A method for detecting enantiomers and diastereomers of mabaloxavir intermediates

Abstract

The present invention belongs to the technical field of drug analysis, and relates to a method for detecting enantiomers and diastereomers of a mabaloxavir intermediate. The present invention uses a high performance liquid chromatography method, a chiral chromatographic column, and a mixed solution of lower alkanes, lower alcohols and additives as a mobile phase to achieve rapid separation of baloxavir intermediate isomers, and can also exclude the interference of its precursor impurities MBL-1 and SM2, thereby filling the technical gap of mabaloxavir intermediate isomer detection. The detection method has the characteristics of practicality, strong specificity and high accuracy, and provides technical support for the quality control of intermediates and final products in the synthesis process of mabaloxavir, thereby improving the safety of baloxavir clinical medication.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis and relates to a method for detecting enantiomers and diastereomers of a mabaloxavir intermediate. Background Art

[0002] Mabaloxavir is an endonuclease that can act on the polymerase acid protein necessary for the transcription of the influenza virus genome, can block the transcription of the virus's own mRNA, and has current antiviral activity and safety.

[0003] Chinese invention patent CN109311911B discloses the following synthetic route:

[0004]

[0005] Chinese invention patent application CN114539285A discloses the following synthesis route:

[0006]

[0007] Chinese invention patent CN116082361B discloses the following synthetic route:

[0008]

[0009] In the above-disclosed mabaloxavir synthesis routes, it is necessary to first synthesize the intermediate (R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-7-(hexyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (mabaloxavir intermediate MBL-2, whose chemical structure is as follows), and then synthesize mabaloxavir.

[0010]

[0011] Mabaloxavir intermediate MBL-2 has two chiral centers. Four optical isomers with different stereo configurations are generated simultaneously during chemical synthesis. These isomers are listed as isomeric impurities of Mabaloxavir intermediate MBL-2, which are:

[0012] Enantiomeric impurity: (S)-12-((R)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-7-(hexyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (MBL-2-Y1).

[0013] Diastereomeric impurities: (S)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-7-(hexyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (MBL-2-Y2) and (R)-12-((R)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-7-(hexyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (MBL-2-Y3).

[0014] The structural formulas of the isomeric impurities are as follows:

[0015]

[0016] The previous compound in the synthesis of the mabaloxavir intermediate MBL-2 is (R)-7-(hexyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (MBL-1) and 7,8-difluoro-6,11-dihydro-dibenzo[B,E]thieno-11-one (SM2). These two compounds may remain in the mabaloxavir intermediate MBL-2, which may affect the detection of mabaloxavir intermediate isomers.

[0017]

[0018] When preparing Mabaloxavir API, it is necessary to control the impurities of its intermediate isomers. Control research on the intermediate isomers of Mabaloxavir will be beneficial to the quality control of the intermediates and final products in the synthesis process of Mabaloxavir, and help improve product quality, thereby ensuring the quality of Mabaloxavir API. At present, there is no literature reporting the detection method of Mabaloxavir intermediate isomers, and there is no research involving the interference of residual related impurities in the synthesis process of intermediate isomers in the detection process. Summary of the invention

[0019] In view of the problems existing in the prior art, the present invention provides a method for detecting enantiomers and diastereomers of a mabaloxavir intermediate, which fills the technical gap in the detection of isomers of a mabaloxavir intermediate. The detection method can quickly detect and separate the isomers of a baloxavir intermediate and eliminate the interference of the precursor impurities MBL-1 and SM2 remaining in the synthesis process thereof, and has the characteristics of practicality, strong specificity, high accuracy, etc., provides technical support for the intermediate control of the mabaloxavir synthesis process and the control of the quality of the final product, and improves the safety of the clinical use of baloxavir.

[0020] To achieve the above object, the technical solution of the present invention is as follows:

[0021] The present invention provides a method for detecting enantiomers and diastereomers of a mabaloxavir intermediate, wherein a high performance liquid chromatography method is used, a chiral chromatographic column is used, and a mixed solution of a lower alkane, a lower alcohol and an additive is used as a mobile phase to detect the mabaloxavir intermediate; the additive is an organic acid or an organic base;

[0022] The enantiomer is (S)-12-((R)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-7-(hexyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (MBL-2-Y1), the structure of which is shown in Formula I;

[0023]

[0024] The diastereomer is (S)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-7-(hexyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (MBL-2-Y2), having a structure as shown in Formula Ⅱ; and (R)-12-((R)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-7-(hexyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (MBL-2-Y3), the structure of which is shown in Formula III;

[0025]

[0026] Furthermore, the lower alkane is selected from C6-C7 alkanes.

[0027] Preferably, the lower alkane is hexane and / or heptane.

[0028] More preferably, the lower alkane is n-hexane and / or n-heptane.

[0029] More preferably, the lower alkane is n-heptane.

[0030] Furthermore, the lower alcohol is selected from C1-C2 alcohols.

[0031] Preferably, the lower alcohol is methanol and / or ethanol.

[0032] More preferably, the lower alcohol is ethanol.

[0033] In some preferred embodiments of the present invention, the lower alkane is n-hexane, and the lower alcohol is ethanol; the volume ratio of n-hexane to the lower alcohol is 60:40.

[0034] Furthermore, the chromatographic column conditions also include: a detection wavelength of 254 nm, and / or a column temperature of 25-35°C.

[0035] Preferably, the column temperature is 35°C.

[0036] In some other preferred embodiments of the present invention, the lower alkane is n-heptane, and the lower alcohol is ethanol; preferably, the volume ratio of n-heptane to lower alcohol is 69:31-70:30; the volume ratio of n-heptane to lower alcohol is preferably 69:31.

[0037] Furthermore, the chromatographic column conditions also include: a detection wavelength of 312 nm, and / or a column temperature of 20-25°C.

[0038] Preferably, the column temperature is 20°C.

[0039] Furthermore, the organic acid includes but is not limited to trifluoroacetic acid.

[0040] Furthermore, the organic base includes but is not limited to ethylenediamine and / or triethylamine; preferably ethylenediamine.

[0041] In some preferred embodiments of the present invention, the additive is an organic base, more preferably ethylenediamine.

[0042] Furthermore, the volume percentage of the additive in the mobile phase is 0.05-0.5%.

[0043] Preferably, the additive accounts for 0.1% by volume of the mobile phase.

[0044] Furthermore, the filler of the chiral chromatographic column is amylose-tris(3,5-dimethylphenylcarbamate).

[0045] Furthermore, the chiral chromatographic column is selected from CHIRALPAK IA, with a specification of 4.6 mm×250 mm, 5 μm, or a chromatographic column with equivalent performance.

[0046] Furthermore, the HPLC conditions also include: a flow rate of 0.5-1.5 mL / min; preferably 0.5-1.0 mL / min; and more preferably 1.0 mL / min.

[0047] Furthermore, the HPLC conditions also include: an injection volume of 5-20 μL; preferably 5-10 μL; and more preferably 10 μL.

[0048] Furthermore, the method can also detect impurities related to the intermediates of Mabaloxavir; the related impurities include: (R)-7-(hexyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione (MBL-1), the structure of which is shown in Formula IV; and / or 7,8-difluoro-6,11-dihydro-dibenzo[B,E]thieno-11-one (SM2), the structure of which is shown in Formula V;

[0049]

[0050] In some specific embodiments of the present invention, the method comprises the following specific steps:

[0051] (1) Preparation of system suitability solution;

[0052] (2) Preparation of test solution;

[0053] (3) Determination: Accurately measure the system suitability solution and the test sample solution and inject them into the high performance liquid chromatograph respectively, and record the chromatogram.

[0054] Furthermore, step (1) is specifically as follows: taking appropriate amounts of the standard mabaloxavir intermediate MBL-2, and its enantiomers MBL-2-Y1, diastereomers MBL-2-Y2 and MBL-2-Y3, adding methanol-ethanol (50:50) and ultrasonically dissolving them to prepare a solution containing 1 mg of MBL-2 and 10 μg of each of MBL-2-Y1, MBL-2-Y2 and MBL-2-Y3 per 1 mL.

[0055] Furthermore, the step (2) is specifically as follows: taking an appropriate amount of the test sample mabaloxavir intermediate, adding methanol-ethanol (50:50) and ultrasonically dissolving it to prepare a solution containing 1 mg of the baloxavir intermediate MBL-2 per 1 mL.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] The present invention fills the technical gap in the detection of mabaloxavir intermediate isomers. The detection method can quickly detect and separate baloxavir intermediate isomers and eliminate the interference of its precursor impurities MBL-1 and SM2, providing technical support for the intermediate control of the mabaloxavir synthesis process and the control of the quality of the final product.

[0058] The present invention further improves the separation of isomers and improves the tailing phenomenon of spectral peaks by optimizing the mobile phase composition and the distribution ratio of each group, as well as adjusting the column temperature and the detection wavelength. It has the characteristics of strong practicality, strong specificity and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is a typical spectrum of blank solution for the detection of mabaloxavir intermediate isomers;

[0060] Figure 2 It is the solution spectrum of the suitability of the mabaloxavir intermediate isomer detection system;

[0061] Figure 3 The HPLC spectrum of the diethylamine additive in Example 2;

[0062] Figure 4 The HPLC spectrum of the trifluoroacetic acid additive in Example 2;

[0063] Figure 5 The HPLC spectrum of the triethylamine additive in Example 2;

[0064] Figure 6 HPLC spectrum of the mobile phase system of system 2 in Example 3;

[0065] Figure 7 The HPLC profile of the mobile phase ratio of system 2 in Example 4;

[0066] Figure 8 The column temperature HPLC spectrum of system 2 in Example 5;

[0067] Fig. 9 The HPLC profile of the mobile phase ratio of system 2 in Example 6;

[0068] Fig.10 The HPLC profile of the mobile phase ratio of system 4 in Example 6;

[0069] Fig.11 The column temperature HPLC spectrum of system 1 in Example 7;

[0070] Fig.12 This is the column temperature HPLC spectrum of system 4 in Example 7. DETAILED DESCRIPTION

[0071] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of 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. It is worth noting that the raw materials used in the present invention are all common commercial products, and their sources are not specifically limited.

[0072] Mabaloxavir intermediate MBL-2 standard product (article number B15902), MBL-2-Y1 standard product (article number B15895), MBL-2-Y3 standard product (article number B41481), and MBL-1 standard product (article number B15901) used in the examples of the present invention were all purchased from SINCO PHARMACHEM; MBL-2-Y2 standard product (article number C4X-210814) was purchased from CATO.

[0073] The test sample, mabaloxavir intermediate (batch number: TMB-2-18-0204), was obtained from Boji Pharmaceutical Co., Ltd.

[0074] Example 1: System suitability test

[0075] (1) Preparation of system suitability solution: Take appropriate amount of standard substances MBL-2, MBL-2-Y1, MBL-2-Y2, MBL-2-Y3 and MBL-1, add methanol-ethanol (50:50) and dissolve by ultrasonication to prepare a solution containing 1 mg of MBL-2 and 10 μg each of MBL-2-Y1, MBL-2-Y2, MBL-2-Y3 and MBL-1 per 1 mL;

[0076] (2) A blank solution (a methanol-ethanol mixed solvent with a volume ratio of 50:50) and the above system suitability solution were respectively injected into a high performance liquid chromatograph for detection. The chromatographic conditions were as follows:

[0077] Chromatographic column: CHIRALPAK IA; 4.6 mm × 250 mm, 5 μm;

[0078] Mobile phase: n-heptane-anhydrous ethanol-diethylamine (69:31:0.1);

[0079] Flow rate: 1.0 mL / min;

[0080] Column temperature: 30°C;

[0081] Detection wavelength: 312nm;

[0082] Injection volume: 10uL.

[0083] Results Figure 1 and Figure 2 It can be seen that the blank solution has no interference with the detection of mabaloxavir intermediates, MBL-2-Y1, MBL-2-Y2, MBL-2-Y3, and MBL-1. Figure 2 The results showed that: MBL-2-Y3 Rt = 7.943 min, MBL-2-Y2Rt = 9.831 min, MBL-1Rt = 10.965 min, MBL-2-Y1 Rt = 13.298 min, MBL-2Rt = 20.709 min, and there was no mutual interference, indicating that the detection method of the present invention has good system applicability.

[0084] Example 2: Selection of mobile phase additives

[0085] (1) Take an appropriate amount of the test sample mabaloxavir intermediate, add methanol-ethanol (50:50) and ultrasonically dissolve it to prepare a solution containing 1 mg of baloxavir intermediate per 1 mL;

[0086] (2) The test solution was tested according to the chromatographic conditions in Table 1. The spectral results of systems 1-3 are shown in Table 1. Figure 3-Figure 5 The retention time and separation results are shown in Table 2.

[0087] Table 1

[0088]

[0089] Table 2

[0090]

[0091]

[0092] The results showed that the type of additives had no significant effect on the retention time, separation and peak shape of baloxavir intermediates and their isomers. Both acidic and basic additives were suitable for the detection of isomers of baloxavir intermediates.

[0093] Example 3: Screening of mobile phase system

[0094] (1) Take an appropriate amount of the test sample mabaloxavir intermediate, add methanol-ethanol (50:50) and ultrasonically dissolve it to prepare a solution containing 1 mg of baloxavir intermediate per 1 mL;

[0095] (2) The test solution was tested according to the chromatographic conditions in Table 3. The spectrum results of the optimal system (system 2) are shown in Figure 6 The retention time and separation results are shown in Table 4.

[0096] Table 3

[0097]

[0098] Table 4

[0099]

[0100] The results showed that when n-heptane was selected as the lower alkane in the mobile phase, the peak shape of MBL-2-Y2 could be significantly improved compared with n-hexane, which would help to improve the accuracy of the analytical method.

[0101] Example 4: Optimization of the ratio of mobile phase (n-hexane-ethanol-diethylamine)

[0102] (1) Take an appropriate amount of the test sample mabaloxavir intermediate, add methanol-ethanol (50:50) and ultrasonically dissolve it to prepare a solution containing 1 mg of baloxavir intermediate per 1 mL;

[0103] (2) The test solution was tested according to the chromatographic conditions in Table 5. The spectrum results of the optimal system (system 2) are shown in Figure 7 The retention time and separation results are shown in Table 6.

[0104] Table 5

[0105]

[0106] Table 6

[0107]

[0108] The results showed that when the mobile phase was a mixture of n-hexane-ethanol-diethylamine, the change in the proportion of ethanol would affect the separation of MBL-2-Y2 from MBL-2-Y1 and MBL-2-Y3. Reducing the proportion of ethanol would help improve the separation of MBL-2-Y2 from MBL-2-Y1. MBL-1 appeared between MBL-2-Y2 and MBL-2-Y1. The proportion of ethanol in the mobile phase would affect the separation of MBL-1 from adjacent peaks. SM2 had a shorter retention time under the above conditions and would not interfere with the detection. MBL-1 had a shorter retention time under the above conditions. When the mobile phase is hexane-ethanol-diethylamine (55:45:0.1), there will be a large overlap with the peak position of MBL-2-Y2. When the mobile phase is n-hexane-ethanol-diethylamine (65:35:0.1), there will be a large overlap with the peak position of MBL-2-Y1. When it is necessary to consider excluding the interference of the starting materials MBL-1 and SM2 on the detection of baloxavir intermediate isomers, when the mobile phase is n-hexane-ethanol-diethylamine, the preferred mobile phase ratio is 60:40:0.1. At this time, MBL-1 has the least interference with the detection of MBL-2-Y2 and MBL-2-Y1.

[0109] In addition, the mobile phase system of system 1 in Example 3 is the same as that of system 2 in Example 4, but the detection wavelength and column temperature are different, and the results are also quite different. Next, a screening experiment is conducted on the column temperature when the mobile phase is n-hexane-ethanol-diethylamine.

[0110] Example 5: Column temperature screening when the mobile phase is n-hexane-ethanol-diethylamine

[0111] (1) Take an appropriate amount of the test sample mabaloxavir intermediate, add methanol-ethanol (50:50) and ultrasonically dissolve it to prepare a solution containing 1 mg of baloxavir intermediate per 1 mL;

[0112] (2) The test solution was tested according to the chromatographic conditions in Table 7. The spectrum results of the optimal system (system 2) are shown in Figure 8 The retention time and separation results are shown in Table 8.

[0113] Table 7

[0114]

[0115] Table 8

[0116]

[0117] The results showed that when the mobile phase system was n-hexane-ethanol-diethylamine, the change of column temperature would affect the separation of MBL-2-Y2 from MBL-2-Y1 and MBL-2-Y3, and lowering the column temperature would help improve the separation of MBL-2-Y2 from MBL-2-Y1. Since MBL-1 peaked between MBL-2-Y2 and MBL-2-Y1, the change of column temperature in the mobile phase would affect the separation of MBL-1 from adjacent peaks, and lowering the column temperature would improve the separation of MBL-1 from MBL-2-Y3. The separation degree of -2-Y1 is reduced, and the separation degree of MBL-2-Y2 is increased. Too high or too low temperature will cause MBL-1 to interfere with the detection of MBL-2-Y2 or MBL-2-Y1. The retention time of SM2 is short under the above conditions and will not interfere with the detection. When it is necessary to consider excluding the interference of the starting materials MBL-1 and SM2 on the detection of baloxavir intermediate isomers, the preferred column temperature is 25-35°C, and the most preferred column temperature is 35°C, which can meet the separation requirements.

[0118] Example 6: Optimization of the ratio of mobile phase (n-heptane-ethanol-diethylamine)

[0119] (1) Take an appropriate amount of the test sample mabaloxavir intermediate, add methanol-ethanol (50:50) and ultrasonically dissolve it to prepare a solution containing 1 mg of baloxavir intermediate per 1 mL;

[0120] (2) The test solution was tested according to the chromatographic conditions in Table 9. The spectral results of system 2 and system 4 are shown in Fig. 9and Fig.10 The retention time and separation results are shown in Table 10.

[0121] Table 9

[0122]

[0123] Table 10

[0124]

[0125]

[0126] The results showed that when the mobile phase was a mixture of n-heptane-ethanol-diethylamine, the ethanol ratio varied within a certain range and had no significant effect on the separation of MBL-2-Y2 from MBL-2-Y1 and MBL-2-Y3. Since MBL-1 peaked between MBL-2-Y2 and MBL-2-Y1, the ethanol ratio in the mobile phase would affect the separation of MBL-1 from adjacent peaks. Reducing the ethanol ratio would improve the separation of MBL-1 from MBL-2-Y2, but would reduce the separation of MBL-1 from MBL-2-Y1. degree, a higher ethanol ratio and a lower ethanol ratio will cause the peak positions of MBL-1 and MBL-2-Y2 or MBL-2-Y1 to overlap, thereby affecting the specificity of the analytical method, while SM2 has a shorter retention time under the above conditions and will not interfere with the detection. If it is necessary to consider excluding the interference of the starting materials MBL-1 and SM2 on the detection of baloxavir intermediate isomers, the mobile phase is n-heptane-ethanol-diethylamine, and the preferred mobile phase ratio is 65:35:0.1-70:30:0.1, and the most preferred ratio is 69:31:0.1.

[0127] Example 7: Column temperature screening when the mobile phase is n-heptane-ethanol-diethylamine

[0128] (1) Take an appropriate amount of the test sample mabaloxavir intermediate, add methanol-ethanol (50:50) and ultrasonically dissolve it to prepare a solution containing 1 mg of baloxavir intermediate per 1 mL;

[0129] (2) The test solution was tested according to the chromatographic conditions in Table 11. The spectral results of system 1 and system 4 are shown in Table 11. Fig.11 and Fig.12 The retention time and separation results are shown in Table 12.

[0130] Table 11

[0131]

[0132] Table 12

[0133]

[0134] The results showed that when the mobile phase system was n-heptane-ethanol-diethylamine, when the column temperature was lowered, the separation between the peaks of MBL-1 and MBL-2-Y1 gradually increased, while the separation between the peaks of MBL-1 and MBL-2-Y2 gradually decreased, and the separation trend of the two peaks was opposite to that of the MBL-1 peak. In the column temperature range of 20℃-25℃, the separation between the adjacent peaks of MBL-2-Y3, MBL-2-Y2, MBL-1, MBL-2-Y1 and MBL-2 was greater than 1.2, but since MBL-1 peaked between MBL-2-Y2 and MBL-2-Y1, the change of column temperature in the mobile phase would affect the separation between MBL-1 and the adjacent peaks. Lowering the column temperature would reduce the separation between MBL-1 and MBL-2-Y2, which was different from that of MBL-2-Y1. The separation degree increases. Both higher and lower column temperatures will cause the peak positions of MBL-1 and MBL-2-Y2 or MBL-2-Y1 to overlap, thereby affecting the specificity of the analytical method. SM2 has a shorter retention time under the above conditions and will not interfere with the detection. When it is necessary to consider excluding the interference of the starting materials MBL-1 and SM2 on the detection of baloxavir intermediate isomers, the preferred column temperature is 20-25°C, and the most preferred is 20°C, which can meet the separation requirements.

[0135] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for detecting enantiomers and diastereomers of a mabaloxavir intermediate, characterized in that: The mabaloxavir intermediate is detected by high performance liquid chromatography using a chiral chromatographic column and a mixture of lower alkanes, lower alcohols and additives as a mobile phase; the additive is an organic acid or an organic base; The enantiomer is (S)-12-((R)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-7-(hexyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione, and the structure is shown in Formula I; The diastereoisomers are (S)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-7-(hexyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione, (R)-12-((R)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-7-(hexyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione, 12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione, the structure of which is shown in Formula II; and (R)-12-((R)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-7-(hexyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione, the structure of which is shown in Formula III; 2. The detection method according to claim 1, characterized in that: The lower alkane is selected from C6-C7 alkanes, including hexane and / or heptane; preferably n-hexane and / or n-heptane; the lower alcohol is selected from C1-C2 alcohols, including methanol and / or ethanol; preferably ethanol.

3. The detection method according to claim 2, characterized in that: The lower alkane is n-hexane, the volume ratio of the lower alkane to the lower alcohol is 60:40, and / or the detection wavelength is 254 nm, and / or the column temperature is 25-35° C.; Preferably, the column temperature is 35°C.

4. The detection method according to claim 2, characterized in that: The lower alkane is n-heptane, the volume ratio of the lower alkane to the lower alcohol is 65:35-70:30, and / or the detection wavelength is 312 nm, and / or the column temperature is 20-25° C.; The volume ratio of the lower alkane to the lower alcohol is preferably 69:31; and / or the column temperature is preferably 20°C.

5. The detection method according to claim 1, characterized in that: The volume percentage of the additive in the mobile phase is 0.05-0.5%.

6. The detection method according to claim 1, characterized in that: The organic acid is selected from trifluoroacetic acid; the organic base is selected from ethylenediamine and / or triethylamine; preferably ethylenediamine.

7. The detection method according to claim 1, characterized in that: The filler of the chiral chromatographic column is amylose-tris(3,5-dimethylphenylcarbamate); the chiral chromatographic column is preferably a chromatographic column of model CHIRALPAK IA.

8. The detection method according to claim 1, characterized in that: The HPLC conditions also include: a flow rate of 0.5-1.5 mL / min; preferably 0.5-1.0 mL / min; and more preferably 1.0 mL / min.

9. The detection method according to claim 1, characterized in that: The HPLC conditions also include: the injection volume is 5-20 μL; preferably 5-10 μL; more preferably 10 μL.

10. The detection method according to any one of claims 1 to 9, characterized in that: The method can also detect impurities related to the intermediate of mabaloxavir; the related impurities include: (R)-7-(hexyloxy)-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyrido[2,1-f][1,2,4]triazine-6,8-dione, the structure of which is shown in Formula IV; and / or 7,8-difluoro-6,11-dihydro-dibenzo[B,E]thieno-11-one, the structure of which is shown in Formula V;

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