Method for analyzing related substances and impurities in reaction process of Mabavir

Abstract

The invention discloses a method for analyzing related substances and impurities in a reaction process of Mabazavir, relates to the field of medicine detection, and aims to provide a high-sensitivity and excellent-stability related substance analysis method for controlling the reaction process of Mabazavir, and the chromatographic conditions are as follows: a C18 chromatographic column (Waters Xselect CSH C18, 4.6 * 100mm, 3.5 mu m) of which the surface is charged and hybridized is adopted as a chromatographic column; a mobile phase A is a 0.05%-0.2% trifluoroacetic acid solution containing 0.1-1.0 mmol / L disodium ethylene diamine tetraacetate, a mobile phase B is acetonitrile, and isocratic elution is carried out according to the ratio of 55: 45; the column temperature is 40-60 DEG C; the flow velocity is 0.5 to 1.0 mL / min; the sample size is 5-20 [mu] l; and the operation time is 20-60 minutes. The method has the advantages of high detection sensitivity, good reproducibility, high stability and the like, and is suitable for detecting related substances controlled in the reaction process of the macavir.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical detection, and in particular to a method for analyzing related substances and impurities in a reaction process of mabaloxavir. Background Art

[0002] Mabaloxavir is a prodrug that is converted into an active metabolite, baloxavir, through hydrolysis, and exerts anti-influenza virus activity, and is active against both influenza A and influenza B viruses. Baloxavir is a selective inhibitor of influenza virus cap-dependent endonucleases, which inhibits influenza viruses from seizing the cap structure at the 5' end of host mRNA from host cells for their own mRNA transcription, thereby making the virus lose its ability to replicate itself, thereby achieving the effect of inhibiting influenza viruses at an early stage.

[0003] Due to different viral protein targets, cross-resistance between baloxavir and neuraminidase (NA) inhibitors or M2 proton pump inhibitors (amantadine) is not expected. Baloxavir is effective against oseltamivir-resistant influenza viruses and may have synergistic effects with neuraminidase inhibitors.

[0004] The detection of related substances in the intermediate reaction process is an important detection item for monitoring the reaction conditions during drug synthesis experiments. Currently, there is no report on the detection method for related substances in the intermediate reaction control of mabaloxavir raw materials. Therefore, developing a method for detecting related substances in the intermediate reaction control of mabaloxavir is of great significance for monitoring the reaction progress during the drug reaction process.

[0005] Mabaloxavir is a chemical drug that produces a certain amount of by-products and impurities during its synthesis, namely, the related substances and impurities controlled by the intermediate reaction described in the present invention. Currently, there are about dozens of related process impurities and degradation impurities available. Summary of the invention

[0006] The purpose of the present invention is to provide a method for analyzing related substances of mabaloxavir intermediate reaction control with high sensitivity and good stability.

[0007] The present invention discloses a method for analyzing related substances and impurities in a mabaloxavir reaction process. The method for analyzing related substances in a mabaloxavir reaction process is to analyze related substances in a mabaloxavir reaction solution by using an HPLC method. The specific operation is as follows:

[0008] Step 1: Solvent preparation

[0009] Mix 0.1-1.0 mmol / L disodium ethylenediaminetetraacetate solution containing 0.05%-0.2% trifluoroacetic acid and acetonitrile in a volume ratio of 1:1;

[0010] Step 2: Preparation of test solution

[0011] Take 40-60 μL of the mabaloxavir reaction solution, place it in a 10 mL volumetric flask, add solvent to dissolve and quantitatively dilute to the scale as the test solution;

[0012] Step 3: Preparation of relevant substance location solution

[0013] Take 5-20 mg of mabaloxavir related substances, place them in 10 mL volumetric bottles, add solvent to dissolve and quantitatively dilute to the scale, as the related substance positioning solution;

[0014] Step 4: Preparation of impurity localization solution

[0015] Take 0.5-2 mg of impurity, place it in a 10 mL volumetric flask, add solvent to dissolve and quantitatively dilute to the scale as the impurity positioning solution;

[0016] Step 5: HPLC detection

[0017] The chromatographic conditions are as follows: the chromatographic column model is a Waters Xselect CSH C18 chromatographic column, 3.0×150mm, 3.5μm, the mobile phase A is a 0.05%~0.2% trifluoroacetic acid solution containing 0.1~1.0mmol / L disodium ethylenediaminetetraacetate, and the mobile phase B is acetonitrile, and the operation is performed isocratically at 45:55; the column temperature is 30~50°C, the flow rate is 0.5~1.0mL / min, the detection wavelength is 220~330nm, the injection volume is 5~20μL, and the running time is 10~30min.

[0018] Furthermore, the C18 chromatographic column includes a CORTECS chromatographic column, a BEH chromatographic column, a HSS chromatographic column or a CSH chromatographic column.

[0019] Furthermore, the relevant substances are

[0020] Related substances 1, chemical structure

[0021] Related substances 2, chemical structure

[0022] Related substances 3, chemical structure Related substances 4, chemical structure Related substances 5, chemical structure Related substances 6, chemical structure Furthermore, the impurities are

[0023] Impurity 1, chemical structure Impurity 2, chemical structure Impurity 3, chemical structure Impurity 4, chemical structure Impurity 5, chemical structure Impurity 6, chemical structure Impurity 7, chemical structure Impurity 8, chemical structure

[0024] Impurity 9, chemical structure

[0025] Impurity 10, chemical structure

[0026] Impurity 11, chemical structure

[0027] Impurity 12, chemical structure

[0028] Impurity 13, chemical structure

[0029] The invention discloses a method for analyzing related substances and impurities in a reaction process of mabaloxavir. The chromatographic conditions are as follows: the chromatographic column model is a Waters Xselect CSH C18 chromatographic column, 4.6×100 mm, 3.5 μm, the mobile phase A is a 0.05% to 0.2% trifluoroacetic acid solution containing 0.1 to 1.0 mmol / mol of disodium ethylenediaminetetraacetate, the mobile phase B is acetonitrile, and the operation is isocratic according to a ratio of 55:45; the column temperature is 40 to 60° C., the flow rate is 0.5 to 1.0 mL per minute, the detection wavelength is 220 to 330 nm, the injection volume is 5 to 20 μL, and the operation time is 20 to 60 minutes.

[0030] Furthermore, the related substance detected by the chromatographic conditions is related substance 6, with a chemical structure

[0031]

[0032] Further, the impurities detected under the chromatographic conditions are

[0033] Impurity 15, chemical structure

[0034] Impurity 16, chemical structure

[0035] Impurity 17, chemical structure

[0036] Impurity 18, chemical structure

[0037] Impurity 19, chemical structure

[0038] Impurity 20, chemical structure

[0039] The present invention discloses a method for analyzing related substances in the reaction process control of mabaloxavir. The method for analyzing related substances in the reaction process control of mabaloxavir utilizes HPLC to analyze related substances 1 to 6 and impurities 1 to 20.

[0040] The present invention has the following beneficial effects:

[0041] The present invention separates process impurities and some degradation impurities that may be generated in the reaction process of mabaloxavir, specifically including the following related substances and impurities: related substance 1 to related substance 6, impurity 1 to impurity 20. A related substance analysis method for controlling the reaction process of mabaloxavir is established for the first time. The present invention has the advantages of high detection sensitivity, good reproducibility, high stability, etc., and is suitable for the inspection of related substances in the control of the reaction process of mabaloxavir.

[0042] After analysis and actual process control, we found that in the reaction process control spectrum, the separation degree between the related substance 6 and its impurity 14 (diastereoisomer impurity) is small, which is not conducive to judging the residue of related substance 5 in the reaction liquid. Therefore, the reaction process control related substance inspection method is optimized by replacing the chromatographic column, increasing the column temperature, changing the mobile phase ratio, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Positioning spectra for related substances 1 to 6;

[0044] Figure 2 Positioning maps for impurities 1 to 6;

[0045] Figure 3 Positioning maps for impurities 7 to 13;

[0046] Figure 4 It is the control map of the reaction process;

[0047] Figure 5 This is the control chart of the reaction process after method optimization;

[0048] Figure 6 This is the localization map of impurities 14 to 20 after method optimization. DETAILED DESCRIPTION

[0049] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

[0050] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clearly understood, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0051] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention but are not intended to limit the present invention.

[0052] Example 1

[0053] A method for analyzing related substances in the reaction process control of mabaloxavir in this embodiment is as follows:

[0054] 1) Solvent preparation

[0055] 0.1mmol / L~1.0mmol / L disodium ethylenediaminetetraacetate solution containing 0.05%~2.0% trifluoroacetic acid: acetonitrile (1:1).

[0056] 2) Preparation of test solution

[0057] Take 40-60 μL of the mabaloxavir reaction solution, place it in a 10 mL volumetric flask, add solvent to dissolve and quantitatively dilute to the scale.

[0058] 3) Preparation of positioning solution

[0059] Take 5-10 mg of Mabaloxavir related substances 1 to 6 respectively, put them in 10 mL volumetric flasks, dilute them to the mark with solvent, and shake well; take 0.5-2 mg of impurities 1 to 13 respectively, put them in 10 mL volumetric flasks, dilute them to the mark with solvent, and shake well.

[0060] The location time of each related substance and impurity is shown in Table 1. The location map and process control map of related substances 1 to 6 and impurities 1 to 13 are shown in Figure 1 to Figure 4 .

[0061] Example 2

[0062] This example is about the optimization of the chromatographic conditions used in the analysis method of related substances in the reaction process control of mabaloxavir.

[0063] pass Figures 1 to 4 It can be determined that under the above chromatographic conditions, during the reaction process of preparing related substance 6, the separation of related substance 6 and impurity 14 (diastereomer) is incomplete, which is not conducive to setting limits and guiding experiments. Therefore, optimization is carried out. The optimized method can completely separate related substance 6 and newly added impurities 14 to 20.

[0064] 1) Optimized chromatographic conditions 2

[0065] Chromatographic column: octadecylsilane bonded silica gel was used as filler (Waters Xselect CSH C18), with specifications of 4.6×100 mm, 3.5 μm; mobile phase A was 0.05% to 2.0% trifluoroacetic acid solution containing 0.1 to 1.0 mmol / L disodium ethylenediaminetetraacetate, and mobile phase B was acetonitrile, with isocratic elution at 55:45; column temperature was 40 to 60°C, flow rate was 0.5 to 1.0 mL per minute, detection wavelength was 220 to 330 nm, injection volume was 5 to 20 μL, and running time was 20 to 60 minutes.

[0066] 2) Preparation of test solution

[0067] Take 40-60 μL of the mabaloxavir reaction solution, place it in a 10 mL volumetric flask, add solvent to dissolve and quantitatively dilute to the scale, and shake well.

[0068] 3) Preparation of related substance location solution

[0069] Take 5-10 mg of mabaloxavir related substance 1 to related substance 6 respectively, put them into 10 mL volumetric flasks, quantitatively dilute to the scale with solvent, and shake well.

[0070] 4) Preparation of related substance location solution

[0071] Take 0.5-2 mg of each of impurities 14 to 20, place them in 10 mL volumetric flasks, dilute to the mark with solvent, and shake well.

[0072] The localization time of each related substance is shown in Table 2. The spectrum of related substances in the reaction process control after method optimization is shown in Figure 5 The localization map of impurities 14 to 20 is shown in Figure 6 .

[0073] Table 1 Location time of impurities 1 to 13 in chromatographic condition 1

[0074]

[0075]

[0076]

[0077]

[0078] Table 2 Localization time of related substances 1-6 and impurities 14-20 in chromatographic condition 2 after optimization

[0079]

[0080]

[0081]

Claims

1. A method for analyzing related substances and impurities in the reaction process of Mabaloxavir, characterized in that The method for analyzing related substances in the mabaloxavir reaction process is to analyze the related substances in the mabaloxavir reaction solution by HPLC, and the specific operation is as follows: Step 1: Solvent preparation Mix 0.1-1.0 mmol / L disodium ethylenediaminetetraacetate solution containing 0.05%-0.2% trifluoroacetic acid and acetonitrile in a volume ratio of 1:1; Step 2: Preparation of test solution Take 40-60 μL of the mabaloxavir reaction solution, place it in a 10 mL volumetric flask, add solvent to dissolve and quantitatively dilute to the scale as the test solution; Step 3: Preparation of relevant substance location solution Take 5-20 mg of mabaloxavir related substances, place them in 10 mL volumetric bottles, add solvent to dissolve and quantitatively dilute to the scale, as the related substance positioning solution; Step 4: Preparation of impurity localization solution Take 0.5-2 mg of impurity, place it in a 10 mL volumetric flask, add solvent to dissolve and quantitatively dilute to the scale as the impurity positioning solution; Step 5: HPLC detection The chromatographic conditions are as follows: the chromatographic column model is a Waters Xselect CSH C18 chromatographic column, 3.0×150mm, 3.5μm, the mobile phase A is a 0.05%~0.2% trifluoroacetic acid solution containing 0.1~1.0mmol / L disodium ethylenediaminetetraacetate, and the mobile phase B is acetonitrile, and the operation is performed isocratically at 45:55; the column temperature is 30~50°C, the flow rate is 0.5~1.0mL / min, the detection wavelength is 220~330nm, the injection volume is 5~20μL, and the running time is 10~30min.

2. The method for analyzing related substances and impurities in the reaction process of mabaloxavir according to claim 1, characterized in that The C18 chromatographic column includes a CORTECS chromatographic column, a BEH chromatographic column, a HSS chromatographic column or a CSH chromatographic column.

3. The method for analyzing related substances and impurities in the reaction process of mabaloxavir according to claim 1, characterized in that The relevant substances are Related substances 1, chemical structure Related substances 2, chemical structure Related substances 3, chemical structure Related substances 4, chemical structure Related substances 5, chemical structure Related substances 6, chemical structure 4. The method for analyzing related substances and impurities in the reaction process of mabaloxavir according to claim 1, characterized in that The impurities are Impurity 1, chemical structure Impurity 2, chemical structure Impurity 3, chemical structure Impurity 4, chemical structure Impurity 5, chemical structure Impurity 6, chemical structure Impurity 7, chemical structure Impurity 8, chemical structure Impurity 9, chemical structure Impurity 10, chemical structure Impurity 11, chemical structure Impurity 12, chemical structure Impurity 13, chemical structure 5. A method for analyzing related substances and impurities in the reaction process of mabaloxavir, characterized in that the chromatographic conditions are: the chromatographic column model is a Waters Xselect CSH C18 chromatographic column, 4.6×100 mm, 3.5 μm, the mobile phase A is a 0.05% to 0.2% trifluoroacetic acid solution containing 0.1 to 1.0 mmol / sodium disodium ethylenediaminetetraacetate, and the mobile phase B is acetonitrile, and the operation is performed isocratically at a ratio of 55:45; the column temperature is 40 to 60° C., the flow rate is 0.5 to 1.0 mL per minute, the detection wavelength is 220 to 330 nm, the injection volume is 5 to 20 μL, and the running time is 20 to 60 minutes.

6. The optimized method for analyzing related substances and impurities in the reaction process of mabaloxavir according to claim 5, characterized in that The related substance detected by the chromatographic conditions is related substance 6, chemical structure 7. The optimized method for analyzing related substances and impurities in the reaction process of mabaloxavir according to claim 5, characterized in that The impurities detected under the chromatographic conditions are Impurity 15, chemical structure Impurity 16, chemical structure Impurity 17, chemical structure Impurity 18, chemical structure Impurity 19, chemical structure Impurity 20, chemical structure

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