Quality control method for related isomeric impurities in an intermediate of a new antiviral drug

Abstract

The present invention relates to the technical field of pharmaceutical analysis, and particularly to a quality control method for related isomeric impurities in an intermediate of a new antiviral drug. The intermediate of the new antiviral drug includes three related isomeric impurities, namely impurity I, impurity II, and impurity III. The related isomeric impurities are detected by high performance liquid chromatography using a gradient elution program. The results show that the quality control method provided by the present invention can effectively separate the related isomeric impurities, accurately determine the content of the isomeric impurities, and effectively solve the problems of difficult separation and detection of the isomeric impurities, thereby ensuring the quality controllability of the intermediate of the new antiviral drug. The quality control method has strong specificity, high sensitivity, and good accuracy, can accurately 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, the new antiviral drug.

Description

Technical Field

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

[0002] The intermediate of the new antiviral drug is a class of small molecule chemical inhibitors of the PA subunit of RNA polymerase, and is currently in the clinical phase III for the treatment of influenza A and B. It is designated as WXSH0208-SM1, and its chemical structural formula is as follows:

[0003]

[0004] WXSH0208-SM1 includes three related isomeric impurities, as shown in the following table:

[0005]

[0006] To ensure the quality of WXSH0208-SM1, it is necessary to qualitatively and quantitatively analyze its related isomeric impurities. Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems existing in the related art. For this purpose, the object of the present invention is to provide a method for quality control of related isomeric impurities in an intermediate of a new antiviral drug.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] A method for quality control of related isomeric impurities in an intermediate of a new antiviral drug, the structural formula of the intermediate of the new antiviral drug is as follows:

[0010] ;

[0011] The related isomeric impurities include one or more of impurity I, impurity II, and impurity III;

[0012] Among them, the structural formula of impurity I is as follows:

[0013] ;

[0014] The structural formula of impurity II is as follows:

[0015] ;

[0016] The structural formula of impurity III is as follows:

[0017] ;

[0018] The relevant isomeric impurities are detected by high performance liquid chromatography using a gradient elution program, and the chromatographic conditions are as follows:

[0019] Chromatographic column: chiral chromatographic column;

[0020] The mobile phase includes mobile phase A and mobile phase B. The initial volume ratio of mobile phase A is 72% - 65%, and the initial volume ratio of mobile phase B is 28% - 35%;

[0021] Among them, mobile phase A is selected from an aqueous solution of diammonium phosphate, and mobile phase B is selected from acetonitrile;

[0022] The initial flow rate of gradient elution is 0.36 ml / min - 0.44 ml / min.

[0023] Further, during the gradient elution process, the volume ratio of mobile phase A and mobile phase B is as follows: constant from 0 min to the 25th min, mobile phase A remains at 65% and mobile phase B remains at 35%; then it changes uniformly to the 45th min, mobile phase A is 55% and mobile phase B is 45%; then it changes uniformly to the 46th min, mobile phase A is 65% and mobile phase B is 35%; from the 46th min to the 60th min, it is constant, mobile phase A remains at 65% and mobile phase B remains at 35%.

[0024] Further, during the gradient elution process, the volume ratio of mobile phase A and mobile phase B is as follows: constant from 0 min to the 25th min, mobile phase A remains at 70% and mobile phase B remains at 30%; then it changes uniformly to the 65th min, mobile phase A is 55% and mobile phase B is 45%; then it changes uniformly to the 66th min, mobile phase A is 70% and mobile phase B is 30%; from the 66th min to the 80th min, it is constant, mobile phase A remains at 70% and mobile phase B remains at 30%.

[0025] Further, during the gradient elution process, the volume ratio of mobile phase A and mobile phase B is as follows: constant from 0 min to the 5th min, mobile phase A remains at 70% and mobile phase B remains at 30%; then it changes uniformly to the 45th min, mobile phase A is 55% and mobile phase B is 45%; then it changes uniformly to the 46th min, mobile phase A is 70% and mobile phase B is 30%; from the 46th min to the 60th min, it is constant, mobile phase A remains at 70% and mobile phase B remains at 30%.

[0026] Further, the specification of the chromatographic column is 4.6×150 mm, 3 μm.

[0027] Further, the column temperature during the detection process is 23°C - 27°C.

[0028] Further, during the detection process, the antiviral new drug intermediate is formulated into an injection solution using methanol as a solvent.

[0029] Further, ammonia water is used to adjust the pH of the diammonium hydrogen phosphate aqueous solution to 8.5 - 9.0.

[0030] Further, the quantitation limit of impurity I is not higher than 0.50 μg / ml, the quantitation limit of impurity II is not higher than 0.40 μg / ml, and the quantitation limit of impurity III is not higher than 0.43 μg / ml.

[0031] Further, the detection limit of impurity I is not higher than 0.15 μg / ml, the quantitation limit of impurity II is not higher than 0.12 μg / ml, and the quantitation limit of impurity III is not higher than 0.13 μg / ml.

[0032] One or more of the above technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0033] The quality control method for related isomeric impurities in the antiviral new drug intermediate 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 problems of difficult separation and detection of isomeric impurities in WXSH0208 - SM1, and thus ensure the quality controllability of WXSH0208 - SM1. This quality control method has strong specificity, high sensitivity, and good accuracy, can accurately quantitatively detect each isomeric impurity in the sample, formulate reasonable control limits, and provides a strong guarantee for the quality control of the final product antiviral new drug.

[0034] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the HLPC chromatogram of the mixed solution of WXSH0208 - SM1 and three impurities provided in Example 1 of the present invention.

[0036] Figure 2 is the HPLC chromatogram of the mixed solution of WXSH0208 - SM1 and three impurities provided in Example 2 of the present invention.

[0037] Figure 3 is the HPLC chromatogram of the mixed solution of WXSH0208 - SM1 and three impurities provided in Example 3 of the present invention.

[0038] Figure 4 is the HPLC chromatogram of the detection limit solution in Example 4 of the present invention.

[0039] Figure 5It is the HPLC chromatogram of the quantitation limit solution in Example 4 of the present invention. Detailed implementation manners

[0040] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to 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.

[0041] In the following embodiments, unless otherwise specified, the experimental methods used are all conventional methods, and the materials, reagents, etc. used are carried out according to the technologies or conditions described in the literature in the art or according to the product specifications. Unless otherwise specified, they can all be obtained from commercial channels.

[0042] In the following embodiments, the drugs, reagents and instruments used are as follows:

[0043] Instrumentation: High performance liquid chromatography (HPLC) instrument (Vanquish-DAD, Thermo Fisher Scientific (China) Co., Ltd.);

[0044] Chromatographic column: OX-3R type chromatographic column (4.6×150 mm, 3 μm), Daicel Chiral Technologies (Shanghai) Co., Ltd.;

[0045] Reagents: Methanol (chromatographically pure, batch number 240506, Tianjin Concord Technology Co., Ltd.); Acetonitrile (chromatographically pure, batch number 240619, Tianjin Concord Technology Co., Ltd.); Diammonium hydrogen phosphate (analytically pure, batch number 20221026, Sinopharm Group); Water (ultrapure water, self-made);

[0046] Reference substance information:

[0047] Impurity I (abbreviation Z-1): Batch number E040639-048-02, Duchuang (Chongqing) Pharmaceutical Technology Co., Ltd.;

[0048] Impurity II (abbreviation Z-2): Batch number WS-ES18228-279-P1, Shanghai Wuxi AppTec New Drug Development Co., Ltd.;

[0049] Impurity III (abbreviation Z-3): Batch number E040639-048-01, Duchuang (Chongqing) Pharmaceutical Technology Co., Ltd.

[0050] Example 1

[0051] Chromatographic column: OX-3R chromatographic column, with a specification of 4.6×150 mm, 3 μm;

[0052] Mobile phase: 0.02 mol / L ammonium hydrogen phosphate aqueous solution (adjusted to pH 8.8 with ammonia water) was used as mobile phase A, and acetonitrile was used as mobile phase B;

[0053] The gradient elution program is shown in the following table:

[0054]

[0055] The flow rate was 0.4 ml / min;

[0056] The column temperature was 25 °C;

[0057] The detection wavelength was 290 nm;

[0058] The injection volume was 5 μl;

[0059] Diluent: 50% acetonitrile aqueous solution;

[0060] Operation steps: Weigh about 10 mg of the WXSH0208-SM1 sample into a 10-ml volumetric flask, add 1 ml of methanol to dissolve it, add Z-1 and Z-2 respectively according to 1% of the mass concentration of the test sample, add Z-3 according to 5% of the mass concentration of the test sample, and then add diluent to dilute to the scale and mix well to obtain a mixed solution of the test sample and related isomeric impurities. The test results of this mixed solution are as Figure 1 shown. The results show that the resolution of Z-1 and Z-2 detected by this method is 1.65.

[0061] Example 2

[0062] Chromatographic column: OX-3R chromatographic column, with a specification of 4.6×150 mm, 3 μm;

[0063] Mobile phase: 0.02 mol / L ammonium hydrogen phosphate aqueous solution (adjusted to pH 8.8 with ammonia water) was used as mobile phase A, and acetonitrile was used as mobile phase B;

[0064] The gradient elution program is shown in the following table:

[0065]

[0066] The flow rate was 0.4 ml / min;

[0067] The column temperature was 25 °C;

[0068] The detection wavelength was 290 nm;

[0069] The injection volume was 5 μl;

[0070] Diluent: 50% acetonitrile aqueous solution;

[0071] 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 respectively at 1% of the mass concentration of the test sample, add Z-3 at 5% of the mass concentration of the test sample, then add diluent to the scale, mix well to obtain a mixed solution of the test sample and related isomeric impurities. The test results of this mixed solution are as Figure 2 shown. The results show that the resolution between Z-1 and Z-2 detected by this method is 1.88, and Z-3 and the adjacent peak reach baseline separation. However, this method takes too long and the detection cost is relatively high, so it is necessary to shorten the method time.

[0072] Example 3

[0073] I. Chromatographic conditions and operation process.

[0074] Chromatographic column: OX-3R chromatographic column, with a specification of 4.6×150 mm, 3 μm;

[0075] Mobile phase: Use 0.02 mol / L diammonium hydrogen phosphate aqueous solution (adjusted to pH 8.8 with ammonia water) as mobile phase A, and acetonitrile as mobile phase B;

[0076] The gradient elution program is shown in the following table:

[0077]

[0078] Flow rate: 0.4 ml / min;

[0079] Column temperature: 25 °C;

[0080] Detection wavelength: 290 nm;

[0081] Injection volume: 5 μl;

[0082] Diluent: 50% acetonitrile aqueous solution;

[0083] 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 respectively at 1% of the mass concentration of the test sample, add Z-3 at 5% of the mass concentration of the test sample, then add diluent to the scale, mix well to obtain a mixed solution of the test sample and related isomeric impurities. The test results of this mixed solution are as Figure 3 shown. The results show that the resolution between Z-1 and Z-2 detected by this method is 1.87, and Z-3 and the adjacent peak also reach baseline separation, and the detection time using this method only takes about 60 min.

[0084] II. Investigation on the durability range of method parameters.

[0085] As shown in the following table, the durability range of this method was investigated from aspects such as the initial proportion of mobile phase B, column temperature, and flow rate:

[0086]

[0087] Initial volume percentage of mobile phase B: 28% - 32%;

[0088] When the initial volume proportions of mobile phase B were 28%, 30%, and 32% respectively, the higher the initial volume proportion of mobile phase B, the earlier the peak emergence time. However, the resolution between Z-1, Z-2, and Z-3 under the three conditions could meet the detection requirements.

[0089] Column temperature: 23°C - 27°C;

[0090] The column temperatures of 23°C, 25°C, and 27°C were investigated in sequence. Under the three column temperature conditions, the resolution between Z-1, Z-2, and Z-3 could meet the detection requirements.

[0091] Flow rate: in the range of 0.36 ml / min - 0.44 ml / min;

[0092] The flow rate conditions of 0.36 ml / min, 0.40 ml / min, and 0.44 ml / min were investigated in sequence. Under the same conditions, the higher the flow rate, the earlier the peak emergence time. Under the three flow rate conditions, the resolution between Z-1, Z-2, and Z-3 could meet the requirements.

[0093] Example 4

[0094] I. Chromatographic conditions and operation process.

[0095] Chromatographic column: OX-3R chromatographic column, with a specification of 4.6×150 mm, 3 μm;

[0096] Mobile phase: 0.02 mol / L diammonium hydrogen phosphate aqueous solution (adjusted to pH 8.8 with ammonia water) was used as mobile phase A, and acetonitrile was used as mobile phase B;

[0097] The gradient elution program is shown in the following table:

[0098]

[0099] Flow rate was 0.4 ml / min;

[0100] Column temperature was 25°C;

[0101] Detection wavelength was 290 nm;

[0102] Sample injection volume was 5 μl;

[0103] Diluent: 50% acetonitrile aqueous solution;

[0104] II. Investigation of Quantitation Limit and Detection Limit.

[0105] Operation steps: Weigh appropriate amounts of Z-1, Z-2, and Z-3 impurity reference substances accurately, dissolve them in methanol and dilute to the mark to obtain impurity reference substance solutions; prepare quantitation limit and detection limit solutions of each impurity by successive dilution, inject them into the liquid chromatograph, record the chromatogram, and the test results are as Figure 4 and Figure 5 shown. The quantitation limit and detection limit results of each impurity are shown in the following table:

[0106]

[0107] From the data provided in the above table, it can be seen that when calculated based on the concentration of the test solution being 2 mg / ml, the detection limits are all less than 0.010%, with relatively high sensitivity, and all meet the detection requirements.

[0108] III. Investigation of Recovery Rate.

[0109] Chromatographic conditions: The same as those in Example 4.

[0110] Purpose: To investigate the recovery rates at 50%, 100%, and 150% levels.

[0111] Reference substance solution: Weigh appropriate amounts of Z-1, Z-2, and Z-3 impurity reference substances accurately, dissolve them in the diluent and dilute to prepare a solution containing approximately Z-1 (50 μg), Z-2 (50 μg), and Z-3 (250 μg) per ml as the mixed reference substance stock solution. Then accurately measure 2 ml of the mixed reference substance stock solution, place it in a 10-ml volumetric flask, add 2 ml of methanol, and then dilute to the mark with the diluent as the reference substance solution.

[0112] Test solution without sample addition: Weigh approximately 20 mg of the WXSH0208-SM1 sample accurately, place it in a 10-ml volumetric flask, add 2 ml of methanol to dissolve it, and then add the diluent to the mark to obtain the sample solution.

[0113] Sample solution with 50% addition: Weigh approximately 20 mg of the WXSH0208-SM1 sample accurately, place it in a 10-ml volumetric flask, add 2 ml of methanol to dissolve it, accurately measure 1 ml of the mixed reference substance stock solution into this flask, and add the diluent to the mark as the sample solution with 50% addition.

[0114] Sample solution with 100% addition: Weigh approximately 20 mg of the WXSH0208-SM1 sample accurately, place it in a 10-ml volumetric flask, add 2 ml of methanol to dissolve it, accurately measure 2 ml of the mixed reference substance stock solution into this flask, and add the diluent to the mark as the sample solution with 100% addition.

[0115] 150% spiking solution: Weigh accurately about 20 mg of WXSH0208-SM1 sample, place it in a 10-ml volumetric flask, dissolve it with 2 ml of methanol, accurately pipette 3 ml of the mixed reference stock solution into the flask, and dilute to the mark with the diluent to obtain the 150% spiking solution.

[0116] Pipette the reference solution, the test solution and the spiking solution respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the recovery rate. The results are shown in the following table:

[0117]

[0118] As can be seen from the above table, for Z-1, Z-2 and Z-3 within the range of 50% - 150%, the recovery rates are all between 90% and 108%, meeting the requirements. This result indicates that the quality control method for the related isomeric impurities in the intermediate of the new antiviral drug provided by the present invention has good accuracy.

[0119] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate 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: OX-3R type chromatographic column, specification is 4.6×150mm, 3μm; 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; In the gradient elution process, the volume ratio of mobile phase A and mobile phase B is selected from any of the following situations: From 0min to 25min, the mobile phase A was kept at 65% and the mobile phase B was kept at 35%; then the mobile phase A was changed to 55% and the mobile phase B was changed to 45min; then the mobile phase A was changed to 65% and the mobile phase B was changed to 35%; from 46min to 60min, the mobile phase A was kept at 65% and the mobile phase B was kept at 35%; From 0min to 25min, the mobile phase A was kept at 70% and the mobile phase B was kept at 30%; then the mobile phase A was changed to 55% and the mobile phase B was 45% at a constant speed until the 65th minute; then the mobile phase A was changed to 70% and the mobile phase B was 30% at a constant speed until the 66th minute; from 66min to 80min, the mobile phase A was kept at 70% and the mobile phase B was kept at 30%; From 0min to 5min, it was constant, with mobile phase A maintained at 70% and mobile phase B maintained at 30%; then it changed uniformly until 45min, with mobile phase A at 55% and mobile phase B at 45%; then it changed uniformly until 46min, with mobile phase A at 70% and mobile phase B at 30%; from 46min to 60min, it was constant, with mobile phase A maintained at 70% and mobile phase B maintained at 30%.

2. 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.

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

4. 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.

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 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.

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 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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