A novel stability-indicating HPLC-DAD method for determination of favipiravir, a potential antiviral drug for COVID-19 treatment; application to degradation kinetic studies and in-vitro dissolution profiling
“…Moreover, the studied drug was subjected in the reported methods to forced degradation study using different conditions resulting in different degradation pathway. (Gökce, 2021 ; Lingabathula, 2021 ; Marzouk et al 2022 ; Mosaad I. Morsy et al 2021 ).…”
It is critical to characterize the degradation products of therapeutic drugs to determine their safety as these degradation products may possess fatal effects on the human physiological system. Favipiravir (FVP), a novel anti-Covid-19 drug, that is recently used all over the world with a great impact on humanity was our target to explore more about its toxicity, the margins of its safety, and its degradants in different degradation conditions. The goal of this study is to identify, characterize, and confirm the structures of FVP oxidative and alkaline breakdown products, as well as to assess their safety utilizing in-vitro SRB cytotoxicity assay on normal human skin fibroblasts (NHSF) cell lines. After oxidative and alkaline degradation of FVP, one degradation product was produced in each condition which was isolated from FVP using flash chromatography, characterized by 1HNMR and LC–MS/MS techniques. A reversed-phase Thermo Fischer Hypersil C18 column (4.6 × 150 mm, 5 m) was used to achieve HPLC chromatographic separation. Acetonitrile-5 mM potassium dihydrogen phosphate (pH 2.5) (50:50, v/v) was employed as the mobile phase, with a flow rate of 1 mL/min. At 332 nm, the column effluent was measured. Over the concentration range of 0.5–100 µg/mL, the calibration curve was linear. The intra-day and inter-day relative standard deviations were less than 2%, and good percentage recoveries were obtained that fulfilled the acceptance criteria of the International Conference on Harmonization (ICH) recommendations. The Plackett–Burman design was used to assess the robustness. Each degradant was isolated single using Flash chromatography and methylene chloride: methanol gradient mobile phase. The chemical structures of the degradation products have been confirmed and compared to the intact FVP using 1H-NMR, and Mass spectroscopy. A postulated mechanism of the degradation process has been depicted and the degradants fragmentation pattern has been portrayed. In addition, the in vitro SRB cytotoxicity assay to evaluate the safety profile of FVP and the degradation end products showed their high safety margin in both conditions with IC50 ˃100 µg/ml with no signs of toxicity upon examination of the treated NHSF cells under the optical microscope
“…Moreover, the studied drug was subjected in the reported methods to forced degradation study using different conditions resulting in different degradation pathway. (Gökce, 2021 ; Lingabathula, 2021 ; Marzouk et al 2022 ; Mosaad I. Morsy et al 2021 ).…”
It is critical to characterize the degradation products of therapeutic drugs to determine their safety as these degradation products may possess fatal effects on the human physiological system. Favipiravir (FVP), a novel anti-Covid-19 drug, that is recently used all over the world with a great impact on humanity was our target to explore more about its toxicity, the margins of its safety, and its degradants in different degradation conditions. The goal of this study is to identify, characterize, and confirm the structures of FVP oxidative and alkaline breakdown products, as well as to assess their safety utilizing in-vitro SRB cytotoxicity assay on normal human skin fibroblasts (NHSF) cell lines. After oxidative and alkaline degradation of FVP, one degradation product was produced in each condition which was isolated from FVP using flash chromatography, characterized by 1HNMR and LC–MS/MS techniques. A reversed-phase Thermo Fischer Hypersil C18 column (4.6 × 150 mm, 5 m) was used to achieve HPLC chromatographic separation. Acetonitrile-5 mM potassium dihydrogen phosphate (pH 2.5) (50:50, v/v) was employed as the mobile phase, with a flow rate of 1 mL/min. At 332 nm, the column effluent was measured. Over the concentration range of 0.5–100 µg/mL, the calibration curve was linear. The intra-day and inter-day relative standard deviations were less than 2%, and good percentage recoveries were obtained that fulfilled the acceptance criteria of the International Conference on Harmonization (ICH) recommendations. The Plackett–Burman design was used to assess the robustness. Each degradant was isolated single using Flash chromatography and methylene chloride: methanol gradient mobile phase. The chemical structures of the degradation products have been confirmed and compared to the intact FVP using 1H-NMR, and Mass spectroscopy. A postulated mechanism of the degradation process has been depicted and the degradants fragmentation pattern has been portrayed. In addition, the in vitro SRB cytotoxicity assay to evaluate the safety profile of FVP and the degradation end products showed their high safety margin in both conditions with IC50 ˃100 µg/ml with no signs of toxicity upon examination of the treated NHSF cells under the optical microscope
“…Pyrazine carboxamide is a derivative that exhibits antiviral activity against RNA 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.…”
Favipiravir finished dosage was approved for emergency use in many countries to treat SARS-CoV-2 patients. A specific, accurate, linear, robust, simple, and stabilityindicating 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 Â 4.6 mm, 5-μm particle) and mobile phase. Mobile phase A contained KH 2 PO 4 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.
“…Thus, the term of pseudo first-order kinetics was given to these reactions. From the slopes of the straight lines, we could calculate the relevant degradation rate constant and half-life (t 1/2 ) for each degradation condition by applying the following equations ( Mourad et al, 2016 ; Gawad and Belal; 2017 ; Marzouk et al, 2022 ): logC t = logC 0 - kt/2.303 t 1/2 = 0.693/k where, C t and C 0 are RMD concentrations estimated at a given time t and at zero time, respectively, k is the apparent first order rate constant and -K/2.303 is the slope of the line. Fig.…”
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