CN113155985A - Analysis and detection method of Favipiravir photodegradation product - Google Patents
Analysis and detection method of Favipiravir photodegradation product Download PDFInfo
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Abstract
The invention provides a method for detecting photodegradation products in a Lavenvir sample, which is characterized in that a HILIC chromatographic column is adopted to carry out high performance liquid chromatography analysis on the Lavenvir sample. Compared with the prior art, the invention has the advantages that the photodegradation product can be properly reserved, and the separation degree of the photodegradation product from the main component and the related impurities is good.
Description
Technical Field
The invention relates to the field of drug analysis, in particular to an analysis and detection method for Favipiravir photodegradation impurities.
Background
Favipiravir (trade name AVIGAN) is a nucleoside broad-spectrum antiviral drug, the action mechanism of which is not completely clear, and the action mechanism is mainly considered at present to be that the drug is converted into Favipiravir ribofuranosyl-5' -inositol triphosphate (Favipiravir RTP) with biological activity by host cell enzyme phosphoribose after being orally absorbed, can compete with purine for viral RNA polymerase, and can be inserted into a viral RNA chain so as to block the replication and transcription of the viral RNA chain. Therefore, the Favipiravir can be considered to have potential antiviral capacity to various RNA viruses from the mechanism.
The medicine was originally developed by fukushan chemical pharmaceutical co-company, japan approved for marketing in 3 months 2014, and is used for treating novel or recurrent influenza. The existing research shows that the RNA virus has good antiviral effect on other various RNA viruses, such as Ebola virus, hemorrhagic fever, bunyavirus and the like. Favipiravir tablets are approved to be marketed in China at 2/15 of 2020, and are indicated for treating adult novel or recurrent influenza and are only used when other anti-influenza virus drugs are ineffective or have poor effects, and drug clinical test kits are obtained to develop clinical research using novel coronavirus pneumonia (COVID-19) as an indication and allow clinical administration of treatment COVID-19 within a small range at the present stage. Worldwide, many countries such as japan, the united states of america, italy, etc. have also initiated fapirovir-related clinical trials.
Favipiravir is shown in the structural formula:
possible degradation products can be evaluated by destructive testing and stability testing. When forced degradation research is carried out in the research and development stage, the content of the Favipiravir is reduced under the illumination condition, but the existing HPLC method cannot observe the photodegradation product when detecting impurities. The existing impurity method is proved to miss detection of the photodegradation product, and if no method is available for detecting the photodegradation product, the research and development progress and the quality control of the product are undoubtedly influenced.
Disclosure of Invention
Through a series of methods, we find that the polarity of photodegradation impurities of the Favipiravir is very strong and is not reserved in common reverse-phase chromatography, so the invention turns to develop a normal-phase method to analyze the Favipiravir. The hydrophilic interaction chromatography mode belongs to a normal phase chromatography variant, has great advantages in the aspects of separating polar and hydrophilic substances compared with other separation modes, can be compatible with various detectors, and has the advantages of simple preparation of a mobile phase, high analysis efficiency and good reproducibility.
Therefore, the invention aims to provide an analysis and detection method for a Favipiravir photodegradation product;
the technical scheme adopted by the invention is as follows:
dissolving Favipiravir or a preparation related to Favipiravir in a proper amount in a diluent to prepare a test solution, carrying out high performance liquid chromatography analysis on the test solution, and recording a chromatogram, wherein a chromatographic column is a HILIC chromatographic column.
Further, the HILIC chromatographic column is selected from an amide bonded stationary phase or a silica gel column. The column temperature is preferably 15 to 40 ℃, and more preferably 30 ℃.
Further, the mobile phase in the high performance liquid chromatography analysis condition is formed by mixing an organic solvent and water, and preferably an acetonitrile-buffer aqueous solution system; it is further preferred that the acetonitrile concentration in the mobile phase is in the range of 40% to 97% by mass, more preferably 95% by mass. The buffer aqueous solution system in the mobile phase is selected from an amine formate buffer salt system, an ammonium acetate buffer salt system, a formic acid aqueous solution system or an acetic acid aqueous solution system. The pH of the aqueous buffer system is in the range of 2.0 to 3.0, preferably 3.0. The flow rate of the mobile phase is in the range of 0.3 to 2mL/min, preferably 0.5 to 1.0 mL/min.
Further, in the method, the diluent is acetonitrile or a mixture of acetonitrile and water with the acetonitrile ratio of more than or equal to 50 percent, and more preferably 100 percent of acetonitrile.
Further, in the conditions of the high performance liquid chromatography, the detector is a diode array detector or an ultraviolet detector, when the diode array detector is selected, the wavelength can be selected to be 190 nm and 400nm, and the wavelength scanning can also set the detection wavelength to be 230 nm; the ultraviolet detector is selected to set the detection wavelength at 230 nm.
Further, in the conditions of the high performance liquid chromatography, the detection sample amount ranges from 5 to 80. mu.L, and preferably 30. mu.L.
Further, in the conditions of the high performance liquid chromatography, the column temperature: 15-40 DEG C
The preferred chromatographic detection method of the invention is as follows:
(1) preparing a test solution: dissolving a proper amount of a photo-damaged favipiravir sample in a diluent to prepare a test solution;
(2) the determination method comprises the following steps: and (2) injecting the sample solution prepared in the step (1) into a high performance liquid chromatograph, performing high performance liquid chromatography analysis on the sample solution according to the chromatographic conditions, and recording a chromatogram.
The sample in the step (1) is a mixture sample of Favipiravir or a Favipiravir related preparation or a Favipiravir and related preparation auxiliary materials.
Further, the conditions of the light irradiation destruction in the step (1) are cumulative light irradiation to white light of 1.2X 106Lux hr and violet light of 200w hr/m 2.
Compared with the prior art, the invention has the advantages that the photodegradation product can be properly reserved, the separation degree of the photodegradation product from main components and related impurities is good, the analysis time is short, the detection is efficient, and the chromatographic condition can be compatible with various detectors. Its compatibility with mass spectrometry makes possible the identification of photodegradation products and thus has the potential to provide information for the analysis of synthetic impurities.
Drawings
FIG. 1 is a graph showing the results of HPLC detection of a white solution in comparative example 1.
FIG. 2 is a graph showing the results of HPLC detection of the control solution in comparative example 1.
FIG. 3 is a graph showing the results of HPLC detection of the light control sample solution in comparative example 1.
FIG. 4 is a graph showing HPLC detection results of an empty white solution in example 1 at 230 nm.
FIG. 5 is a graph showing the HPLC detection results of the control solution in example 1 at 230 nm.
FIG. 6 is a graph showing the results of HPLC detection of a light-irradiated sample solution in example 1 at 230 nm.
FIG. 7 is a graph showing HPLC detection results of an empty white solution in example 2 at 230 nm.
FIG. 8 is a graph showing the result of HPLC detection of the control solution in example 2 at 230 nm.
FIG. 9 is a graph showing the results of HPLC detection of a light-irradiated sample solution in example 2 at 230 nm.
FIG. 10 is a graph showing HPLC detection results of an empty white solution in example 3 at 230 nm.
FIG. 11 is a graph showing the HPLC detection results of the control solution in example 3 at 230 nm.
FIG. 12 is a graph showing the results of HPLC detection of a light irradiation sample solution in example 3 at 230 nm.
FIG. 13 is a graph showing HPLC detection results of an empty white solution in example 4 at 230 nm.
FIG. 14 is a graph showing the result of HPLC detection of the control solution in example 4 at 230 nm.
FIG. 15 is a graph showing HPLC detection results of a light irradiation sample solution in example 4 at 230 nm.
Detailed Description
Illumination sample information: the mixed sample of the raw material medicine and the Huahai preparation prescription auxiliary material is placed under the illumination condition for irradiation, and the accumulated illumination reaches 1.2 multiplied by 106Lux hr of white light and 200w hr/m2 of purple light.
Comparative example 1
According to the literature: the HPLC method of related substances in Lapiver is used for measuring [ J ]. Chinese pharmacist, 2018, 21 (4): 739 carrying out the detection method of step 742.
Chromatographic conditions are as follows:
the instrument comprises the following steps: diode array detector equipped for high performance liquid chromatograph
A chromatographic column: agilent Zorbax SB-C18, 250mm 4.6mm, 5.0 μm
Mobile phase: phosphate buffer (1.4 g potassium dihydrogen phosphate, water to 1000mL, pH adjusted to 4.0 + -0.05 with phosphoric acid) -acetonitrile (90:10)
Detection wavelength: 238nm
Flow rate: 1.0mL/min
Column temperature: 30 deg.C
Sample introduction amount: 20 μ L
Diluting liquid: water (W)
Control solution: weighing a proper amount of the raw materials into a proper volumetric flask, and preparing a diluent into a solution containing 0.2mg Favipiravir per 1 mL.
Preparing a photo-destructive sample solution: weighing a proper amount of illumination samples into a proper volumetric flask, adding acetonitrile to prepare a solution containing 1mg Falalavir in each 1mL, transferring a proper amount of the solution into a proper amount of the flask, and adding a diluent to dilute the solution into a solution containing 0.2mg Falalavir in each 1 mL.
The detection and analysis are carried out according to the detection conditions, the sample injection results of the blank solution, the control solution and the photo-damage sample solution are respectively shown in figures 1-3, and the photo-degradation product of the method appears as a peak at a solvent peak.
Example 1
Chromatographic conditions are as follows:
the instrument comprises the following steps: diode array detector equipped for high performance liquid chromatograph
A chromatographic column: waters XBridge Amide, 150mm 4.6mm, 3.5 μm (triple bond-linked Amide groups based on ethylene bridge hybrid particles (BEH))
Mobile phase: acetonitrile: 100mmol/L pH3.0 ammonium formate aqueous solution 95:5(v/v)
Flow rate: 0.8mL/min
Sample introduction volume: 30 μ L of
Column temperature: 30 deg.C
Operating time: 30min
Diluting liquid: acetonitrile
Preparing a blank solution: the same as the diluent.
Control solution: weighing a proper amount of the raw materials into a proper volumetric flask, and preparing a diluent into a solution containing 1mg Favipiravir per 1 mL.
Preparing a photo-destructive sample solution: weighing a proper amount of the illumination sample into a proper volumetric flask, and adding the diluent to prepare a solution containing 1mg Favipiravir per 1 mL.
The detection and analysis are carried out according to the detection conditions, the sample injection results of the blank solution, the control solution and the photo-damage sample solution are respectively shown in figures 4-6, and the photo-degradation product in the method is properly reserved and well separated.
Example 2
The instrument comprises the following steps: diode array detector equipped for high performance liquid chromatograph
A chromatographic column: luna 5. mu. HILIC 200A, 250mm 4.6mm, 5 μm (ultrapure silica gel modified cross-linked diol)
Mobile phase: acetonitrile: 100mmol/L pH3.0 ammonium formate aqueous solution 95:5(v/v)
Flow rate: 0.5mL/min
Sample introduction volume: 30 μ L of
Column temperature: 30 deg.C
Operating time: 30min
Diluting liquid: acetonitrile
Preparing a blank solution: the same as the diluent.
Control solution: weighing a proper amount of the raw materials into a proper volumetric flask, and preparing a diluent into a solution containing 1mg Favipiravir per 1 mL.
Preparing a photo-destructive sample solution: weighing a proper amount of the illumination sample into a proper volumetric flask, and adding the diluent to prepare a solution containing 1mg Favipiravir per 1 mL.
The detection and analysis are carried out according to the detection conditions, the sample injection results of the blank solution, the control solution and the photo-damage sample solution are respectively shown in figures 7-9, and the method can be seen that the photo-degradation product is properly reserved and the separation condition is good.
Example 3
The instrument comprises the following steps: diode array detector equipped for high performance liquid chromatograph
A chromatographic column: ACE 5 μm HILIC-N, 250mm 4.6mm (ultrapure silica gel bonded hydroxy)
Mobile phase: acetonitrile: 20mmol/L pH3.0 ammonium formate aqueous solution 95:5(v/v)
Flow rate: 0.8mL/min
Sample introduction volume: 30 μ L of
Column temperature: 30 deg.C
Operating time: 30min
Diluting liquid: acetonitrile
Preparing a blank solution: the same as the diluent.
Control solution: weighing a proper amount of the raw materials into a proper volumetric flask, and preparing a diluent into a solution containing 1mg Favipiravir per 1 mL.
Preparing a photo-destructive sample solution: weighing a proper amount of the illumination sample into a proper volumetric flask, and adding the diluent to prepare a solution containing 1mg Favipiravir per 1 mL.
The detection and analysis are carried out according to the detection conditions, the sample injection results of the blank solution, the control solution and the photo-damage sample solution are respectively shown in figures 10-12, and the method can be seen that the photo-degradation products are properly reserved and the separation condition is good.
Example 4
The instrument comprises the following steps: diode array detector equipped for high performance liquid chromatograph
A chromatographic column: ACE 5 μm HILIC-N, 250mm 4.6mm (ultrapure silica gel bonded hydroxy)
Mobile phase: acetonitrile: ph3.0 aqueous formic acid 95:5(v/v)
Flow rate: 0.8mL/min
Sample introduction volume: 30 μ L of
Column temperature: 30 deg.C
Operating time: 30min
Diluting liquid: acetonitrile
Preparing a blank solution: the same as the diluent.
Control solution: weighing a proper amount of the raw materials into a proper volumetric flask, and preparing a diluent into a solution containing 1mg Favipiravir per 1 mL.
Preparing a photo-destructive sample solution: weighing a proper amount of the illumination sample into a proper volumetric flask, and adding the diluent to prepare a solution containing 1mg Favipiravir per 1 mL.
The detection and analysis are carried out according to the detection conditions, the sample injection results of the blank solution, the control solution and the photo-damage sample solution are respectively shown in figures 13-15, and the method can be seen that the photo-degradation product is properly reserved and the separation condition is good.
The above description is only for the details of the preferred embodiments of the present invention, and the present invention is not limited to the above embodiments, and any changes and modifications of the present invention fall within the protection scope of the present invention.
Claims (11)
1. An analysis and detection method for a Favipiravir photodegradation product comprises the steps of dissolving a proper amount of Favipiravir or a preparation related to the Favipiravir in a diluent to prepare a test solution, carrying out high performance liquid chromatography analysis on the test solution, and recording a chromatogram, wherein a chromatographic column is a HILIC chromatographic column.
2. The process as claimed in claim 1, wherein the stationary phase for HILIC chromatography column is amide-bonded stationary phase or silica gel column.
3. The method of claim 2, wherein the mobile phase of the hplc analysis conditions consists of an organic solvent-water mixture, preferably an acetonitrile-buffered aqueous solution system, further preferably the acetonitrile mass concentration in the mobile phase is selected from the range of 40% to 97%, and even more preferably 95%.
4. A process according to claim 3, wherein the pH of the aqueous buffered solution system is in the range of 2.0 to 3.0, preferably 3.0.
5. The method of claim 3, wherein the aqueous buffered solution system is an amine formate buffered salt system, an ammonium acetate buffered salt system, an aqueous formic acid solution system, or an aqueous acetic acid solution system.
6. A process according to claim 3, wherein the mobile phase flow rate is in the range of 0.3-2mL/min, preferably 0.5-1.0 mL/min.
7. The process of claim 1, wherein the diluent is acetonitrile or a mixture of acetonitrile and water, preferably acetonitrile, at a ratio of acetonitrile to water of 50% or more.
8. The method of claim 1, wherein the high performance liquid chromatography is performed using a diode array detector or an ultraviolet detector.
9. The method according to claim 8, wherein the detection wavelength is 190-400nm, preferably 230 nm.
10. The method of claim 1, wherein the amount of sample is in the range of 5-80 μ L, preferably 30 μ L.
11. The method according to claim 1, wherein the HPLC analysis is performed under a column temperature of 15 to 40 ℃, preferably 30 ℃.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104914185A (en) * | 2015-06-10 | 2015-09-16 | 山东省药学科学院 | HPLC method for measuring related substances in Favipiravir |
| US20160000754A1 (en) * | 2004-01-06 | 2016-01-07 | Paul Edward Stamets | Antiviral Activity from Medicinal Mushrooms and their Active Constituents |
| CN106588786A (en) * | 2015-10-14 | 2017-04-26 | 山东省药学科学院 | Preparation method of high purity favipiravir impurity |
| CN111471025A (en) * | 2020-03-26 | 2020-07-31 | 兰州康寓信生物科技有限公司 | Favipiravir intermediate and synthesis method of favipiravir |
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- 2020-12-19 CN CN202011509294.2A patent/CN113155985A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160000754A1 (en) * | 2004-01-06 | 2016-01-07 | Paul Edward Stamets | Antiviral Activity from Medicinal Mushrooms and their Active Constituents |
| CN104914185A (en) * | 2015-06-10 | 2015-09-16 | 山东省药学科学院 | HPLC method for measuring related substances in Favipiravir |
| CN106588786A (en) * | 2015-10-14 | 2017-04-26 | 山东省药学科学院 | Preparation method of high purity favipiravir impurity |
| CN111471025A (en) * | 2020-03-26 | 2020-07-31 | 兰州康寓信生物科技有限公司 | Favipiravir intermediate and synthesis method of favipiravir |
Non-Patent Citations (1)
| Title |
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| 刘葵葵等: "法匹拉韦中有关物质的HPLC法测定", 《中国药师》 * |
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