Method for simultaneously detecting three isomer impurities in macloxvir raw material and preparation of macloxvir raw material and application of macloxvir raw material

Abstract

The invention belongs to the technical field of drug analysis and detection, and particularly relates to a method for simultaneously detecting three isomer impurities in a macloxvir raw material and a preparation thereof and application. The method can be used for simultaneously detecting three isomer impurities in the macarovir raw material and the preparation thereof under one chromatographic condition, the separation degree of each chromatographic peak is relatively good, the detection limit and the quantitation limit also meet the qualitative and quantitative analysis requirements of medicines, the sensitivity is high, the precision and the stability are relatively high, the reproducibility is good, the accuracy is high, and the method is suitable for industrial production. The method can be used for monitoring the impurities in the production process of the macloxvir, and is beneficial to control and improvement of the quality of the macloxvir; meanwhile, operation is easy, detection time is short, and the method is very suitable for quality monitoring of raw material medicines in large-scale industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis and detection, and more specifically, relates to a method and application for simultaneously detecting three isomeric impurities in mabaloxavir raw materials and preparations thereof. Background Art

[0002] Baloxavir marboxil, chemical name: [[(R)-12-[(S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]-thiapiprolin] [-11-yl]-6,8-dioxo-3,4,6,8,12,12a-hexahydro-1H-[1,4]azinol[3,4-c]pyrido[2,1-f][1,2,4]triazin-7-yl]oxy]methyl carbonate is a new anti-influenza virus drug with a new mechanism of action that was first developed in the past 20 years. It was jointly developed by Shionogi Pharmaceutical Co., Ltd. of Japan and Roche of Switzerland. It was launched in Japan in February 2018 and in the United States in October of the same year. It is used for single-dose treatment of acute, non-high-risk adult and adolescent influenza A and B patients with symptoms for no more than 48 hours and aged 12 years or older. Mabaloxavir dipyroxetine is its prodrug, which is rapidly converted into the active form of mabaloxavir by hydrolysis of esterases in the intestinal epithelium, liver and blood.

[0003] Mabaloxavir contains two chiral centers in its structure, and mainly contains four isomers, of which the (R, S) type has pharmacological effects, and the other three isomers are impurities. These three isomer impurities may be introduced during the production process of mabaloxavir raw materials:

[0004]

[0005]

[0006] Generally speaking, drugs containing chiral centers have large differences in efficacy between different configurations, and affect the yield of the target configuration drug synthesis, etc. The isomeric impurities of the target product need to be studied and controlled for quality. At the same time, the Chinese Pharmacopoeia's impurity research guidelines also clearly stipulate that other enantiomers and diastereomers that may coexist in single enantiomer drugs should be checked as impurities. The production and storage of mabaloxavir raw materials and preparations may lead to the conversion of chiral configurations. In order to ensure the chiral purity of mabaloxavir and the quality and safety of its preparations, it is necessary to establish corresponding isomeric impurity control methods.

[0007] When testing mabaloxavir, it was found that the mabaloxavir isomer 2 impurity often co-eluted with mabaloxavir under conventional reverse-phase HPLC conditions, and the other two mabaloxavir isomers 2 and 3 impurities also co-eluted, making it impossible to accurately quantify the content of a single impurity.

[0008] At present, chromatography is the main method for separating chiral drugs of the mabaloxavir class. For example, Zhou Lixin et al. (Zhou Lixin, Peng Jiajuan, Jia Zhongtian, Zhu Xueyan, Zhang Peng. Optimization of the synthesis process of the new anti-influenza virus drug mabaloxavir [J]. Chinese Journal of Pharmaceutical Industry, 2021, 52(02): 190-197. DOI: 10.16522 / j.cnki.cjph.2021.02.005.) disclosed a method for determining mabaloxavir and its isomers. The method uses a Waters Sunfire C 18 A column (4.6 mm × 250 mm, 5 μm) was used as a chromatographic column, 0.1% trifluoroacetic acid solution was used as mobile phase A, and acetonitrile was used as mobile phase B to perform chromatographic determination of mabaloxavir and isomer 1; in addition, a CHIRALPAK IE column (4.6 mm × 250 mm, 5 μm) was used as a chromatographic column, n-hexane: ethanol (30: 70) was used as a mobile phase, the detection wavelength was 254 nm, the flow rate was 0.5 mL / min, and the column temperature was 30°C. The isomers 2 and 3 of mabaloxavir were determined under the detection conditions, and there was only one peak. It can be seen that this method requires the setting of detection conditions of both positive and reverse phase systems to detect the isomers of mabaloxavir step by step, and it is impossible to use the same detection condition to simultaneously separate and detect mabaloxavir and its isomers. Summary of the invention

[0009] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the prior art that the three isomeric impurities in the raw material of Mabaloxavir and its preparations cannot be simultaneously determined under the same HPLC detection conditions, and to provide a method for simultaneously detecting the three isomeric impurities in the raw material of Mabaloxavir and its preparations.

[0010] The object of the present invention is to provide application of the method in quality monitoring of mabaloxavir.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention protects a method for simultaneously detecting three isomeric impurities in mabaloxavir raw materials and preparations thereof, which is detected by high performance liquid chromatography, and the conditions of the high performance liquid chromatography are:

[0013] A CHIRALPAK AD-H chromatographic column was used, and a mixed solution of n-hexane, ethanol, and diethylamine in a volume ratio of (68-72):(28-32):(0.1) was used as the mobile phase. After isocratic elution, detection was performed.

[0014] According to the different polarities of the mobile phase and the stationary phase, HPLC can be divided into normal phase chromatography and reversed phase chromatography. The stationary phase of reversed phase HPLC is composed of non-polar compounds, such as octadecylsilane, C 18 , C8 and other organic compounds, and the mobile phase is polar. Therefore, compounds with high polarity are eluted first, and compounds with low polarity or no polarity are eluted last. HPLC is mostly used to analyze drugs, foods, biochemical molecules and other substances, which are non-polar substances in nature. Therefore, reversed-phase high-performance liquid chromatography is often used to measure these substances.

[0015] Chiral HPLC is a separation method that introduces chiral drugs into a chiral environment to make enantiomers show differences in physical characteristics. It can be divided into chiral reagent derivatization (CDR), chiral mobile phase (CMP) and chiral stationary phase (CSP) for separation: chiral reagent derivatization is to use a chiral reagent to pre-column derivatize the enantiomeric mixture to form a pair of diastereomers, and then separate them with a conventional stationary phase. The chiral mobile phase method is to add a chiral reagent to the mobile phase to form a diastereomeric complex with the chiral drug to achieve separation; the chiral stationary phase method is to bond the chiral reagent to the stationary phase, react with the drug enantiomer to form diastereomers to achieve separation. However, the existing technology cannot simultaneously detect mabaloxavir and its isomeric impurities under the same detection conditions.

[0016] After a lot of preliminary research, the inventor creatively discovered that according to the characteristics of the drug, the chiral column, the mobile phase and other key detection conditions were studied in a targeted manner by combining the chiral HPLC method, and a method for quickly and accurately detecting three isomeric impurities in the raw material of Mabaloxavir and its preparations was summarized. Furthermore, the overall influence of the chiral stationary phase (chiral column), the mobile phase and other detection parameters was continuously studied, and the optimized conditions and methods for more quickly and accurately detecting three isomeric impurities in the raw material of Mabaloxavir and its preparations were summarized, which were applied to the detection and quality control of the raw material of Baloxavir and its preparations.

[0017] The structural formulas of the three isomers are shown below, and isomers 1 to 3 are (R, S) type, (S, S) type and (S, R) type impurities, respectively:

[0018]

[0019] Preferably, when determining mabaloxavir, the test solution is prepared with ethanol.

[0020] More preferably, the CHIRALPAK AD-H chromatographic column has a specification of 4.6 mm×250 mm, 5 μm.

[0021] Preferably, in the high performance liquid chromatography conditions, the detection wavelength is 250 to 260 nm.

[0022] Preferably, in the HPLC conditions, the column temperature is 20-30°C.

[0023] Preferably, under the conditions of high performance liquid chromatography, the injection volume is 10 to 30 μL.

[0024] Preferably, in the HPLC conditions, the flow rate of the mobile phase is 0.9 to 1.1 mL / min.

[0025] Furthermore, the limits of quantification of the three isomers are all 0.02%.

[0026] Furthermore, the detection limits of the three isomers are all 0.01%.

[0027] The present invention also protects the application of the method in quality monitoring of mabaloxavir.

[0028] The present invention has the following beneficial effects:

[0029] The detection method of the present invention can simultaneously detect three isomeric impurities in the raw material of mabaloxavir and the preparation thereof under one chromatographic condition, the separation degree of each chromatographic peak is good, the detection limit and the quantification limit also meet the requirements of qualitative and quantitative analysis of drugs, the sensitivity is high, and the method has high precision and stability, good reproducibility and high accuracy, and can be used for monitoring impurities in the production process of mabaloxavir, which is beneficial to controlling and improving the quality of mabaloxavir; meanwhile, the method is simple to operate and the detection time is short, and the method is very suitable for quality monitoring of raw materials in large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the chromatogram of blank reagent in Example 1.

[0031] Figure 2 The chromatogram of the test solution in Example 1.

[0032] Figure 3 This is the chromatogram of the spiked test solution in Example 1.

[0033] Figure 4 It is the chromatogram of the blank auxiliary material in Example 2.

[0034] Figure 5 This is the chromatogram of the spiked test solution in Example 2.

[0035] Figure 6This is the chromatogram of the spiked test solution in Comparative Example 1.

[0036] Figure 7 This is the chromatogram of the spiked test solution in Comparative Example 2.

[0037] Figure 8 This is the chromatogram of the spiked test solution in Comparative Example 3.

[0038] Fig. 9 This is the chromatogram of the spiked test solution in Comparative Example 4.

[0039] Fig.10 This is the chromatogram of the spiked test solution in Comparative Example 5.

[0040] Fig.11 This is the chromatogram of the spiked test solution in Comparative Example 6.

[0041] Fig.12 This is the chromatogram of the spiked test solution in Comparative Example 7.

[0042] Fig.13 This is the chromatogram of the spiked test solution in Comparative Example 8. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0044] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0045] The impurity reference substances in the examples and comparative examples were all sourced from Xiamen Hailejing Biochemical Co., Ltd.

[0046] Mabaloxavir API: Sourced from Nanchang Changyou Pharmaceutical Technology Co., Ltd., batch number BLX-705-211006, content is 100.0%.

[0047] Mabaloxavir tablets: sourced from Hunan Qianjin Xiangjiang Pharmaceutical Co., Ltd.; batch number: 20230305.

[0048] Isomer impurity 1: from Xiamen Hailejing Biochemical Co., Ltd.; batch number: 210701; purity ≥ 90.0%.

[0049] Isomer impurity 2: from Xiamen Hailejing Biochemical Co., Ltd.; batch number: 210701; purity ≥ 90.0%.

[0050] Isomer impurity 3: from Xiamen Hailejing Biochemical Co., Ltd.; batch number: 210701; purity ≥ 90.0%.

[0051] Example 1 A method for simultaneously detecting three isomeric impurities in mabaloxavir raw materials and preparations thereof

[0052] (1) Liquid chromatography conditions:

[0053] Chromatographic column: CHIRALPAK AD-H amylose silica chiral column, 4.6 mm × 250 mm, 5 μm.

[0054] The mobile phase was n-hexane-ethanol-diethylamine (70:30:0.1), the detection wavelength was 254 nm, the column temperature was 25 °C, the flow rate was 1.0 mL per minute, and the injection volume was 10 μL.

[0055] Sample preparation solvent: ethanol.

[0056] (2) Sample preparation:

[0057] Test solution: Take an appropriate amount of Mabaloxavir API, weigh accurately, add an appropriate amount of ethanol and sonicate to dissolve it to make a solution containing about 2 mg per 1 mL, and shake well.

[0058] Control solution: Accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, add solvent to dilute to the scale, and shake well (each 1 mL contains approximately 20 μg of solution); then accurately measure 3 mL, place it in a 20 mL volumetric flask, add solvent to dilute to the scale, shake well to make a solution containing approximately 3 μg per 1 mL.

[0059] Mixed solution of isomeric impurities: Take appropriate amounts of each of the isomeric impurities of Mabaloxavir, dissolve and dilute with ethanol to prepare a mixed solution containing approximately 10 μg of each isomeric impurity per 1 mL.

[0060] Spiked test solution: Take about 40 mg of mabaloxavir raw material, accurately weigh, and place in a 20 mL volumetric flask. Accurately measure 3 mL of the isomeric impurity mixed solution, place in the same volumetric flask, add an appropriate amount of ethanol and ultrasonically dissolve it to make a mixed solution containing about 2 mg of mabaloxavir and about 3 μg of each isomeric impurity per 1 mL.

[0061] (3) Testing and results of spiked test solution and reference substance

[0062] Precisely measure the blank solvent (ethanol), the test solution, and the spiked test solution for testing. The test results are shown in Table 1. Figure 1 (Blank reagent), Figure 2 (test solution) and Figure 3 (Spiked test solution) as shown.

[0063] Table 1 Separation results of spiked test solution

[0064] name Retention time / min Separation Isomer 3 23.422 / Isomer 2 31.173 2.55 Mabaloxavir 34.940 1.62 Isomer 1 57.846 5.44

[0065] Note: 2.55 in the separation data in Table 1 represents the separation between the chromatographic peak of isomer 2 and the chromatographic peak of isomer 3; 1.62 represents the separation between the chromatographic peak of mabaloxavir and the chromatographic peak of isomer 2; 5.44 represents the separation between the chromatographic peak of isomer 1 and the chromatographic peak of mabaloxavir.

[0066] The results showed that the blank solvent did not interfere with the detection of the three isomeric impurities; in the spiked test samples, the separation between the main peak of mabaloxavir and the three isomeric impurities met the requirement of separation > 1.5 stipulated in the "Chinese Pharmacopoeia", which can achieve the separation of mabaloxavir and its isomeric impurities, and subsequent quantification can accurately quantify the content of a single impurity.

[0067] (4) Determination of limit of quantification and limit of detection

[0068] In order to analyze the detection ability of this method for isomeric impurities, the isomeric impurity mixed solution was diluted step by step to investigate the detection limit (LOD) and quantification limit (LOQ) of the impurities. The results are shown in Tables 2 and 3.

[0069] Table 2 Quantitation limit results

[0070] Impurities S / N Limit of quantitation / % Isomer 3 13.9 0.02 Isomer 2 12.5 0.02 Isomer 1 10.6 0.02

[0071] Table 3 Detection limit results

[0072] Impurities S / N Detection limit / % Isomer 3 5.5 0.01 Isomer 2 4.8 0.01 Isomer 1 3.6 0.01

[0073] As can be seen from the table, the detection method of the present invention has lower detection limit and quantification limit for the three isomers, higher detection sensitivity and better method accuracy.

[0074] (5) Method stability study

[0075] To analyze the stability of the solution in this method, a control solution and a spiked test solution were prepared according to the preparation method in step (2) above, placed at room temperature for 0 h, 4 h, 8 h, and 12 h, respectively, injected into a liquid chromatograph, and the chromatogram was recorded. The relative change rate of the peak area of ​​each impurity at each time point compared with the peak area at 0 h was measured and calculated. The results are shown in Table 4.

[0076] Table 4 Solution stability results

[0077]

[0078]

[0079] The results showed that compared with 0h, after the control solution and the spiked test solution were placed at room temperature for 12 hours, the peak area change rates of the three isomeric impurities did not exceed the range of ±5%, indicating that the solution was stable within 12 hours at room temperature.

[0080] (6) Method repeatability study

[0081] To analyze the repeatability of this method, the control solution and the spiked test solution were prepared according to the preparation method in step (2) above, and the same sample was injected into the liquid chromatograph 6 times, the chromatogram was recorded, and the RSD of the peak area of ​​each peak of the 6 injections was calculated. The results showed that the RSD was less than 5.0%, which met the requirements.

[0082] The above verification process and verification results prove that the invention can accurately and effectively detect the contents of three isomeric impurities in mabaloxavir raw materials and preparations, and that the method has strong specificity, high sensitivity, stability and reliability.

[0083] Example 2 A method for simultaneously detecting three isomeric impurities in mabaloxavir raw materials and preparations thereof

[0084] The detection method of Example 2 is different from that of Example 1 in that Example 1 detects the raw material of Mabaloxavir, while Example 2 detects Mabaloxavir tablets (i.e., Mabaloxavir preparations), and the preparation steps thereof are as follows:

[0085] Test solution: Take an appropriate amount of Mabaloxavir tablets (equivalent to 40 mg of Mabaloxavir), accurately weigh, place in a 20 mL volumetric flask, add an appropriate amount of ethanol, sonicate for 15 minutes to dissolve, dilute to the scale with ethanol, shake well, filter, and take the filtrate;

[0086] Mixed solution of isomeric impurities: Take appropriate amounts of each of the isomeric impurities of Mabaloxavir, dissolve and dilute with ethanol to prepare a mixed solution containing approximately 10 μg of each isomeric impurity per 1 mL.

[0087] Spiked test solution: Take an appropriate amount of Mabaloxavir tablets (equivalent to 40 mg of Mabaloxavir), accurately weigh, and place in a 20 mL volumetric flask. Accurately measure 3 mL of the isomer impurity mixed solution, place in the same volumetric flask, add an appropriate amount of ethanol, sonicate for 15 minutes to dissolve, dilute to the scale with ethanol, shake well, filter, and take the filtrate;

[0088] Blank excipient solution: Take an appropriate amount of blank excipient (according to the prescription ratio, approximately equivalent to the amount of excipients contained in the above-mentioned Mabaloxavir tablets), accurately weigh, place in a 20mL volumetric flask, add an appropriate amount of ethanol and sonicate for 15 minutes to dissolve, dilute to the scale with ethanol, shake well, filter, and take the filtrate.

[0089] The remaining parameters refer to Example 1.

[0090] In order to analyze the interference of excipients in the preparation on isomeric impurities, blank excipient solution and spiked test solution were tested. The results are as follows: Figure 4 (blank excipient solution), Figure 5 (spiked test solution) and shown in Table 5.

[0091] Table 5 Separation results of spiked test solution

[0092] name Retention time / min Separation Isomer 3 23.460 / Isomer 2 31.168 3.13 Mabaloxavir 34.927 1.64 Isomer 1 57.966 5.57

[0093] Note: 3.13 in the separation data in Table 5 represents the separation between the chromatographic peak of isomer 2 and the chromatographic peak of isomer 3; 1.64 represents the separation between the chromatographic peak of mabaloxavir and the chromatographic peak of isomer 2; 5.57 represents the separation between the chromatographic peak of isomer 1 and the chromatographic peak of mabaloxavir.

[0094] Combining Table 1 and Table 5, it can be seen that the separation data obtained after adding excipients is not much different from the separation data without excipients, which meets the requirement of separation > 1.5 stipulated in the "Chinese Pharmacopoeia". The separation degree even increases after adding excipients, which indicates that the presence of excipients does not interfere with the detection of the three isomeric impurities.

[0095] Example 3 A method for simultaneously detecting three isomeric impurities in mabaloxavir raw materials and preparations thereof

[0096] The detection method of Example 3 is different from that of Example 1 in that, in step (1), the flow rate is 0.9 or 1.1 mL / min.

[0097] Other steps and parameters refer to Example 1.

[0098] In order to analyze the effect of flow rate on the detection separation of mabaloxavir and its three isomeric impurities, the spiked test solution was taken for detection. The results are shown in Tables 6 and 7.

[0099] Table 6 Separation results of spiked test solution - 0.9 mL / min

[0100] name Retention time / min Separation Isomer 3 24.872 / Isomer 2 32.568 2.62 Mabaloxavir 36.318 1.72 Isomer 1 59.350 5.64

[0101] Note: 2.62 in the separation data in Table 6 represents the separation between the chromatographic peak of isomer 2 and the chromatographic peak of isomer 3; 1.72 represents the separation between the chromatographic peak of mabaloxavir and the chromatographic peak of isomer 2; 5.64 represents the separation between the chromatographic peak of isomer 1 and the chromatographic peak of mabaloxavir.

[0102] Table 7 Separation results of spiked test solution -1.1mL / min

[0103] name Retention time / min Separation Isomer 3 21.874 / Isomer 2 29.562 2.32 Mabaloxavir 33.383 1.55 Isomer 1 56.215 5.21

[0104] Note: 2.32 in the separation data in Table 7 represents the separation between the chromatographic peak of isomer 2 and the chromatographic peak of isomer 3; 1.55 represents the separation between the chromatographic peak of mabaloxavir and the chromatographic peak of isomer 2; 5.21 represents the separation between the chromatographic peak of isomer 1 and the chromatographic peak of mabaloxavir.

[0105] The results showed that the peak separation of mabaloxavir and its three isomeric impurities in the detection method met the requirement of separation > 1.5 stipulated in the Chinese Pharmacopoeia, which indicates that mabaloxavir and its three isomeric impurities can be accurately determined under the flow rate conditions of the present invention.

[0106] Example 4 A method for simultaneously detecting three isomeric impurities in mabaloxavir raw materials and preparations thereof

[0107] The detection method of Example 4 is different from that of Example 1 in that, in step (1), the column temperature is 20 or 30°C.

[0108] Other steps and parameters refer to Example 1.

[0109] In order to analyze the effect of column temperature on the detection separation of mabaloxavir and its three isomeric impurities, the spiked test solution was taken for detection. The results are shown in Tables 8 and 9.

[0110] Table 8 Separation results of spiked test solution -20℃

[0111] name Retention time / min Separation Isomer 3 24.263 / Isomer 2 31.956 2.68 Mabaloxavir 35.731 1.65 Isomer 1 58.526 5.52

[0112] Note: 2.68 in the separation data in Table 8 represents the separation between the chromatographic peak of isomer 2 and the chromatographic peak of isomer 3; 1.55 represents the separation between the chromatographic peak of mabaloxavir and the chromatographic peak of isomer 2; 5.21 represents the separation between the chromatographic peak of isomer 1 and the chromatographic peak of mabaloxavir.

[0113] Table 9 Separation results of spiked test solution -30℃

[0114]

[0115]

[0116] Note: 2.29 in the separation data in Table 9 represents the separation between the chromatographic peak of isomer 2 and the chromatographic peak of isomer 3; 1.57 represents the separation between the chromatographic peak of mabaloxavir and the chromatographic peak of isomer 2; 5.19 represents the separation between the chromatographic peak of isomer 1 and the chromatographic peak of mabaloxavir.

[0117] The results show that the peak separation of mabaloxavir and its three isomeric impurities in the detection method meets the requirement of separation > 1.5 stipulated in the Chinese Pharmacopoeia, which indicates that mabaloxavir and its three isomeric impurities can be accurately determined under the column temperature conditions of the present invention.

[0118] Example 5 A method for simultaneously detecting three isomeric impurities in mabaloxavir raw materials and preparations thereof

[0119] The detection method of Example 5 is different from that of Example 1 in that, in step (1), the mobile phase is n-hexane-ethanol-diethylamine (72:28:0.1).

[0120] Other steps and parameters refer to Example 1.

[0121] In order to analyze the effect of the change in the mobile phase ratio on the detection separation of mabaloxavir and its three isomeric impurities, the spiked test solution was taken for detection. The results are shown in Table 10.

[0122] Table 10 Separation results of spiked test solution

[0123] name Retention time / min Separation Isomer 3 26.682 / Isomer 2 34.165 3.57 Mabaloxavir 38.012 1.75 Isomer 1 62.094 5.34

[0124] Note: 3.57 in the separation data in Table 10 represents the separation between the chromatographic peak of isomer 2 and the chromatographic peak of isomer 3; 1.75 represents the separation between the chromatographic peak of mabaloxavir and the chromatographic peak of isomer 2; 5.34 represents the separation between the chromatographic peak of isomer 1 and the chromatographic peak of mabaloxavir.

[0125] The results showed that the peak separation of mabaloxavir and its three isomeric impurities in the detection method met the requirement of separation > 1.5 stipulated in the Chinese Pharmacopoeia, which indicates that mabaloxavir and its three isomeric impurities can be accurately determined under the mobile phase conditions of the present invention.

[0126] Example 6 A method for simultaneously detecting three isomeric impurities in mabaloxavir raw materials and preparations thereof

[0127] The detection method of Example 6 is different from that of Example 1 in that, in step (1), the mobile phase is n-hexane-ethanol-diethylamine (68:32:0.1).

[0128] Other steps and parameters refer to Example 1.

[0129] In order to analyze the effect of the change in the mobile phase ratio on the detection separation of mabaloxavir and its three isomeric impurities, the spiked test solution was taken for detection. The results are shown in Table 11.

[0130] Table 11 Separation results of spiked test solution

[0131] name Retention time / min Separation Isomer 3 20.052 / Isomer 2 28.013 3.18 Mabaloxavir 31.850 1.57 Isomer 1 53.459 4.26

[0132] Note: 3.18 in the separation data in Table 11 represents the separation between the chromatographic peak of isomer 2 and the chromatographic peak of isomer 3; 1.57 represents the separation between the chromatographic peak of mabaloxavir and the chromatographic peak of isomer 2; 4.26 represents the separation between the chromatographic peak of isomer 1 and the chromatographic peak of mabaloxavir.

[0133] The results showed that the peak separation of mabaloxavir and its three isomeric impurities in the detection method met the requirement of separation > 1.5 stipulated in the Chinese Pharmacopoeia, which indicates that mabaloxavir and its three isomeric impurities can be accurately determined under the mobile phase conditions of the present invention.

[0134] Comparative Example 1 A method for detecting isomeric impurities in mabaloxavir raw materials and preparations thereof

[0135] The detection method of Comparative Example 1 is different from that of Example 1 in that, in step (1), the mobile phase is n-hexane-ethanol-isopropanol-diethylamine (60:20:20:0.1).

[0136] Other steps and parameters refer to Example 1.

[0137] The test results of the spiked test solution are detailed in Figure 6 and Table 12.

[0138] Table 12 Separation results of spiked test solution

[0139] name Retention time / min Separation Mabaloxavir 13.534 / Isomer 2 + Isomer 3 18.424 3.58 Isomer 1 24.491 3.64

[0140] Note: The separation data of 3.58 in Table 12 indicates the separation between the chromatographic peak of isomer 2 and isomer 3 and the chromatographic peak of mabaloxavir; 3.64 indicates the separation between the chromatographic peak of mabaloxavir and the chromatographic peak of isomer 1.

[0141] The results showed that isomers 2 and 3 co-emitted peaks and could not be separated, which did not meet the requirements. This indicated that the addition of isopropanol to the mobile phase had a significant impact on the determination of mabaloxavir and its three isomeric impurities, and the separation of mabaloxavir and its isomeric impurities could not be achieved.

[0142] Comparative Example 2 A method for detecting isomeric impurities in mabaloxavir raw materials and preparations thereof

[0143] The detection method of Comparative Example 2 is different from that of Example 1 in that, in step (1), the chromatographic column is a CHIRALPAK AS-H amylose silica chiral column, 4.6 mm×250 mm, 5 μm.

[0144] Other steps and parameters refer to Example 1.

[0145] The test results of the spiked test solution are detailed in Figure 7 and Table 13.

[0146] Table 13 Separation results of spiked test solution

[0147] name Retention time / min Separation Isomer 1 5.820 / Isomer 2 + Isomer 3 10.414 No display Mabaloxavir 12.807 No display

[0148] Note: No display means that the chromatographic peaks cannot be separated and the resolution cannot be calculated.

[0149] The results showed that isomers 2 and 3 co-emitted peaks and the peaks could not be separated, which did not meet the separation requirements and could not accurately quantify the content of a single isomer impurity. This indicates that not all chiral columns can achieve the separation of mabaloxavir and its isomer impurities.

[0150] Comparative Example 3 A method for detecting isomeric impurities in mabaloxavir raw materials and preparations thereof

[0151] The detection method of Comparative Example 3 is different from that of Example 1 in that, in step (1), the mobile phase is methanol-methyl tert-butyl ether-diethylamine (74:26:0.2).

[0152] Other steps and parameters refer to Example 1.

[0153] The test results of the spiked test solution are detailed in Figure 8 and Table 14.

[0154] Table 14 Separation results of spiked test solution

[0155]

[0156]

[0157] Note: No display means that the chromatographic peaks cannot be separated and the resolution cannot be calculated.

[0158] The results showed that isomers 1, 2 and 3 co-existed and the peaks could not be separated, which did not meet the separation requirements and could not accurately quantify the content of a single isomer impurity. This indicated that a specific mobile phase was required to achieve the separation of mabaloxavir and its isomer impurities.

[0159] Comparative Example 4 A method for detecting isomeric impurities in mabaloxavir raw materials and preparations thereof

[0160] The detection method of Comparative Example 4 is different from that of Example 1 in that, in step (1), the mobile phase is ethanol-ethyl acetate-diethylamine (40:60:0.1).

[0161] Other steps and parameters refer to Example 1.

[0162] The test results of the spiked test solution are detailed in Fig. 9 and Table 15.

[0163] Table 15 Separation results of spiked test solution

[0164] name Retention time / min Separation Isomer 1 4.921 / Mabaloxavir 5.499 2.07 Isomer 2 + Isomer 3 6.885 3.69

[0165] Note: The separation data of 2.07 in Table 15 indicates the separation between the chromatographic peak of isomer 1 and the chromatographic peak of mabaloxavir; 3.64 indicates the separation between the chromatographic peak of mabaloxavir and the chromatographic peak of isomers 2 and 3.

[0166] The results showed that isomers 2 and 3 co-emitted and could not be separated, which did not meet the requirements, and the content of isomers 2 and 3 impurities could not be accurately quantified. This shows that a specific mobile phase is needed to achieve the separation of mabaloxavir and its isomeric impurities.

[0167] Comparative Example 5 A method for detecting isomeric impurities in mabaloxavir raw materials and preparations thereof

[0168] The detection method of Comparative Example 5 is different from that of Example 1 in that, in step (1), the mobile phase is n-hexane-ethanol-ethyl acetate-diethylamine (27:22:51:0.1).

[0169] Other steps and parameters refer to Example 1.

[0170] The test results of the spiked test solution are detailed in Fig.10 and Table 16.

[0171] Table 16 Separation results of spiked test solution

[0172] name Retention time / min Separation Isomer 1 7.877 / Mabaloxavir 10.216 3.84 Isomer 2 + Isomer 3 15.301 5.39

[0173] Note: The separation data of 3.84 in Table 16 indicates the separation between the chromatographic peak of isomer 1 and the chromatographic peak of mabaloxavir; 5.39 indicates the separation between the chromatographic peak of mabaloxavir and the chromatographic peak of isomers 2 and 3.

[0174] The results showed that isomers 2 and 3 co-emitted and could not be separated, which did not meet the requirements, and the content of isomers 2 and 3 impurities could not be accurately quantified. This shows that a specific mobile phase is needed to achieve the separation of mabaloxavir and its isomeric impurities.

[0175] Comparative Example 6 A method for detecting isomeric impurities in mabaloxavir raw materials and preparations thereof

[0176] The detection method of Comparative Example 6 is different from that of Example 1 in that the chromatographic conditions in step (1) are: using silica gel coated with cellulose-tri(4-methylbenzoate) as a filler (CHIRALPAK OJ-RH 4.6 mm×150 mm, 5 μm, or a chromatographic column of equivalent performance); using acetonitrile-water (30:70) as a mobile phase, a detection wavelength of 254 nm, a flow rate of 0.4 mL per minute; a column temperature of 25° C.; and an injection volume of 10 μL.

[0177] Solvent: acetonitrile-water (30:70).

[0178] Other steps and parameters refer to Example 1.

[0179] The test results of the spiked test solution are shown in Table 17 and Fig.11 shown.

[0180] Table 17 Separation results of spiked test solution

[0181] Spiked test product RT / min RRT Separation (with front peak) Isomer 3 23.832 0.91 / Mabaloxavir + Isomer 2 26.311 1.00 1.75 Isomer 1 32.469 1.23 2.43

[0182] Note: The 1.75 in the resolution data in Table 17 indicates the resolution between the chromatographic peak of isomer 3 and the chromatographic peak of mabaloxavir and isomer 2; 2.43 indicates the resolution between the chromatographic peak of isomer 1 and the chromatographic peak of mabaloxavir and isomer 2.

[0183] The results showed that under this system, mabaloxavir and isomer 2 co-eluted and the two could not be separated, which did not meet the requirements. Therefore, this method could not meet the needs of simultaneously detecting three isomer impurities.

[0184] Comparative Example 7 A method for detecting isomeric impurities in mabaloxavir raw materials and preparations thereof

[0185] The detection method of Comparative Example 7 is different from that of Example 1 in that, in step (1), the mobile phase is n-hexane, ethanol and diethylamine in a volume ratio of 40:60:0.1.

[0186] Other steps and parameters refer to Example 1.

[0187] The test results of the spiked test solution are shown in Table 18 and Fig.12 shown.

[0188] Table 18 Separation results of spiked test solution

[0189] Spiked test product RT / min RRT Separation (with front peak) Isomer 1 5.820 0.45 / Isomer 2 + Isomer 3 10.414 0.81 No display Main Peak 12.807 1.00 No display

[0190] The detection results of the spiked test solution showed that isomers 2 and 3 co-emitted peaks and the peaks could not be separated, which did not meet the separation requirements and could not accurately quantify the content of isomers 2 and 3 impurities. This shows that the ratio of the mobile phase needs to be within a specific range to achieve the separation of mabaloxavir and its isomeric impurities.

[0191] Comparative Example 8 A method for detecting isomeric impurities in mabaloxavir raw materials and preparations thereof

[0192] The detection method of Comparative Example 8 is different from that of Example 1 in that, in step (1), the mobile phase is n-hexane, ethanol and diethylamine in a volume ratio of 60:40:0.1.

[0193] Other steps and parameters refer to Example 1.

[0194] The test results of the spiked test solution are shown in Table 19 and Fig.13 shown.

[0195] Table 19 Separation results of spiked test solution

[0196] Spiked test product RT / min RRT Separation (with front peak) Main Peak 11.367 1.00 / Isomer 2 + Isomer 3 14.684 1.29 3.48 Isomer 1 19.384 1.71 3.93

[0197] The test results of the spiked test solution showed that isomers 2 and 3 co-emitted and could not be separated, which did not meet the requirements and could not accurately quantify the content of isomers 2 and 3 impurities. This shows that the ratio of the mobile phase needs to be within a specific range to achieve the separation of mabaloxavir and its isomeric impurities.

[0198] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for simultaneously detecting three isomeric impurities in mabaloxavir raw materials and preparations thereof, characterized in that: The detection was performed by high performance liquid chromatography, and the conditions of the high performance liquid chromatography were as follows: A CHIRALPAK AD-H column was used, and a mixed solution of n-hexane, ethanol, and diethylamine in a volume ratio of (68-72): (28-32): (0.1) was used as the mobile phase. After isocratic elution, detection was performed; Wherein, the structural formulas of the three isomers are as follows:

2. The method according to claim 1, characterized in that: When determining the mabaloxavir, the test solution is prepared with ethanol.

3. The method according to claim 1, characterized in that: In the high performance liquid chromatography conditions, the CHIRALPAK AD-H chromatographic column has a specification of 4.6 mm×250 mm, 5 μm.

4. The method according to claim 1, characterized in that: In the high performance liquid chromatography conditions, the detection wavelength is 250-260 nm.

5. The method according to claim 1, characterized in that: In the high performance liquid chromatography conditions, the column temperature is 20-30°C.

6. The method according to claim 1, characterized in that: Under the conditions of HPLC, the injection volume was 10 to 30 μL.

7. The method according to claim 1, characterized in that: In the high performance liquid chromatography conditions, the flow rate of the mobile phase was 0.9 to 1.1 mL / min.

8. The method according to any one of claims 1 to 7, characterized in that: The quantification limits of the three isomers were all 0.02%.

9. The method according to any one of claims 1 to 7, characterized in that: The detection limits of the three isomers were all 0.01%.

10. Use of the method according to any one of claims 1 to 7 in quality control of mabaloxavir.

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