Method for detecting related substances of macloxvir tablets

Abstract

The invention relates to a method for detecting related substances of a macloxvir tablet, which belongs to the technical field of pharmaceutical analysis, and adopts a high performance liquid chromatography, a reversed-phase liquid chromatographic column taking octadecyl bonded silica gel as a filler, a mixed solution of a trifluoroacetic acid solution containing ion pairs and acetonitrile as a mobile phase A and acetonitrile as a mobile phase B, a gradient elution mode is adopted. According to the method, elution conditions and detection conditions of high performance liquid chromatography are optimized through scientific screening, effective separation and detection of nine known impurities which are similar in structure and difficult to separate and seven or more unknown impurities contained in the macavir tablets are achieved at the same time, splitting of a detection method is avoided, time cost can be saved, economic benefits are improved, and the method is suitable for industrial production. The method has a positive effect and practical application value on detection of related substances of the macloxvir tablets.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting related substances of baloxavir marboxil tablets. Background Art

[0002] Baloxavir marboxil is a novel anti-influenza virus drug targeting viral replication. It selectively inhibits cap-dependent endonuclease (CEN), shows potent inhibitory effects on viral replication, is active against influenza A and B viruses and related subtypes (including influenza resistant to existing antiviral drugs and influenza with high pathogenic and pandemic potential), and existing non-clinical research data show that it is also active against the avian influenza subtype A / H7N9 resistant to oseltamivir (WHO 2017). At the same time, it only requires a single dose, with better convenience and treatment compliance. The chemical name is: [[(12aR)-12-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiepin-11-yl]-3,4,6,8,12,12a-hexahydro-6,8-dioxo-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazin-7-yl]oxymethyl carbonate, and its structure is shown as follows: -11-yl]-3,4,6,8,12,12a-hexahydro-6,8-dioxo-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazin-7-yl]oxymethyl carbonate, and its structure is shown as follows:

[0003]

[0004] Baloxavir marboxil tablets have relatively many impurities. The impurities of baloxavir marboxil tablets mainly come from process impurities and degradation impurities (impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity H, impurity I, and impurity IN6) introduced during the production process of the active pharmaceutical ingredient. Among them, impurity B, impurity F, and impurity IN6 belong to degradation impurities.

[0005] Table 1 List of Impurities in Baloxavir Marboxil Tablets

[0006]

[0007]

[0008] Baloxavir marboxil tablets involve many impurities, and there are few patents on the liquid phase method for baloxavir marboxil tablets in the existing public domain. It mainly involves the following: CN112858534A discloses an HPLC detection method for baloxavir marboxil intermediates and their related substances, using an octadecylsilane-bonded silica column, and the mobile phase is a mixed solution of phosphoric acid solution and methanol. The baloxavir marboxil intermediates and their related substances are 7,8-difluoro-6,11-dihydro-dibenzo[b,e]thiopheno-11-ol, 3,4-difluoro-2-methylbenzoic acid, methyl 3,4-difluoro-2-methylbenzoate, methyl 3,4-difluoro-2-bromomethylbenzoate, methyl 3,4-difluoro-2-phenylsulfanylmethylbenzoate, 3,4-difluoro-2-phenylsulfanylmethylbenzoic acid, 7,8-difluorodibenzo[b,e]thiepin-11(6H)-one. However, this method can only separate and detect a limited number of impurities, and some other key intermediates and impurities involved in the preparation of baloxavir marboxil still cannot be effectively separated.

[0009] CN116381068A discloses a high performance liquid chromatography method for separating and detecting related substances in baloxavir marboxil. The stationary phase of the chromatographic column is an octadecylsilane-bonded silica column, and the mobile phase is an acid-containing solution composed of phase A and phase B. The related substances are selected from any one or a combination of related substances 1 - related substances 11. However, this method is also not applicable to the impurity separation of baloxavir marboxil tablets based on a comprehensive impurity profile analysis.

[0010] To ensure the quality of drugs and the safety and effectiveness of clinical medication, it is necessary to control the attribution of impurities in drugs. Due to the large number of impurities involved, it is extremely important to attribute and separate the related impurities in baloxavir marboxil tablets. Currently, there is no literature report on the detection method for related substances in baloxavir marboxil tablets based on a comprehensive impurity profile analysis. Summary of the Invention

[0011] The object of the present invention is to address the lack of a detection method for related substances in baloxavir marboxil tablets based on a comprehensive impurity profile analysis in the prior art. By using high performance liquid chromatography technology to develop and optimize the composition of the mobile phase, the selection of the chromatographic column, and the elution gradient, a detection method for related substances in baloxavir marboxil tablets is invented.

[0012] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0013] A detection method for related substances in baloxavir marboxil tablets, using high performance liquid chromatography, with a reverse-phase liquid chromatographic column filled with octadecyl-bonded silica as the filler, a mixed solution of trifluoroacetic acid solution containing an ion pair and acetonitrile as mobile phase A, and acetonitrile as mobile phase B. Set the column temperature, flow rate, and detection wavelength, and the elution method is gradient elution. This detection method can simultaneously detect 9 known and 7 unknown related substances that may exist in baloxavir marboxil tablets.

[0014] Preferably, in terms of volume percentage, the conditions for gradient elution are as follows:

[0015] Time (min) Mobile phase A (%) Mobile phase B (%) 0 77 23 26 55 45 45 12 88 55 12 88 56 77 23 70 77 23 .

[0016] The gradient of the present invention is selected from different mobile phase ratios and different elution times, considering the influence of each gradient on the resolution between impurities and the main peak. Through research, it is found that different gradients have a great influence on the separation of impurities. In the high performance liquid chromatography method disclosed in the present invention, through the optimization and screening of the gradient elution program, a gradient elution program with high resolution of each impurity and appropriate retention time of the main peak is selected as the gradient elution program of the present invention. Through research, this gradient can effectively separate various impurities of marboxilavir, thus ensuring the quality control of marboxilavir tablets.

[0017] Preferably, the trifluoroacetic acid solution containing an ion pair is a trifluoroacetic acid solution containing disodium ethylenediaminetetraacetate. More preferably, the trifluoroacetic acid solution containing an ion pair is a trifluoroacetic acid solution containing 0.5 mmol / L disodium ethylenediaminetetraacetate.

[0018] Preferably, in the high performance liquid chromatography method disclosed in the present invention, Waters XSelect CSH C18 with a specification of 150 mm × 3.0 mm, 3.5 μm and ACE Excel 3 Super C18 with a specification of 150 mm × 4.6 mm, 3 μm chromatographic columns are screened. Considering the influence of each chromatographic column on the resolution of each impurity, it is found that the ACE Excel 3 Super C18 type chromatographic column can be used and can obtain better impurity separation. The chromatographic column of the detection method disclosed in the present invention is preferably ACE Excel 3 Super C18, with a specification of 150 mm × 4.6 mm, 3 μm.

[0019] Preferably, in the high performance liquid chromatography method disclosed in the present invention, the trifluoroacetic acid solution is a 0.1%-0.2% (V / V) trifluoroacetic acid solution, and more preferably a 0.15% (V / V) trifluoroacetic acid solution.

[0020] Preferably, in the high performance liquid chromatography method disclosed in the present invention, the volume ratio of the trifluoroacetic acid solution containing an ion pair to acetonitrile is selected as 90:10.

[0021] Preferably, in the high performance liquid chromatography method disclosed in the present invention, the column temperature is selected as 33-37 °C, the flow rate is selected as 0.6-1.0 mL / min, and the detection wavelength is selected as 259 nm.

[0022] Preferably, the column temperature is selected as 35 °C and the flow rate is selected as 0.8 mL / min.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The present invention optimizes the elution conditions and detection conditions of high-performance liquid chromatography through scientific screening, achieving the effective separation and detection of nine known impurities with similar structures and difficult to separate and more than seven unknown impurities in baloxavir marboxil tablets. It avoids the splitting of detection methods, can save time costs, improve economic benefits, and has positive effects and practical application values for the detection of related substances in baloxavir marboxil tablets.

[0025] (2) The method of the present invention has strong specificity. Each related substance does not interfere with the blank solvent, and the resolution between each component peak is greater than 1.5. In the chromatogram of the system suitability solution, the elution order of each component is unknown impurity 1, unknown impurity 2, impurity B, unknown impurity 3, impurity F, unknown impurity 4, impurity A, impurity IN6, impurity I, unknown impurity 5, impurity E, baloxavir marboxil, impurity D, impurity C, unknown impurity 6, unknown impurity 7, and impurity H peak, and the resolution between each component is not less than 1.5. Description of the Drawings

[0026] Figure 1 Shown is the high-performance liquid chromatogram of the blank solvent in Example 1.

[0027] Figure 2 Shown is the high-performance liquid chromatogram of the system suitability of related substances in baloxavir marboxil tablets in Example 1.

[0028] Figure 3 Shown is the high-performance liquid chromatogram of the system suitability of related substances in baloxavir marboxil tablets in Example 2.

[0029] Figure 4 Shown is the high-performance liquid chromatogram of the system suitability of related substances in baloxavir marboxil tablets in Example 3.

[0030] Figure 5 Shown is the high-performance liquid chromatogram of the system suitability of related substances in baloxavir marboxil tablets in Example 4. Detailed Embodiments

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. Where the present invention is not described in detail is the prior art.

[0032] Among them, the present invention does not have special restrictions on the sources of all raw materials, and they can be commercially available.

[0033] Example 1

[0034] (1) The related substances system solution of baloxavir marboxil tablets contains 9 known impurities (impurity B, impurity F, impurity A, impurity IN6, impurity I, impurity E, impurity D, impurity C and impurity H) and 7 unknown impurities;

[0035] (2) Brand model of the high performance liquid chromatograph: Waters Arc HPLC

[0036] (3) Detector: DAD

[0037] (4) Chromatographic column: Waters XSelect CSH C18 (150mm×3.0mm, 3.5μm)

[0038] (5) Mobile phase A: 0.1% trifluoroacetic acid solution containing 0.5 mmol / L disodium edetate, mobile phase B: acetonitrile, gradient elution as follows:

[0039]

[0040]

[0041] (6) Flow rate: 0.6 mL / min;

[0042] (7) Detection wavelength: 259 nm;

[0043] (8) Column temperature: 35 °C;

[0044] (9) Injection volume: 5 μL;

[0045] (10) Figure 1 The blank solvent does not interfere with the detection of related substances, Figure 2 In the system suitability solution, the chromatographic peak with a retention time of 16.242 min is the chromatographic peak of baloxavir marboxil, and the remaining chromatographic peaks are the chromatographic peaks of each impurity of baloxavir marboxil. It can be seen from the figure that baloxavir marboxil and the adjacent impurities fail to achieve baseline separation. The resolution between unknown impurity 4 and unknown impurity 5 (retention times of 10.863 min and 11.157 min) is only 1.29, and the resolution between impurity I and impurity IN6 is 1.43, which does not meet the requirements of the Chinese Pharmacopoeia.

[0046] Example 2

[0047] (1) The related substances system solution of baloxavir marboxil tablets contains 9 known impurities (impurity B, impurity F, impurity A, impurity IN6, impurity I, impurity E, impurity D, impurity C and impurity H) and 7 unknown impurities;

[0048] (2) Brand model of the high performance liquid chromatograph: Waters Arc HPLC

[0049] (3) Detector: DAD

[0050] (4) Chromatographic column: Waters XSelect CSH C18 (150 mm × 3.0 mm, 3.5 μm)

[0051] (5) Mobile phase A: 0.1% trifluoroacetic acid solution - acetonitrile (90:10) containing 0.5 mmol / L disodium ethylenediaminetetraacetate, mobile phase B: acetonitrile, gradient elution as follows:

[0052] Time (min) Mobile phase A (%) Mobile phase B (%) 0 77 23 26 55 45 45 12 88 55 12 88 56 77 23 70 77 23

[0053] (6) Flow rate: 0.6 mL / min;

[0054] (7) Detection wavelength: 259 nm;

[0055] (8) Column temperature: 35 °C;

[0056] (9) Injection volume: 5 μL;

[0057] (10) Figure 3 The chromatographic peak with a retention time of 15.121 min is the chromatographic peak of maribavir, and the remaining chromatographic peaks are the chromatographic peaks of various impurities of maribavir. It can be seen from the figure that the two peaks of unknown impurity 4 and unknown impurity 5 coincide (retention time 9.003 min), and the resolution between impurity C and unknown impurity 6 is only 1.22, which does not meet the requirements of the Chinese Pharmacopoeia.

[0058] Example 3

[0059] (1) The related substance system solution of maribavir tablets contains 9 known impurities (impurity B, impurity F, impurity A, impurity IN6, impurity I, impurity E, impurity D, impurity C and impurity H) and 7 unknown impurities;

[0060] (2) Brand model of the high performance liquid chromatograph: Waters Arc HPLC

[0061] (3) Detector: DAD

[0062] (4) Chromatographic column: Waters XSelect CSH C18 (150 mm × 3.0 mm, 3.5 μm)

[0063] (5) Mobile phase A: 0.15% trifluoroacetic acid solution - acetonitrile (90:10) containing 0.5 mmol / L disodium ethylenediaminetetraacetate, mobile phase B: acetonitrile, gradient elution as follows:

[0064] Time (min) Mobile phase A (%) Mobile phase B (%) 0 77 23 26 55 45 45 12 88 55 12 88 56 77 23 70 77 23

[0065] (6) Flow rate: 0.6 mL / min;

[0066] (7) The detection wavelength is 259 nm;

[0067] (8) The column temperature is 35 °C;

[0068] (9) The injection volume is 5 μl;

[0069] (10) Figure 4 The chromatographic peak with a retention time of 14.971 min is the chromatographic peak of marboxilavir, and the remaining chromatographic peaks are the chromatographic peaks of various impurities of marboxilavir. It can be seen from the figure that the resolution between marboxilavir and most impurities has been improved, but the resolution between impurity C and unknown impurity 6 (retention time 19.959 min) is 1.19, which does not meet the requirements of the Chinese Pharmacopoeia.

[0070] Example 4

[0071] (1) The related substance system solution of marboxilavir tablets contains 9 known impurities (impurity B, impurity F, impurity A, impurity IN6, impurity I, impurity E, impurity D, impurity C and impurity H) and 7 unknown impurities;

[0072] (2) Brand model of the high performance liquid chromatograph: Waters Arc HPLC

[0073] (3) Detector: DAD

[0074] (4) Chromatographic column: ACE Excel 3 Super C18 (150 mm × 4.6 mm, 3 μm)

[0075] (5) Mobile phase A: 0.15% trifluoroacetic acid solution containing 0.5 mmol / L disodium ethylenediaminetetraacetate - acetonitrile (90:10), mobile phase B: acetonitrile, gradient elution is carried out as follows:

[0076] Time (min) Mobile phase A (%) Mobile phase B (%) 0 77 23 26 55 45 45 12 88 55 12 88 56 77 23 70 77 23

[0077] (6) The flow rate is 0.8 mL / min;

[0078] (7) The detection wavelength is 259 nm;

[0079] (8) The column temperature is 35 °C;

[0080] (9) The injection volume is 10 μl;

[0081] (10) Figure 5 The chromatographic peak with a retention time of 24.070 min is the chromatographic peak of marboxilavir, and the remaining chromatographic peaks are the chromatographic peaks of various impurities of marboxilavir. It can be seen from the figure that the resolution of various impurities of marboxilavir meets the requirements, and the retention time of the main peak is appropriate, meeting the requirements of the Chinese Pharmacopoeia.

[0082] The method of the present invention can effectively control the related substances of baloxavir marboxil tablets. The resolution between each impurity peak and the adjacent main peak is greater than 1.5, and the peak purity of the main peak and each impurity peak meets the requirements. The analysis process is shown in Example 4, and the results are shown in Table 2.

[0083] Table 2 Results of Chromatographic Separation Parameters of Baloxavir Marboxil and Each Known Impurity

[0084]

[0085]

[0086] It can be seen from the test results that a method for detecting related substances of baloxavir marboxil tablets provided by the present invention is a method for separating and detecting baloxavir marboxil and 9 known impurities (Impurity B, Impurity F, Impurity A, Impurity IN6, Impurity I, Impurity E, Impurity D, Impurity C and Impurity H) and 7 unknown impurities under chromatographic conditions different from those of the prior art. It can be seen from the HPLC chromatogram that there is a certain distance between them, and the related substances of baloxavir marboxil tablets can be accurately determined, and the 9 known impurities (Impurity B, Impurity F, Impurity A, Impurity IN6, Impurity I, Impurity E, Impurity D, Impurity C and Impurity H) and 7 unknown impurities can be effectively separated.

[0087] Finally, it should be noted that the above-mentioned embodiments are only specific embodiments of the present invention, which are used to illustrate the technical solutions of the present invention, rather than to limit them. The protection scope of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: any person skilled in the art within the technical scope disclosed by the present invention can still modify the technical solutions recorded in the foregoing embodiments or can easily think of changes, or perform equivalent replacements on some of the technical features; and these modifications, changes or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be covered by the protection scope of the present invention.

Claims

1. A method for detecting related substances of baloxavir marboxil tablets, which uses high performance liquid chromatography, and is characterized in that, An RP-HPLC column packed with octadecylsilane is used, and a mixed solution of trifluoroacetic acid solution containing an ion pair and acetonitrile is used as mobile phase A, and acetonitrile is used as mobile phase B, and gradient elution is adopted.

2. The detection method for related substances of baloxavir marboxil tablets according to claim 1, wherein In terms of volume percentage, the conditions for gradient elution are as follows: 。 3. The detection method for related substances of baloxavir marboxil tablets according to claim 1, wherein The trifluoroacetic acid solution containing an ion pair is a trifluoroacetic acid solution containing disodium ethylenediaminetetraacetate.

4. The detection method for related substances of baloxavir marboxil tablets according to claim 1, wherein The trifluoroacetic acid solution containing an ion pair is a trifluoroacetic acid solution containing 0.5 mmol / L disodium ethylenediaminetetraacetate.

5. The detection method of related substances of baloxavir marboxil tablets according to claim 1, characterized in that, The RP-HPLC column packed with octadecylsilane as the packing is selected from Waters XSelect CSH C18, ACE Excel3Super C18 or a chromatographic column with the same performance.

6. The detection method of related substances of baloxavir marboxil tablets according to claim 1, characterized in that, The trifluoroacetic acid solution is a trifluoroacetic acid solution with a volume percentage concentration of 0.1% - 0.2%.

7. The detection method for related substances of baloxavir marboxil tablets according to claim 1 or 4, characterized in that, The trifluoroacetic acid solution is a trifluoroacetic acid solution with a volume percentage concentration of 0.15%.

8. The detection method for related substances of baloxavir marboxil tablets according to claim 1, characterized in that, The volume ratio of the trifluoroacetic acid solution containing an ion pair to acetonitrile is 90:

10.

9. The detection method of the related substances of baloxavir marboxil tablets according to claim 1, characterized in that, The column temperature of the HPLC is 33°C - 37°C, the flow rate of the mobile phase is 0.6 - 1.0 mL / min, and the detection wavelength is 259 nm.

10. The detection method of the related substances of baloxavir marboxil tablets according to claim 1 or 9, characterized in that, The column temperature of the HPLC is 35°C, and the flow rate of the mobile phase is 0.8 mL / min.

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