z-3 impurity reference substances, accurately weigh them, dissolve them in diluent and dilute them to make a solution containing about Z-1 (50μg), Z-2 (50μg), and Z-3 (250μg) per ml as mixed reference substance stock solution. Accurately measure 2ml of mixed reference substance stock solution, place it in a 10ml volumetric flask, add 2ml of methanol, and then add diluent to dilute to the scale as reference substance solution.

[0039] Unsampled test solution: Accurately weigh about 20 mg of WXSH0208-SM1 sample, place it in a 10 ml volumetric flask, add 2 ml of methanol to dissolve it, and then add diluent to the scale to obtain the sample solution.

[0040] 50% horizontal loading solution: accurately weigh about 20 mg of WXSH0208-SM1 sample, place it in a 10 ml volumetric flask, add 2 ml of methanol to dissolve it, accurately measure 1 ml of the mixed reference stock solution into the flask, add diluent to the scale, and use it as the 50% horizontal loading solution.

[0041] 100% horizontal loading solution: accurately weigh about 20 mg of WXSH0208-SM1 sample, place it in a 10 ml volumetric flask, add 2 ml of methanol to dissolve it, accurately measure 2 ml of mixed reference stock solution into the flask, add diluent to the scale, and use it as the 100% horizontal loading solution.

[0042] 150% horizontal loading solution: accurately weigh about 20 mg of WXSH0208-SM1 sample, place it in a 10 ml volumetric flask, add 2 ml of methanol to dissolve it, accurately measure 3 ml of the mixed reference stock solution into the flask, add diluent to the scale, and use this as the 150% horizontal loading solution.

[0043] Take reference solution, test solution and sample solution respectively, inject them into liquid chromatograph, record the chromatogram, calculate the recovery rate, and the results are shown in the following table: It can be seen from the above table that the recovery rates of Z-1, Z-2 and Z-3 are all between 90% and 108% in the range of 50% to 150%, which meets the requirements. The results show that the quality control method for related isomeric impurities in the new antiviral drug intermediates provided by the present invention has good accuracy.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A quality control method for related isomeric impurities in an antiviral new drug intermediate, characterized in that: The structural formula of the new antiviral drug intermediate is as follows: ； Related isomeric impurities include one or more of impurities I, II and III; Among them, the structural formula of impurity I is as follows: ； The structural formula of impurity II is as follows: ； The structural formula of impurity III is as follows: ； The related isomeric impurities were detected by high performance liquid chromatography using a gradient elution procedure, and the chromatographic conditions were as follows: Chromatographic column: chiral column; The mobile phase includes mobile phase A and mobile phase B, the initial volume proportion of mobile phase A is 72% to 65%, and the initial volume proportion of mobile phase B is 28% to 35%; Wherein, mobile phase A is selected from an aqueous solution of diammonium phosphate, and mobile phase B is selected from acetonitrile; The initial flow rate of gradient elution was 0.36 ml / min to 0.44 ml / min.

2. The quality control method for related isomeric impurities in the intermediate of the new antiviral drug according to claim 1, characterized in that: During the gradient elution process, the volume ratios of mobile phase A and mobile phase B were as follows: constant from 0 min to 25 min, with mobile phase A maintained at 65% and mobile phase B maintained at 35%; then changed at a constant rate until the 45 min, with mobile phase A at 55% and mobile phase B at 45%; then changed at a constant rate until the 46 min, with mobile phase A at 65% and mobile phase B at 35%; constant from 46 min to 60 min, with mobile phase A maintained at 65% and mobile phase B maintained at 35%.

3. The quality control method for related isomeric impurities in the intermediate of the new antiviral drug according to claim 1, characterized in that: During the gradient elution process, the volume ratios of mobile phase A and mobile phase B were as follows: constant from 0 min to 25 min, with mobile phase A maintained at 70% and mobile phase B maintained at 30%; then changed at a constant rate to the 65th min, with mobile phase A at 55% and mobile phase B at 45%; then changed at a constant rate to the 66th min, with mobile phase A at 70% and mobile phase B at 30%; constant from 66 min to 80 min, with mobile phase A maintained at 70% and mobile phase B maintained at 30%.

4. The quality control method for related isomeric impurities in the intermediate of the new antiviral drug according to claim 1, characterized in that: During the gradient elution process, the volume proportions of mobile phase A and mobile phase B were as follows: constant from 0 min to 5 min, with mobile phase A maintained at 70% and mobile phase B maintained at 30%; then changed at a constant speed until the 45th min, with mobile phase A at 55% and mobile phase B at 45%; then changed at a constant speed until the 46th min, with mobile phase A at 70% and mobile phase B at 30%; constant from 46th min to 60th min, with mobile phase A maintained at 70% and mobile phase B maintained at 30%.

5. The quality control method for related isomeric impurities in the intermediate of the new antiviral drug according to claim 1, characterized in that: The specifications of the chromatographic column are 4.6×150 mm, 3 μm.

6. The quality control method for related isomeric impurities in the intermediate of the new antiviral drug according to claim 1, characterized in that: The column temperature during the detection process was 23°C to 27°C.

7. The quality control method for related isomeric impurities in the intermediate of the new antiviral drug according to claim 1, characterized in that: During the detection process, the new antiviral drug intermediate is prepared into an injection solution using methanol as a solvent.

8. The quality control method for related isomeric impurities in the intermediate of the new antiviral drug according to claim 1, characterized in that: The pH of the diammonium hydrogen phosphate aqueous solution was adjusted to 8.5 to 9.0 using aqueous ammonia.

9. The method for quality control of related isomeric impurities in the intermediate of the new antiviral drug according to claim 1, characterized in that: The limit of quantification of impurity I is not higher than 0.50µg / ml, the limit of quantification of impurity II is not higher than 0.40µg / ml, and the limit of quantification of impurity III is not higher than 0.43µg / ml.

10. The quality control method for related isomeric impurities in the intermediate of the new antiviral drug according to claim 1, characterized in that: The detection limit of impurity I is not higher than 0.15µg / ml, the detection limit of impurity II is not higher than 0.12µg / ml, and the detection limit of impurity III is not higher than 0.13µg / ml.

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