Favipiravir (SARS‐CoV‐2) degradation impurities: Identification and route of degradation mechanism in the finished solid dosage form using LC/LC–MS method

Abstract Favipiravir finished dosage was approved for emergency use in many countries to treat SARS‐CoV‐2 patients. A specific, accurate, linear, robust, simple, and stability‐indicating HPLC method was developed and validated for the determination of degradation impurities present in favipiravir film‐coated tablets. The separation of all impurities was achieved from the stationary phase (Inert sustain AQ‐C18, 250 × 4.6 mm, 5‐μm particle) and mobile phase. Mobile phase A contained KH2PO4 buffer (pH 2.5 ± 0.05) and acetonitrile in the ratio of 98:2 (v/v), and mobile phase B contained water and acetonitrile in the ratio of 50:50 (v/v). The chromatographic conditions were optimized as follows: flow rate, 0.7 mL/min; UV detection, 210 nm; injection volume, 20 μL; and column temperature, 33°C. The proposed method was validated per the current International Conference on Harmonization Q2 (R1) guidelines. The recovery study and linearity ranges were established from the limit of quantification to 150% optimal concentrations. The method validation results were found to be between 98.6 and 106.2% for recovery and r 2 = 0.9995–0.9999 for linearity of all identified impurities. The method precision results were achieved below 5% of relative standard deviation. Forced degradation studies were performed in chemical and physical stress conditions. The compound was sensitive to chemical stress conditions. During the study, the analyte degraded and converted to unknown degradation impurities, and its molecular mass was found using the LC–MS technique and established degradation pathways supported by reaction of mechanism. The developed method was found to be suitable for routine analysis of research and development and quality control.

infected with this illness with mild to moderate symptoms. Pyrazine carboxamide is a derivative that exhibits antiviral activity against RNA viruses. It is known as favipiravir. The drug can help lower the severity of symptoms caused by SARS-CoV-2 in humans. It has also been shown to reduce the duration of illness in some people. The chemical name of favipiravir is 6-fluoro-3-oxo-3,4-dihydro-2-pyrazinecarboxamide, its molecular formula is C 5 H 4 FN 3 O 2 , and its mass is 157.104 amu. The chemical structure of favipiravir is shown Figure 1. The development of the process for favipiravir production was carried out by identifying the impurities, namely impurities A, B, and C.
A literature survey revealed that no stability-indicating method has been reported yet for the quantitative determination of favipiravir impurities. A few analytical methods were found; those various experiments were performed in human plasma using LC-MS/MS and HPLC techniques (Balu & Paresh, 2021;Eryavuz et al., 2021;Hailat et al., 2021;Ibrahim et al., 2021;Katharina et al., 2021;Marzouk et al., 2022;Mikhail et al., 2021;Mohamed et al., 2021;Nakayama & Ryo, 2021;Nazifa Sabir Ali et al., 2021;Nippes et al., 2021;Pallavi & Kamalkishor, 2021;Ramarao & Abhinandana, 2021;Rezk et al., 2021;Srinivas, et al., 2021) (Table 1). The literature does not report any HPLC method that accurately and quantitatively assesses the relative and degradation impurities of favipiravir. It is therefore necessary to develop a new stability-indicating method for the determination and estimation of favipiravir and its related impurities. The mass of major degradation impurities was identified using LC-MS techniques. Therefore, a reproducible, stability-indicating LC method was developed to determine favipiravir and its impurities. This method was successfully validated according to the current International Conference on Harmonization (ICH) guidelines. The proposed method can be used to estimate the known and degradation impurities of favipiravir in the finished dosage and drug substance. In addition, it is user friendly and cost effective.

| Chemicals and reagents
Favipiravir standard (99.9% purity) and its related known impurities (impurities A, B, and C) were provided by Laurus Labs (Hyderabad, India

| Chromatographic conditions
Chromatographic separation was achieved from a suitable stationary phase (Inert sustain AQ-C18, 250 Â 4.6 mm, 5-μm particle), and gradient program was set as time/%B: 0.0/0, 40/30, 60/55, 62/00, and 70/00. Mobile phase A consisted of phosphate buffer (pH 2.5) and acetonitrile in the ratio of 98:2 (v/v), and mobile phase B comprised acetonitrile and water in the ratio of 50:50 (v/v). The measurements were performed as follows: injection volume, 20 μL; flow rate, 0.7 mL/min; column temperature, 33 C; and UV detection, 210 nm. The diluent was prepared using water and acetonitrile in the ratio of 98:2 (v/v). All impurities were well separated in these conditions.

| LC-MS conditions
The LC-MS studies were performed using a Waters TQD triple Quadrupole mass spectrometer [12][13][14][15]. The source voltage was kept at 5000 V and capillary temperature at 400 C. Mass range was kept at

| Impurity stock solution preparation
About 25  Then the remaining volume was diluted to volume with the diluent.
For further dilution, 5 mL of the filtered sample solution was transferred into a 50-mL volumetric flask and made up to volume with the diluent. 3. In this trial, impurity B merged with the main peak 4. Impurity A eluted at the void 5. Unknown impurity eluted at impurity C; for details see Figure 3 Rejected 8. All impurities are well separated 9. All impurities possess good peak shapes 10. No interference was observed at known impurities and degradation impurities; for details see Figure 4 Approved VEMURI ET AL.  were injected in the current LC method. All chromatography data were reviewed, no interference was observed at the retention time of known and degradation impurities, and peaks of favipiravir and known impurities were found to be spectrally pure.

| Forced degradation studies
This study plays a significant role in identifying the stability-indicating method and could identify the molecular behavior in the different stress reagents. Any LC method must undergo this study to be reviewed by the U.S. Food and Drug Administration, the European Union, and other regulatory bodies. Forced degradation studies confirm that the method is stability indicating in nature. Before the experiment was started, we obtained molecule solubility and polarity information and some stress reagent concentrations and conditions from regulatory guidelines (Blessy et al., 2014;Murthy et al., 2013;Saranjit et al., 2013). The results are presented in Table 3 and Figures 6, 10, 11, and 12. For further dilution, 5 mL of the filtered sample solution was transferred into a 50-mL volumetric flask and then made up to volume with the diluent. When the degradation data were reviewed, one unknown degradation impurity was found at R T 11.816 min. The LC-MS technique identified the impurity mass value and found a molecular structure with proper reaction mechanism, and the results are presented in Table 3 Table 3 and Figures 8, 10-12.  Table 3 and Figures 9, 10, 11, and 12.

| Water hydrolysis degradation sample
Based on the chemical degradation study results, this compound is very sensitive to acid, base, peroxide, and water hydrolysis conditions.

| Solid-state degradation studies
Physical degradation study plays an essential role in degradation study. It could identify the degradation pathway of impurities formed in stability studies. Solid-state degradation includes heat, humidity, and light stress studies. Before experimental testing, the samples were kept in respective storage conditions. Therefore, the drug product samples were stored in a photostability chamber and were exposed to 200 Wh/m 2 of UV light and 1.2 million lux hours of visible light (per ICH Q1B, UV 200 Wh/m 2 , 1.2 million lux hours) in humidity conditions (85% Relative Humidity) for 7 days and exposed to heat (105 C) for 10 days. After time intervals, the samples were removed, and the degradation solutions were prepared and immediately injected into the LC instrument. No major degradation impurities were observed in all degradation sample chromatograms; good results were achieved within limits.
The results show that the compound is stable at heat, humidity, and light environmental conditions. The results are presented in Table 3.  Table 4 and Figures 13 and 14.

| Accuracy
This study was tested by the standard addition method by spiking the known impurities at the limit of quantification (LOQ) level: 50, 100, and 150% of the specification concentrations, that is, 0.09, 0.15, 0.3, and 0.45 μg/mL with respective test concentrations. Each spiked sample determination was carried out thrice, and the corresponding data are presented in Table 4 and Figures 15 and 16. The recovery of impurities at four levels (LOQ: 50, 100, and 150%) was 98.2-105.1% F I G U R E 1 1 Mass fragmentation of oxidation degradation peak 2 F I G U R E 1 2 The route of degradation reaction occurred in acid, water, and oxidation environment conditions

T A B L E 4 Method validation results
Parameters Details Acceptance criteria

Results
System suitability n = 6 (favipiravir standard 0.5%, with respect to sample concentration) Tailing factor for favipiravir should not be more than 1.5 Plate count for favipiravir should not be less than 5000 %RSD of favipiravir peak from six injections of standard solution should not be more than 2.0%  Table 5.

| Determination of LOD and LOQ
This study plays an essential role in the related substances method development, from which the method of sensitivity could be identi- signal-to-noise ratio method. The LOQ and LOD solutions were prepared from the impurity standard stock solution. In the process, the concentrations of all impurity solutions were decreased successively to obtain an LOQ, such that the yield signal-to-noise ratio was 10.5 for impurity A, 10.8 for impurity B, and 10.9 for impurity C. The LOD was calculated using the formula LOD = LOQ/3.3, and the LOQ solution was diluted thrice, and the LOD solution was injected into the HPLC instrument. The experimental and theoretical LOD values are comparable, which is good. The LOQ and LOD for all known impurities were 0.09 and 0.027 μg/mL, respectively. The results are presented in Table 4.

| Solution stability
This study is more beneficial to analytical scientists. Thus, the standard and spiked sample solutions should confirm how many hours they are stable at refrigerator and room temperatures. The stability study was evaluated by injecting the standard and spiked sample solutions at 2-8 C and room temperature 25 C. At refrigerator condition, target samples were injected at initial, 24, and 48 h, yielding results within limits. At room temperature, as the impurities are sensitive, this study was performed at initial, 1, 2, 4, and 5 h. The results are presented in Table 5.

| Robustness
This study could achieve method sensitivity. The optimized test method was examined by intentional changes in flow rate, column temperature, and mobile phase pH. The actual flow rate of the mobile phase was 0.7 mL/min, which was changed (0.6-0.8 mL/min). The effect of column temperature on analysis was 31 and 35 C. The optimized mobile phase pH was 2.5, which was changed by ±0.2.
Standard and spiked samples were injected into the LC instrument. All the aforementioned parameters did not show any significant differences in identified impurities. The experimental data are presented in Table 4.

| CONCLUSION
A high-sensitive, accurate, linear, specific, and robust analytical method was developed and validated to determine favipiravir degradation and identified impurities in the tablet dosage form. The mass values of degradation impurities were identified using the LC-MS technique. The proposed method has been validated per current ICH guidelines. The forced degradation studies identified three unknown degradation impurities in the presence of acid, base, water, and oxidation conditions. Therefore, it is sensitive to chemical stress conditions. Per solid-state degradation study results, no degradation occurred. The molecule was found to be stable at heat, humidity, and UV light conditions. According to method validation results, this method is typically able to estimate favipiravir impurities in the finished dosage forms and drug substances. Now in this pandemic situation, this drug product is more helpful to treat SARS-CoV-2 patients. Therefore, in the present pandemic situation, this research work contributes more to developing the efficient drug product of favipiravir. Therefore, for quality products, this method can be used.
The current method is cost effective and simple for quality control analysis.