Stability Indicating RP-HPLC Method for the estimation of Favipiravir in API and Pharmaceutical Dosage Forms (Tablets)

 

N S Yamani, Mukthinuthalapati Mathrusri Annapurna*

Department of Pharmaceutical Analysis,

GITAM School of Pharmacy, Gandhi Institute of Technology and Management, Visakhapatnam, India.

 

ABSTRACT:

Favipiravir is an anti-viral agent used for the treatment of a large number of RNA viruses including influenza and was considered for the treatment of COVID-19. A new stability indicating RP-HPLC has been developed for the estimation of Favipiravir and the method was validated. Hypersil BDS C18 column was used for the chromatographic study with a flow rate 1ml/min (Detection wavelength 230nm). The LOD and LOQ were found to be 0.2918μg/mL and 0.9207μg/mL respectively. Linearity was observed over the concentration 1-300μg/mL with linear regression equation, y = 28444x + 21853 (R² = 09998) and the method is precise, accurate and robust. Stress degradation studies were performed and the method was found to be selective and specific.

 

 

INTRODUCTION:

Favipiravir (CAS no. 259793-96-9) is an oral pyrazine carboxamide derivative and chemically 6-fluoro-3-hydroxy-2-pyrazine-2-carboxamide¹. Favipiravir (Figure 1) has the molecular formula C₅H₄FN₃O₂ with molecular weight 157.104g/mol. It is a potent RNA-dependent RNA polymerase inhibitor and effective against RNA viruses it was approved² for the influenza treatment and currently used for the treatment of COVID-19. Several analytical methods such as UPLC-MS/MS, LC-MS/MS, HPLC, have been published in the literature for the quantification of Favipiravir in pharmaceutical formulations as well as biological fluids. Mamdouh et al., developed a UPLC-MS/MS method³ for the determination of Favipiravir in human plasma using Xevo TQD LC–MS/MS and Acquity UPLC® HSS C18 column operated under the multiple-reaction monitoring mode (ESI) in presence of an internal standard Lamivudine.

 

 

Mobile mixture of ammonium formate and methanol was pumped with a flow rate of 0.35ml/min on gradient mode and the linearity was observed the concentration range 0.25-16μg/ml. Abd Allah et al., developed a UPLC-MS/MS method⁴ for the determination of Favipiravir in human plasma using triple quadrupole tandem mass spectrometer and Acquity UPLCr BEH HILIC column in presence of an internal standard Valproic acid. Mobile mixture of acetonitrile and 0.005% ammonia in water (75:25) was pumped with a flow rate of 0.25ml/min on isocratic mode and the linearity was observed the concentration range 50-10,000ng/ml. Morsy et al., developed a LC-MS/MS method⁵ for the determination of Favipiravir for the emergency treatment of SARS-CoV-2 virus in human plasma in Egyptian human volunteers using API4500 triple quadrupole tandem mass spectrometer and Eclipse plus C₁₈ column operated under the multiple-reaction monitoring mode (ESI) in presence of an internal standard Pyrazinamide. Mobile mixture of methanol and 0.2% acetic acid (20:80) was used with a flow rate of 0.6 ml/min on isocratic elution mode and the linearity was observed the concentration range 100.0-20000.0ng/mL. Eryavuz Onmaz et al., developed a LC-MS/MS method⁶ for the determination of Favipiravir in human serum using RP Phenomenex C18 column operated under the positive and negative multiple-reaction monitoring mode (ESI). Mobile mixture of 0.1% formic acid in water and 0.1% formic acid in methanol was pumped with a flow rate of 0.35ml/min on gradient mode and the linearity was observed the concentration range 0.048-50µg/mL (in negative ionization mode) and 0.062-50µg/mL (in positive ionization mode). Divya Kumar et al., developed a stability indicating HPLC method on gradient mode and a LC-MS/MS techniques7 for the identification of two unknown impurities found during the degradation studies of Favipiravir film-coated tablets. The LC-MS technique was used with Electrospray positive ionization and the structures of major degradation impurities were identified. The impurities were separated using the HPLC technique with a mobile phase mixture of A and B. The mobile phase A consists of KH2PO4 buffer (pH 2.5±0.05) and acetonitrile (98:2) and mobile phase B consists of water and acetonitrile (50:50) with 0.7mL/min flow rate (UV detection, 210nm). The LC-MS studies were performed using a Waters TQD triple Quadrupole mass spectrometer in positive ionization mode keeping mass range at m/z 90–500. Mobile phase A consisting of 0.01 M ammonium acetate (pH 2.5) and acetonitrile (98:2) and mobile phase B consisting of acetonitrile and water (1:1) were used. İbrahim developed a HPLC method⁸ for the quantification of Favipiravir in pharmaceutical formulations using Inertsil ODS-3V C18 column with mobile mixture of 50mM potassium dihydrogen phosphate (pH 2.3) and acetonitrile (90:10) at a flow rate of 1ml/min (Detection wavelength 323 nm) on isocratic mode and the linearity was observed the concentration range 10-100μg/ml. In the present study the authors have developed a new simple, precise and accurate stability indicating method for the quantification of Favipiravir in tablets and the method was validated as per ICH guidelines.

 

Figure 1: Chemical structure of Favipiravir

 

MATERIALS AND METHODS:

Dilute o-phosphoric acid solution was prepared by diluting 10mL of Conc. o-phosphoric acid with water in a 100mL volumetric flask and sonicated.

 

The buffer solution was prepared by accurately transferring 1.5grams of Ammonium di hydrogen phosphate and 6.8grams of Tetra butyl ammonium hydrogen sulphate into a 1000ml volumetric flask and diluted with water with the addition of 5ml of Tri ethyl amine. The pH was adjusted to 5.0 with dilute o-phosphoric acid solution. The resulting solution was filtered through 0.45µm nylon membrane filter and sonicated for degassing which is stable for about 2 days.

The diluent solution was prepared by accurately transferring 1.5grams of Ammonium di hydrogen phosphate and 5ml of Tri ethyl amine into a 1000ml volumetric flask and diluted with water. The pH was adjusted to 7.0 with dilute o-phosphoric acid solution. The resulting solution was filtered through 0.45µm nylon membrane filter and sonicated for degassing.

 

Instrumentation and Chromatographic conditions

Thermo Fisher HPLC system equipped with PDA detector and Hypersil BDS C₁₈ column (250mm × 4.6 mm, 5μm) was employed for the study. Mobile phase consisting of methanol and a mixture of Tetra butyl ammonium hydrogen sulphate with Ammonium di hydrogen phosphate (pH adjusted to 5.0 with dilute o-phosphoric acid) was chosen with a flow rate 1.0 mL/min for the chromatographic study and 10μl solution was injected and the run time was 25 minutes.

 

Preparation of stock solution of Favipiravir

25mg of Favipiravir was accurately weighed and transferred in to a 25mL volumetric flask and dissolved in HPLC grade methanol (1000μg/mL) and all other solutions were made from this stock solution with diluent and all the solutions were filtered through 0.45 μm membrane filter prior to injection.

 

Method validation⁹

Linearity, Precision, Accuracy and Robustness

A series of 1.0-300µg/mL Favipiravir solutions were prepared from the stock solution using the diluent and 10μl of each of these solutions were injected (n=3) into the HPLC system and the chromatograms were observed. The area under curve for each solution (n=3) was noted at the retention time and the mean peak area was calculated. Calibration curve was drawn by plotting the concentration of Favipiravir solutions on the x-axis and the corresponding mean peak area on the y-axis. Precision of the method was evaluated intra-day and inter-day precision studies. Three different concentration solutions (50, 100 and 200µg/mL) of Valsartan were prepared within the linearity range on the same day (intra-day precision) and on three consecutive days (inter-day precision) and the chromatographic study was performed. The mean peak area (n=3) and thereby the % RSD was calculated.

 

Accuracy of the method was measured by spiking the drug formulation solution (50, 100, 150%) with a known concentration of standard drug (n=3). These studies were carried out and finally the % RSD was calculated from the values obtained followed by the percentage recovery from the linear regression equation. The robustness of the method was proved by incorporating a very small changes in the optimized chromatographic conditions such as pH, mobile phase composition (± 2% v/v) and flow rate (± 0.1 mL) and detection wavelength (± 2 nm).

 

Stress degradation studies¹⁰

Stress degradation studies were performed to determine the stability of Favipiravir towards stress conditions such as acidic hydrolysis, basic hydrolysis, oxidation and thermal degradation and also to identify the degradation pathway. Acidic degradation was performed by treating Favipiravir drug solution (1mg/ml) with 2 mL of 1.0 N HCl solution at room temperature (25⁰C) for 4 hours. The stressed sample was then neutralized with 2mL of 1.0 N sodium hydroxide solution, sonicated for 20 minutes, filtered through 0.45µm nylon filter and then diluted with the diluent as per the requirement and 10µl of the solution was injected in to the HPLC system. Alkaline degradation was performed by treating Favipiravir drug solution (1mg/1ml) with 2mL of 5.0 N sodium hydroxide solution at room temperature (25⁰ C) for 4 hours. The stressed sample was then neutralized with 2mL of 5.0 N HCl solution, sonicated for 20 minutes, filtered through 0.45µm nylon filter and then diluted with the diluent as per the requirement and 10µl of the solution was injected in to the HPLC system. Thermal degradation was performed by heating Favipiravir drug solution (1mg/1ml) was kept in oven at 60⁰C for 4 hours. The stressed sample was then sonicated for 20 minutes, filtered through 0.45µm nylon filter and then diluted with the diluent as per the requirement and 10µl of the solution was injected in to the HPLC system. Oxidative degradation was performed by treating Favipiravir drug solution (1mg/1ml) with 2 mL of 10% H₂O₂ solution at room temperature (25⁰ C) for 4 hours. The stressed sample was then sonicated for 20 minutes, filtered through 0.45µm nylon filter and then diluted with the diluent as per the requirement and 10µl of the solution was injected in to the HPLC system. Neutral stress degradation was performed by treating Favipiravir drug solution (1mg/1ml) with 2ml of water and was kept at room temperature (25⁰C) for 4 hours. The stressed sample was then sonicated for 20 minutes, filtered through 0.45µm nylon filter and then diluted with the diluent as per the requirement and 10µl of the solution was injected in to the HPLC system. Sunlight stress degradation was performed by exposing Favipiravir drug solution (1mg/1ml) to sunlight for 4 hours. The stressed sample was then sonicated for 20 minutes, filtered through 0.45µm nylon filter and then diluted with the diluent as per the requirement and 10µl of the solution was injected in to the HPLC system.

 

Assay of Favipiravir tablets

Favipiravir is available as tablets with brand names Fabi flu (Label claim: 200mg) (Glenmark Pharmaceuticals Pvt. Ltd), Ciplenza (Label claim: 200mg) (Cipla Ltd), Favicovid 200 (Label claim: 200 mg) and favicovid 800 (Label claim: 800 mg) (Optimus Drugs Pvt Ltd) and Feravir-200 (Label claim: 200 mg) & Feravir-400 (Label claim: 400mg) (Xenon Pharma) in India. Twenty tablets of Favipiravir were powdered and powder equivalent to 25mg of Favipiravir was accurately weighed from two different brands and extracted in to two different 25ml volumetric flasks with methanol. The resulting mixture was sonicated, filtered and then diluted with the methanol. The dilutions were made with diluent and 10 µL of each of these two branded solutions were injected (n=3) in to the HPLC system and the peak area was noted from the resultant chromatogram. The mean peak area was calculated and the assay was quantified from the calibration curve.    

 

RESULTS AND DISCUSSION:

Favipiravir is an anti-viral drug. A new stability indicating RP-HPLC method has been proposed for the estimation of Favipiravir in pharmaceutical dosage forms i. e. tablets. A brief review of the literature on the analytical methods so far developed was given in Table 1.

 

Table 1: Literature survey of Favipiravir          

Reagent / Mobile phase (v/v)	Linearity (µg/mL)	Observations	Ref
Ammonium formate: Methanol (Gradient mode); Lamivudine (Internal standard)	0.25-16	UPLC-MS/MS Human plasma	3
Acetonitrile: 0.005% Ammonia in water (75:25) (Isocratic mode); Valproic acid (Internal standard)	0.05-10	UPLC-MS/MS Human plasma	4
Methanol: 0.2 % acetic acid (20:80) (75:25) (Isocratic mode) Pyrazinamide (Internal standard)	0.1-20	LC-MS/MS Human plasma	5
0.1% Formic acid in water: 0.1% Formic acid in Methanol (Gradient mode)	0.048-50 (Negative ionization mode) 0.062-50 (Positive ionization mode)	LC-MS/MS Human serum	6
Mobile phase A:  Phosphate buffer (pH 2.5): Acetonitrile (98:2) Mobile phase B: Water: Acetonitrile (50:50)   Mobile phase A: 0.01 M Ammonium acetate (pH 2.5): Acetonitrile (98:2) Mobile phase B: Acetonitrile: Water (1:1)	-   -	RP-HPLC   LC-MS	7
50 mM Phosphate buffer (pH 2.3): Acetonitrile (90:10) (Isocratic mode)	10-100	RP-HPLC	8
Mobile phase A: Ammonium di hydrogen phosphate: Tetra butyl ammonium hydrogen sulphate: Tri ethyl amine (pH 5.0 adjusted with o-phosphoric acid) Mobile phase B: Methanol	0.1-50	RP-HPLC Stability indicating	Present method

 

Method optimization:

Initially a 10µg/mL of Favipiravir was injected in to the HPLC system with Hypersil BDS C18 column with mobile phase mixture Ammonium di hydrogen phosphate: Methanol where Favipiravir was eluted at 11.389 min within the run time of 25 min. Then the ion pairing reagent, Tetra butyl ammonium hydrogen sulphate was incorporated in to the mobile phase mixture and the system was monitored by injecting the drug solution. The theoretical plates as well as the tailing factors were not within the acceptable criteria and therefore the pH adjustment was done using o-phosphoric acid (pH 5.0) and thereby Favipiravir was eluted at 5.665min. Finally, the optimised chromatographic conditions are:

 

Mobile phase A: Ammonium di hydrogen phosphate: Tetra butyl ammonium hydrogen sulphate: Tri ethyl amine (pH 5.0 adjusted with o-phosphoric acid); Mobile phase B: Methanol; flow rate 1.0ml/min and detection wavelength 230nm. The characteristic chromatograms of the placebo and Favipiravir standard were shown in Figure 2.

 

A) Placebo

B) Favipiravir (API) (Rt 5.665 min) Theoretical plates 16365 & Tailing factor 0.95

C) Favipiravir (Tablets) (Rt 5.657 min) Theoretical plates 16258 & Tailing factor 0.98

Figure 2: Typical chromatograms A) Placebo B) Favipiravir standard C) Favipiravir tablets (Labelled claim: 200 mg)

 

Method validation:

Favipiravir obeys Beer-Lambert’s law over the concentration range of 1-300 µg/mL (% RSD 0.22-0.61) (Table 2) and the linear regression equation was found to be y = 28444x + 21853 with correlation coefficient 0.9998 (Figure 3). The LOD and LOQ were found to be 0.2918 μg/mL and 0.9207μg/mL respectively. The % RSD was found to be 0.0022-0.0373 (Intraday) (Table 3) and 0.0039-0.0759 (Inter-day) (Table 4) in precision studies which is less than 2.0 indicating that the method is precise. In accuracy study the % recovery was found to be 98.99-99.88% and the % RSD was 0.72-1.01 (< 2%) (Table 5) indicating that the method is accurate. The % RSD in robustness study was found to be 0.42-1.03 which was less than 2% indicating that the method is robust (Table 6).

 

Table 2: Linearity study

*Mean of three replicates

 

Figure 3: Calibration curve of Favipiravir

 

Table 3: Intraday precision study

*Mean of three replicates

 

Table 4: Inter day precision study

*Mean of three replicates

 

Table 5: Accuracy study

*Mean of three replicates

 

 

 

 

 

 

Table 6: Robustness study

*Mean of three replicates

 

Assay of Favipiravir tablets:

The assay of Favipiravir was performed with two different marketed brands of two different pharmaceutical industries using the optimised chromatographic conditions and the percentage of purity was found to be 98.99-99.32 (Table 7) in tablet formulations. The individual chromatograms observed for the placebo and the Favipiravir tablet formulation were shown in Figure 2C.

 

Table 7:  Assay of Favipiravir tablets

*Mean of three replicates

 

Stress degradation studies:

A sharp peak of Favipiravir (API) was eluted at 5.665 min with theoretical plates 16365 (> 2000) and tailing factor 0.95 (<1.5) indicating that system suitability parameters are within acceptable criteria. During the acidic degradation Favipiravir was eluted at 5.664 min and has shown 1.38% degradation with theoretical plates 16245 (> 2000) and tailing factor 0.98. During the alkaline degradation Favipiravir was eluted at 5.660 min and has shown 10.76% degradation with theoretical plates 16342 (> 2000) and tailing factor 0.96. During the oxidative degradation Favipiravir was eluted at 5.660 min and has shown 4.54% degradation with theoretical plates 16239 (> 2000) and tailing factor 0.93. During the neutral degradation Favipiravir was eluted at 5.661 min and has shown 0.39% degradation with theoretical plates 16322 (> 2000) and tailing factor 0.91. During the thermal degradation Favipiravir was eluted at 5.664 min and has shown 0.02% degradation with theoretical plates 16349 (> 2000) and tailing factor 0.98. During the sunlight exposure Favipiravir was eluted at 5.660 min and has shown 4.68% degradation with theoretical plates 16328 (> 2000) and tailing factor 0.96. The % recovery during the degradation studies was shown in Table 8 and the respective chromatograms were shown in Figure 4.

Table 8: Stress degradation studies of Favipiravir (200μg/mL)

Stress condition (Temp 0C / Time min)	Rt (min)	% Recovery	% Drug degradation	Theoretical plates	Tailing factor
Standard drug	5.665	100	-----	16365	0.95
Acidic degradation 1.0N HCl/ Room temp 250/ 4 Hrs	5.664	98.62	1.38	16245	0.98
Alkaline degradation 5.0N NaOH/ Room temp 250/ 4 Hrs	5.660	89.24	10.76	16342	0.96
Oxidative degradation 10% H2O2/ Room temp 250/ 4 Hrs	5.660	95.46	4.54	16239	0.93
Neutral Stressed sample Room temp 250/ 4 Hrs	5.661	99.61	0.39	16322	0.91
Thermal degradation/ 600/ 4 Hrs	5.664	99.98	0.02	16349	0.98
Sunlight Exposure/ 4 Hrs	5.660	95.32	4.68	16328	0.96

*Mean of three replicates

 

 

A

 

 

B

 

 

C

 

 

D

 

 

E

 

 

F

Figure 4: Representative chromatograms of Favipiravir (200µg/mL) A) Acidic degradation B) Alkaline degradation C) Oxidative degradation D) Neutral degradation E) Thermal degradation F) Sunlight degradation

 

 

CONCLUSION:

The proposed RP-HPLC method for the estimation of Favipiravir was validated and was found to be precise, accurate and robust. The method was successfully applied for the estimation of Favipiravir tablets and the % recovery is good. The method is specific and during the degradation study Favipiravir peak was not interfered with any other degradants peak and the system suitability parameters were within the acceptable criteria. Favipiravir is more stable towards all stress degradation studies and less than 15% degradation was reported.

 

ACKNOWLEDGEMENT:

The authors are grateful to Glenmark Pharmaceuticals Pvt. Ltd, India for providing the gift samples of Favipiravir. There is no conflict of interest.

 

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Accepted on 14.12.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(12):5700-5706.