A method for determining related substances of Baloxavir marboxil

Abstract

The present invention relates to a separation and detection method of related substances, belonging to the field of analytical chemistry. The method specifically relates to separation and determination of Baloxavir marboxil related substances by liquid chromatography, characterized in that a chromatographic column with octadecyl combined with pentafluorophenyl as filler, a buffer salt in a certain pH range as phase A, and a mixed solution of acetonitrile and lower alcohol as phase B as mobile phase. The method of the present invention is simple, rapid and accurate.

Description

Technical Field

[0001] The invention relates to the field of analytical chemistry, and in particular to a method for determining baloxavir marboxil-related substances. Background Art

[0002] XOFLUZA (baloxavir marboxil) is an antiviral PA endonuclease inhibitor that can inhibit the initiation of mRNA synthesis and can be used to treat influenza A and B in adults and children. The chemical name of Baloxavir marboxil is ({(12aR)-12-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiazol-11-yl]-6,8-dioxo-3,4,6,8,12,12a-hexahydro-1H-[1,4]oxoazino[3,4-c]pyrido[2,1-f][1,2,4]triazin-7-yl}oxy)methyl carbonate, with a molecular weight of 571.55 and a molecular formula of C 27 H 23 F 2 N 3 O 7 S, chemical structure is shown below:

[0003]

[0004] There are 2 chiral centers in the Baloxavir marboxil molecule, and 4 optical isomers with different stereo configurations can be generated simultaneously in chemical synthesis, and these isomers are listed as impurities of Baloxavir marboxil; quality control is required in the production of Baloxavir marboxil, but the United States Pharmacopoeia USP, the European Pharmacopoeia EP and the Chinese Pharmacopoeia Ch.P. do not include Baloxavir marboxil at present, and the Japanese Pharmacopoeia JP includes a method for detecting related substances of Baloxavir marboxil, using a 0.1% formic acid-acetonitrile system and a Kinetex XB-C18 (4.6x100mm, 2.6um) chromatographic column, which has poor detection capability for the impurities of the Baloxavir marboxil sample of the present invention, and impurities are wrapped in the main peak. In order to better and more accurately control the related substances in the product and ensure the quality of the bulk drug, the present invention independently develops an analytical method suitable for the determination of related substances of Baloxavir marboxil. The method of the present invention can effectively separate Baloxavir marboxil from other 9 impurities, has good specificity, high sensitivity and reliable accuracy, and can be used for quality control of Baloxavir marboxil. Summary of the invention

[0005] The object of the present invention is to provide a method for separating and determining related substances of Baloxavir marboxil using a chiral chromatographic column, thereby achieving separation and determination of related substances of Baloxavir marboxil.

[0006] A method for separating and determining baloxavir marboxil related substances by liquid chromatography, characterized in that it comprises: using a chromatographic column with octadecyl combined with pentafluorophenyl as filler, using a buffer salt in a certain pH range as phase A, and using a mixed solution of acetonitrile and lower alcohol as phase B as mobile phase.

[0007] In some embodiments, the chromatographic column can be selected from ACE Excel 3, C18-PFP, 4.6x100mm, 3μm or C18 combined with pentafluorophenyl column. In some embodiments, the chiral chromatographic column is ACE Excel 3, C18-PFP, 4.6x100mm, 3μm column; the sales manufacturer is Pheromon.

[0008] In some embodiments, the buffer salt in phase A includes a 5% acetonitrile solution of potassium dihydrogen phosphate, sodium phosphate, ammonium phosphate or a combination thereof. In certain embodiments, the buffer salt in phase A is a 5% acetonitrile solution of potassium dihydrogen phosphate.

[0009] In some embodiments, the certain pH value range in phase A is a pH value of 2.0-6.2. In some embodiments, the pH value in phase A is adjusted to 2.0-6.2 with phosphoric acid; in some embodiments, the pH value is adjusted to 2.0-3.0 with phosphoric acid; in some embodiments, the pH value is adjusted to 2.0 with phosphoric acid.

[0010] In some embodiments, the lower alcohol in phase B is methanol or ethanol or a combination thereof; in certain embodiments, the lower alcohol is methanol.

[0011] In some embodiments, the volume ratio of acetonitrile to lower alcohol in the mobile phase is 1: 1 to 9: 1. In some embodiments, the volume ratio of acetonitrile to lower alcohol in the mobile phase is 3: 1 to 7: 1. In some embodiments, the volume ratio of acetonitrile to lower alcohol in the mobile phase is 3: 1, which is more conducive to separation and determination.

[0012] In some embodiments, the separation and determination method described in the present invention can be implemented according to the following steps:

[0013] 1) Take an appropriate amount of Baloxavir marboxil or a sample containing Baloxavir marboxil, and use a certain amount of a mixed solution of acetonitrile and water as a diluent to dissolve the sample;

[0014] 2) Set the instrument parameters: flow rate of the mobile phase, detection wavelength, and column box temperature of the chromatographic column;

[0015] 3) taking a certain amount of the solution in step 1) and injecting it into a high performance liquid chromatograph to complete the separation and determination of the related substances of Baloxavir marboxil.

[0016] The Baloxavir marboxil in step 1) may be of any purity.

[0017] The Baloxavir marboxil in step 1) may be of any optical purity.

[0018] Step 1) The volume ratio (V / V) of acetonitrile to water in the diluent is 10:90 to 100:0. In some embodiments, in the diluent, the volume ratio (V / V) of acetonitrile to water is 50:50.

[0019] The diluent contains 0.3mg to 5mg of the Baloxavir marboxil sample per 1ml of the diluent. In some embodiments, the diluent in step 1) contains 1.0mg of the Baloxavir marboxil sample per 1ml of the diluent.

[0020] The flow rate of the mobile phase is 0.5 ml / min to 2.0 ml / min. In some embodiments, the flow rate of the mobile phase is 0.5 ml / min; in some embodiments, the flow rate of the mobile phase is 0.8 ml / min; in some embodiments, the flow rate of the mobile phase is 1.0 ml / min.

[0021] The detection wavelength is 200 nm to 230 nm. In some embodiments, the detection wavelength is 210 nm.

[0022] The column box temperature of the chromatographic column is 40° C. to 70° C. In some embodiments, the column box temperature of the chromatographic column is 40° C.; in some embodiments, the column box temperature of the chromatographic column is 60° C.; in some embodiments, the column box temperature of the chromatographic column is 70° C.

[0023] The sample solution injection volume is 4 μl to 20 μl. In some embodiments, the sample solution injection volume is 5 μl. In some embodiments, the sample solution injection volume is 10 μl.

[0024] In some embodiments, the separation and determination method described in the present invention can be implemented as follows:

[0025] 1) Take an appropriate amount of Baloxavir marboxil or a sample containing Baloxavir marboxil, use a certain amount of a mixed solvent of acetonitrile and water as a diluent to dissolve the sample, and prepare a sample solution containing 0.3mg to 5mg of Baloxavir marboxil per 1ml;

[0026] 2) Set the flow rate of the mobile phase to 0.5 ml / min to 2.0 ml / min, the detection wavelength to 205 nm to 270 nm, and the column box temperature to 40°C to 70°C;

[0027] 3) Take 4 μl to 20 μl of the sample solution of step 1) and inject it into a high performance liquid chromatograph to complete the separation and determination of baloxavirmarboxil related substances.

[0028] In the method provided by the present invention, the high performance liquid chromatograph can be an American Agilent 1260 high performance liquid chromatography system and workstation.

[0029] In some embodiments, the separation and determination method described in the present invention uses a chromatographic column of ACE Excel 3, C18-PFP, 4.6x100mm, 3μm, and the mobile phase A is a buffer salt with a certain pH value range, and the B phase is a mixed solution of acetonitrile and lower alcohol; the pH value range of phase A is 2.0 to 6.2, the buffer salt is a 5% acetonitrile solution of potassium dihydrogen phosphate, sodium phosphate, ammonium phosphate or potassium phosphate or a combination thereof, and the volume ratio (V / V) of acetonitrile and lower alcohol in phase B is approximately 1:9 to 9:1.

[0030] In some embodiments, the separation and determination method described in the present invention uses a chromatographic column of ACE Excel 3, C18-PFP, 4.6x100mm, 3μm, and the mobile phase A is a 5% acetonitrile solution of potassium dihydrogen phosphate with a concentration of 2.0 to 6.2, and the phase B is a mixed solution of acetonitrile and lower alcohol; the volume ratio (V / V) of acetonitrile and lower alcohol in phase B is approximately 1:9 to 9:1.

[0031] In some embodiments, the separation and determination method described in the present invention uses a chromatographic column of ACE Excel 3, C18-PFP, 4.6x100mm, 3μm, and the mobile phase A is a 5% acetonitrile solution of potassium dihydrogen phosphate with a pH value of 2.0 to 6.2, and the phase B is a mixed solution of acetonitrile and methanol; the volume ratio (V / V) of acetonitrile and methanol in phase B is approximately 1:9 to 9:1.

[0032] In some embodiments, the separation and determination method described in the present invention uses a chiral chromatographic column of ACE Excel3, C18-PFP,

[0033] 4.6x100mm, 3μm, mobile phase A is a 5% acetonitrile solution of potassium dihydrogen phosphate with a pH value of 2.0 to 6.2, and phase B is a mixed solution of acetonitrile and methanol; the volume ratio (V / V) of acetonitrile and methanol in phase B is about 3:1.

[0034] By adopting the separation method of the present invention, the time for separating and detecting the related substances of Baloxavir marboxil is within 50 minutes.

[0035] In the above or below, regardless of whether the words "about" or "approximately" are used, all the numbers disclosed herein are approximate values. The value of each number may differ by 1%, 2%, 5%, 7%, 8% or 10%.

[0036] The invention adopts a chromatographic column with octadecyl combined with pentafluorophenyl as filler, a buffer salt in a certain pH range as phase A, and a mixed solution of acetonitrile and lower alcohol as phase B as mobile phase, so as to effectively separate Baloxavir marboxil from other 9 impurities, with a separation degree of 1.5 or more, 2.5 or more, or 5 or more, and complete baseline separation. The method of the invention has good specificity, high sensitivity, good accuracy, and good stability indication ability, so as to accurately and effectively control the quality of related substances of Baloxavir marboxil.

[0037] In the present invention, impurity BVM0207 represents ((12aR)-12-((11S)-7,8-difluoro-5-oxo-6,11-dihydrodibenzo[b,e]thiazin-11-yl)-6,8-dioxo-3,4,6,8,12,12a-hexahydro-1H-[1,4]oxazine[3,4-c]pyridinium[2,1-f][1,2,4]triazine-7-yl)methyl carbonate;

[0038] In the present invention, impurity BVM0211 represents (R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-7-methoxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyridine[2,1-f][1,2,4]triazine-6,8-dione;

[0039] In the present invention, impurity BVM0206A represents (((R)-12-((S)-7-fluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-6,8-dioxo-3,4,6,8,12,12a-hexahydro-1H-[1,4]oxazine[3,4-c]pyridinium[2,1-f][1,2,4]triazin-7-yl)oxy)methyl carbonate;

[0040] In the present invention, impurity BVM0212 represents (R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-6,8-dioxo-3,4,6,8,12,12a-hexahydro-1H-[1,4]oxazine[3,4-c]pyridinium[2,1-f][1,2,4]triazine-7-yl methyl carbonate;

[0041] In the present invention, impurity BVM0213 represents (((R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-6,8-dioxo-3,4,6,8,12,12a-hexahydro-1H-[1,4]oxazine[3,4-c]pyridin[2,1-f][1,2,4]triazin-7-yl)oxy)methyl carbonate;

[0042] In the present invention, the impurity BVM diastereomer represents (((S)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-6,8-dioxo-3,4,6,8,12,12a-hexahydro-1H-[1,4]oxazine[3,4-c]pyridin[2,1-f][1,2,4]triazin-7-yl)oxy)methyl carbonate ((((S)-12-((R)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-6,8-dioxo-3,4,6,8,12,12a-hexahydro-1H-[1,4]oxazine[3,4-c]pyridin[2,1-f][1,2,4]triazin-7-yl)oxy)methyl carbonate);

[0043] In the present invention, impurity BVM01 represents (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]pyridine[2,1-f][1,2,4]triazine-6,8-dione;

[0044] In the present invention, impurity BVM02 represents (R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyridine[2,1-f][1,2,4]triazine-6,8-dione;

[0045] In the present invention, benzyl chloride means chloromethylbenzene. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The HPLC chromatogram of the blank solution of Baloxavir marboxil in Example 1;

[0047] Figure 2 HPLC chromatogram of the spiked sample of the Baloxavir marboxil test product in Example 1;

[0048] Figure 3 HPLC chromatogram of the blank solution of Baloxavir marboxil in Example 2;

[0049] Figure 4 HPLC chromatogram of the spiked sample of the Baloxavir marboxil test product in Example 2;

[0050] Figure 5 The HPLC chromatogram of the blank solution of Baloxavir marboxil in Example 3;

[0051] Figure 6 HPLC chromatogram of the spiked sample of the Baloxavir marboxil test product in Example 3;

[0052] Figure 7 The HPLC chromatogram of the blank solution of Baloxavir marboxil in Example 4;

[0053] Figure 8 HPLC chromatogram of the spiked sample of the Baloxavir marboxil test product in Example 4;

[0054] In the attached figure, the abscissa represents the retention time, in minutes (min); the ordinate represents the electrical signal, in mAU. DETAILED DESCRIPTION

[0055] The embodiment of the present invention discloses a method for separating and detecting related substances of Baloxavir marboxil. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method of the present invention has been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0056] In order to further understand the present invention, the present invention is described in detail below in conjunction with embodiments.

[0057] Specifications of instruments and chromatographic columns: American Agilent 1260 high performance liquid chromatography system and workstation; automatic injection;

[0058] Chromatographic columns Specification filler ACE Excel 3, C18-PFP, 4.6x100mm, 3μm Octadecyl-bonded pentafluorophenyl

[0059] Example 1-4: Separation and Detection of Baloxavir Marboxil Related Substances Using ACE Excel 3, C18-PFP, Chromatographic Column

[0060] Example 1

[0061] Instruments and conditions

[0062] Column: ACE Excel 3, C18-PFP, 4.6x100mm, 3μm;

[0063] Detector: DAD (ultraviolet detector), detection wavelength 210nm;

[0064] Flow rate: 1.0 mL / min;

[0065] Column temperature: 60°C;

[0066] Injection volume: 5 μL;

[0067] Mobile phase A: Buffer salt (1.36 g / L potassium dihydrogen phosphate, pH adjusted to 2.0 with phosphoric acid)

[0068] Mobile phase B: acetonitrile: methanol = 3:1 (V / V)

[0069] Gradient elution:

[0070] Time (min) Phase A (%) Phase B (%) 0 97 3 13 63 37 28 59 41 38 20 80 43 20 80 43.1 97 3 48 97 3

[0071] Running time: 50min;

[0072] Diluent / blank solution: acetonitrile: water = 50:50 (V:V);

[0073] Experimental procedures

[0074] Take about 25 mg of Baloxavir marboxil (hereinafter referred to as BVM) test sample, accurately weigh it into a 25 mL brown volumetric flask, dissolve it with a diluent by ultrasonic, take appropriate amounts of impurities BVM0207, impurity BVM0211, impurity BVM0206A, impurity BVM0212, impurity BVM0213, BVM diastereomers, BVM01, BVM02, and benzyl chloride and add them to the volumetric flask, dilute to the scale with a diluent, shake well, and use this as the test spiked sample solution.

[0075] Take the blank solution and the spiked sample respectively, perform HPLC analysis according to the above conditions, record the chromatogram, and the results are shown in Figure 1 , Figure 2 .

[0076] Figure 2The chromatographic peak with a retention time of 26.93 minutes is the chromatographic peak of BVM, and the retention times of impurities BVM0207, BVM0211, BVM0206A, BVM0212, BVM0213, BVM diastereomers, BVM01, BVM02, and benzyl chloride are 16.07min, 21.84min, 23.62min, 25.25min, 32.15min, 25.59min, 37.35min, 22.96min, and 21.15min, respectively. The separation degree between the main peak of BVM and the impurity of BVM0121 is 4.04.

[0077] Example 2

[0078] Instruments and conditions

[0079] Agilent 1260 high performance liquid chromatography system and workstation from the United States; with automatic sample injector;

[0080] Column: ACE Excel 3, C18-PFP, 4.6x100mm, 3μm;

[0081] Detector: DAD (ultraviolet detector), detection wavelength 210nm;

[0082] Flow rate: 1.0 mL / min;

[0083] Column temperature: 60°C;

[0084] Injection volume: 5 μL;

[0085] Mobile phase A: Buffer salt (1.36 g / L potassium dihydrogen phosphate, pH adjusted to 3.0 with phosphoric acid)

[0086] Mobile phase B: acetonitrile: methanol = 3:1 (V / V)

[0087] Gradient elution:

[0088] Time (min) Phase A (%) Phase B (%) 0 97 3 13 63 37 28 59 41 38 20 80 43 20 80 43.1 97 3 48 97 3

[0089] Running time: 50min;

[0090] Diluent / blank solution: acetonitrile: water = 50:50 (V:V);

[0091] Experimental procedures

[0092] Take about 25 mg of Baloxavir marboxil (hereinafter referred to as BVM) test sample, accurately weigh it into a 25 mL brown volumetric flask, dissolve it with a diluent by ultrasonic, take appropriate amounts of impurities BVM0207, impurity BVM0211, impurity BVM0206A, impurity BVM0212, impurity BVM0213, BVM diastereomers, BVM01, BVM02, and benzyl chloride and add them to the volumetric flask, dilute to the scale with a diluent, shake well, and use this as the test spiked sample solution.

[0093] Take the blank solution and the spiked sample respectively, perform HPLC analysis according to the above conditions, record the chromatogram, and the results are shown in Figure 3 , Figure 4 .

[0094] Figure 4 The chromatographic peak with a retention time of 26.89 minutes is the chromatographic peak of BVM, and the retention times of impurities BVM0207, BVM0211, BVM0206A, BVM0212, BVM0213, BVM diastereomers, BVM01, BVM02, and benzyl chloride are 16.16min, 21.91min, 23.65min, 25.18min, 32.11min, 28.51min, 37.51min, 22.97min, and 20.99min, respectively. The separation degree between the main peak of BVM and the impurity of BVM0121 is 4.06.

[0095] Example 3

[0096] Instruments and conditions

[0097] Agilent 1260 high performance liquid chromatography system and workstation from the United States; with automatic sample injector;

[0098] Column: ACE Excel 3, C18-PFP, 4.6x100mm, 3μm;

[0099] Detector: DAD (ultraviolet detector), detection wavelength 210nm;

[0100] Flow rate: 1.0 mL / min;

[0101] Column temperature: 40°C;

[0102] Injection volume: 5 μL;

[0103] Mobile phase A: Buffer salt (1.36 g / L potassium dihydrogen phosphate, pH adjusted to 2.0 with phosphoric acid)

[0104] Mobile phase B: acetonitrile: methanol = 3:1 (V / V)

[0105] Gradient elution:

[0106] Time (min) Phase A (%) Phase B (%) 0 97 3 13 63 37 28 59 41 38 20 80 43 20 80 43.1 97 3 48 97 3

[0107] Running time: 50min;

[0108] Diluent / blank solution: acetonitrile: water = 50:50 (V:V);

[0109] Experimental procedures

[0110] Take about 25 mg of Baloxavir marboxil (hereinafter referred to as BVM) test sample, accurately weigh it into a 25 mL brown volumetric flask, dissolve it with a diluent by ultrasonic, take appropriate amounts of impurities BVM0207, impurity BVM0211, impurity BVM0206A, impurity BVM0212, impurity BVM0213, BVM diastereomers, BVM01, BVM02, and benzyl chloride and add them to the volumetric flask, dilute to the scale with a diluent, shake well, and use this as the test spiked sample solution.

[0111] Take the blank solution and the spiked sample respectively, perform HPLC analysis according to the above conditions, record the chromatogram, and the results are shown in Figure 5 , Figure 6 .

[0112] Figure 6 The chromatographic peak with a retention time of 24.47 minutes is the chromatographic peak of BVM, and the retention times of impurities BVM0207, BVM0211, BVM0206A, BVM0212, BVM0213, BVM diastereomers, BVM01, BVM02, and benzyl chloride are 15.46min, 20.40min, 21.74min, 23.20min, 28.69min, 25.15min, 37.51min, 20.95min, and 19.77min, respectively. The separation degree between the main peak of BVM and the impurity of BVM0121 is 3.40.

[0113] Example 4

[0114] Instruments and conditions

[0115] Agilent 1260 high performance liquid chromatography system and workstation from the United States; with automatic sample injector;

[0116] Column: ACE Excel 3, C18-PFP, 4.6x100mm, 3μm;

[0117] Detector: DAD (ultraviolet detector), detection wavelength 210nm;

[0118] Flow rate: 0.8 mL / min;

[0119] Column temperature: 60°C;

[0120] Injection volume: 5 μL;

[0121] Mobile phase A: Buffer salt (1.36 g / L potassium dihydrogen phosphate, pH adjusted to 2.0 with phosphoric acid)

[0122] Mobile phase B: acetonitrile: methanol = 3:1 (V / V)

[0123] Gradient elution:

[0124] Time (min) Phase A (%) Phase B (%) 0 97 3 13 63 37 28 59 41 38 20 80 43 20 80 43.1 97 3 48 97 3

[0125] Running time: 50min;

[0126] Diluent / blank solution: acetonitrile: water = 50:50 (V:V);

[0127] Experimental procedures

[0128] Take about 25 mg of Baloxavir marboxil (hereinafter referred to as BVM) test sample, accurately weigh it into a 25 mL brown volumetric flask, dissolve it with a diluent by ultrasonic, take appropriate amounts of impurities BVM0207, impurity BVM0211, impurity BVM0206A, impurity BVM0212, impurity BVM0213, BVM diastereomers, BVM01, BVM02, and benzyl chloride and add them to the volumetric flask, dilute to the scale with a diluent, shake well, and use this as the test spiked sample solution.

[0129] Take the blank solution and the spiked sample respectively, perform HPLC analysis according to the above conditions, record the chromatogram, and the results are shown in Figure 7 , Figure 8 .

[0130] Figure 8 The chromatographic peak with a retention time of 23.97 minutes is the chromatographic peak of BVM, and the retention times of impurities BVM0207, BVM0211, BVM0206A, BVM0212, BVM0213, BVM01, BVM02, and benzyl chloride are 16.39min, 20.67min, 21.49min, 22.88min, 27.59min, 35.77min, 21.04min, and 19.74min, respectively. The separation degree between the main peak of BVM and the impurity of BVM0121 is 3.39.

Claims

1. A method for separating and determining related substances of Baloxavir marboxil by liquid chromatography, characterized in that: A chromatographic column with octadecyl combined with pentafluorophenyl as filler is used, a buffer salt with a certain pH value range is used as phase A, and a mixed solution of acetonitrile and lower alcohol is used as phase B as mobile phase; The mobile phase consists of phase A and phase B, phase A is a 5% acetonitrile solution of potassium dihydrogen phosphate in a certain pH range, and phase B is a mixed solvent of acetonitrile and methanol; In the phase A, the pH value range is from 2.0 to 6.2, and in the phase B, the volume ratio of acetonitrile to methanol is from 3:1 to 7:1; The related substances include impurity BVM0207, impurity BVM0211, impurity BVM0206A, impurity BVM0212, impurity BVM0213, impurity BVM diastereomers, impurity BVM01, impurity BVM02, benzyl chloride, wherein impurity BVM0207 represents ((12aR)-12-((11S)-7,8-difluoro-5-oxy-6,11-dihydrodiphenyl impurity BVM0211 represents (R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-7-methyl Oxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyridinium[2,1-f][1,2,4]triazine-6,8-dione, impurity BVM0206A represents (((R)-12-((S)-7-fluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-6,8-dioxo-3,4,6,8,12,12a-hexahydro-1H- [1,4]oxazine[3,4-c]pyridinium[2,1-f][1,2,4]triazin-7-yl)oxy)methyl carbonate, impurity BVM0212 represents (R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-6,8-dioxo-3,4,6,8,12,12a-hexahydro-1H-[1,4]oxazine[3,4-c]pyridinium [2,1-f][1,2,4]triazine-7-yl methyl carbonate, impurity BVM0213 represents (((R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-6,8-dioxo-3,4,6,8,12,12a-hexahydro-1H-[1,4]oxazine[3,4-c]pyridin[2,1-f][1,2,4]triazine-7-yl)oxy) methyl carbonate, impurity BVM non-para Enantiomers represent (((S)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-6,8-dioxo-3,4,6,8,12,12a-hexahydro-1H-[1,4]oxazin[3,4-c]pyridin[2,1-f][1,2,4]triazin-7-yl)oxy)methyl carbonate impurity BVM01 represents (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]pyridin[2,1-f][1,2,4]triazine-7-yl)oxy)methyl carbonate. ,4]oxazine[3,4-c]pyridine[2,1-f][1,2,4]triazine-6,8-dione, impurity BVM02 represents (R)-12-((S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11-yl)-7-hydroxy-3,4,12,12a-tetrahydro-1H-[1,4]oxazine[3,4-c]pyridine[2,1-f][1,2,4]triazine-6,8-dione, benzyl chloride represents chloromethylbenzene; The method comprises taking appropriate amounts of impurity BVM0207, impurity BVM0211, impurity BVM0206A, impurity BVM0212, impurity BVM0213, BVM diastereomers, BVM01, BVM02, and benzyl chloride into a volumetric flask, diluting to scale with a diluent, and shaking well to use the solution as a spiked sample solution.

2. The method according to claim 1, wherein the buffer salt is potassium dihydrogen phosphate, sodium phosphate, ammonium phosphate or potassium phosphate.

3. according to the arbitrary described method of claim 1-2, in the described mobile phase, the volume ratio of acetonitrile and lower alcohol is 3:1 to 7:

1.

4. The method according to claim 3, characterized in that: The method comprises the following steps: 1) Take an appropriate amount of Baloxavir marboxil or a sample containing Baloxavir marboxil, and use a mixed solvent of a certain amount of acetonitrile and a lower alcohol as a diluent to dissolve the sample; 2) Set the instrument parameters: flow rate of mobile phase, detection wavelength, and column oven temperature; 3) taking a certain amount of the solution in step 1) and injecting it into a high performance liquid chromatograph to complete the separation and determination of the related substances of Baloxavir marboxil.

5. The method according to claim 4, wherein the volume ratio of acetonitrile to lower alcohol in the diluent in step 1) is 3:1 to 7:

1.

6. The method according to claim 4, wherein the flow rate of the mobile phase is 0.5 ml / min to 2.0 ml / min; the detection wavelength is 205 nm to 270 nm; and the column box temperature of the chromatographic column is 40°C to 70°C.

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