INTRODUCTION:

Favipiravir is a pyrazine analogue¹ (Fig.1). Favipiravir (T-705) is a synthetic prodrug that was initially identified when testing the antiviral efficacy of substances in Toyama Chemical's chemical library that were effective against the influenza virus². The RNA-dependent RNA polymerase (RdRp) enzyme uses this molecule as a substrate; however, the enzyme misinterprets it for a purine nucleotide, which inhibits the enzyme's activity and stops the creation of viral proteins³.

Favipiravir is a virucidal medication since it also causes lethal mutagenesis in vitro when the influenza virus is infected⁴. As of right now, COVID-19 and other infections that pose a serious threat to human life are being treated with favipiravir⁵. It was one of the widely used medications for COVID 19 infection, used in conjunction with other treatments such remedisvir, azithromycin, plasma therapy, stem inhalation, gargles, and the seven point healthy lifestyle⁶.

There are very few methods documented for determining Favipiravir in pharmaceutical formulation by, UV spectrophotometry⁷, and there is no official method accessible in the Pharmacopoeia for the estimation of Favipiravir in Pharmaceutical dosage form. UV spectrophotometric method is simple, accurate, precise and economic⁸. Thus, validated UV spectrophotometric techniques are still required to identify Favipiravir in pharmaceutical formulations. Thus, in the current work, an effort is made to establish a simple, specific, and accurate analytical approach for estimating favipiravir using UV spectrophotometric techniques.

Chemical names of Favipiravir:^9-10

6-fluoro-3-hydroxypyrazine-2-carboxamide

6-fluoro-3-oxo-3,4-dihydopyrazine-2-carboxamide avigan

5-fluoro-2-oxo-1H-pyrazine-3-carboxamide

6-fluoro-3-hydroxy-2-pyrazinecarboxamide

Molecular Formula: C₅H₄FN₃O₂

Molecular Weight: 157.10

Fig 1: Structure of Favipiravir

MATERIALS AND METHODS:

Chemicals:

Favipiravir standard (Gift sample from Hetero Pharmaceuticals, Hyderabad), Analytical grade Ethanol (SD fine chemicals, Mumbai).

Instrumentation:

Double beam UV spectrophotometer; Model: SL 210; Make: ELICO.The data was obtained using Spectra Treats 3.11.01Rel 2b software, CONTECH electronic balance.

Method:

Solvent Selection:

Favipiravir is soluble in ethanol and partially soluble in water. So 90% Ethanol and water system was selected as a solvent for analysis of Favipiravir.

Preparation of Standard stock solution:

Standard stock solution was prepared by dissolving 100 mg of drug in 100ml of ethanol to get concentration of 1mg/ml (1000µg/ml) solution.

Preparation of working Standard:

The prepared stock solution was further diluted to get working standard solution of 100µg/ml of Favipiravir. The stock solution was used to prepare various concentration from 0.5-25µg/ml by dilution with water.

Selection of wavelength for analysis of Favipiravir:

Appropriate volume of 2ml of working standard solution of Favipiravir was transferred into 10ml volumetric flask, diluted with water up to the mark to give a concentration 20µg/ml. The resulting solution was scanned between 200-400nm. Absorption maxima of 228 nm is selected for further analysis (Figure 2).

Validation of UV Spectroscopy Method:

Standard Calibration Curve (Linearity):

A calibration curve was plotted over a concentration range of 0.5-25μg/ml for Favipiravir at 228nm. Accurately measured volume of working stock solution of Favipiravir at 228nm were transferred to separate series of 10ml volumetric flask and diluted up to the mark with water to obtain a concentration series of 0.5-25μg/ml. The absorbance of all solution was taken at 228 nm wavelength. The calibration curve was constructed by plotting concentration against absorbance (Figure. 3).

Figure 2: UV Spectrum of Favipiravir

Method Validation:

Present study was conducted to obtain a new, affordable, cost-effective and convenient method for spectroscopic determination of Favipiravir. The method was validated for the parameters like linearity, accuracy, precision and robustness as per ICH guidelines¹¹.

Estimation of Favipiravir in commercial formulation:

Ten tablets of Commercial brand (FABIFLU 400) were weighed, powdered and a quantity of formulation equivalent to 10mg of drug was weighed and transferred to 10ml volumetric flask, dissolved in 100ml of analytical grade ethanol. The solution was filtered through Whatmann filter paper and it is suitably diluted with distilled water to obtain the concentration of 10μg/ml. The absorbance of the solution was measured at a Wavelength of 228nm. The % purity of Favipiravir in the tablet was calculated from regression equation obtained from the calibration curve.

RESULTS:

Linearity:

Standard stock solution of drug was prepared by dissolving 10mg of drug in 10ml of ethanol and future volume was made up with water till 10ml to get concentration of 1000µg/ml. Further 1ml was taken and diluted to 10ml with water in volumetric flask to get 100 μg/ml. This solution was further diluted with water to get range of solution containing different concentrations 0.5-25μg/ml. Absorbance was noted at λ max 228nm. The equation of calibration curve by UV- Spectroscopy was found to be y = 0.0749 x - 0.0044 with R² = 0.9995. The absorbance of drug was plotted against the corresponding concentrations to obtain the calibration curve as shown in Figure 3.

Figure 3: Calibration Curve of Favipiravir

Precision:

The intraday and interday precision of the proposed method were performed by analyzing the corresponding responses 6 times on the same day and on 6 different days of same concentration of standard solutions of Favipiravir without changing the parameters for the method. The results were reported in terms of relative standard deviation (% RSD). The results obtained for intraday and inter-day variations by UV-Spectroscopy are shown in Table 1.

Table 1: Intra-Day Precision and Inter-Day Precision of the developed Method

Sample No	Absorbance (nm)	Day	Absorbance (nm)
1	0.7768	Day 1	0.7768
2	0.7821	Day 2	0.7789
3	0.7802	Day 3	0.7753
4	0.7726	Day 4	0.7762
5	0.7765	Day5	0.7765
6	0.779	Day6	0.7769
Mean	0.7778	Mean	0.7767
SD	0.003038	SD	0.001089
%RSD	0.003906%	%RSD	0.001402%

Accuracy:

To check the accuracy of the method, recovery studies were carried out by addition of standard drug solution to preanalysed sample solution at three different levels 50%, 100% and 150%. The drug concentrations and % recovery was determined from linear equation. Results obtained are shown in Table 2.

Table 2: Accuracy of Developed Method

S. No	Level	Concentration in µg/ml		Mean	% Recovery
S. No	Level	Spiked	Sample	Mean	% Recovery
1	50%	5	10	1.1439	99.7%
2	100%	10	10	1.5506	99.7%
3	150%	15	10	1.9133	99.8%

Robustness:

Robustness of the method was determined by carrying out the analysis under conditions during which detection wavelength (±2nm) was altered and the effect on the absorbance was noted. The method was found to be robust. The result of the robustness for Favipiravir is shown in Table 3.

Table 3: Robustness of Developed Method

S. No	Wave length	Percentage Recovery	% RSD
1.	227	99.35 ± 0.0004	0.053
2.	229	99.53± 0.001	0.136

Ruggedness:

Ruggedness of an analytical method is the degree of reproducibility of test results obtained by the analysis of the same samples in different laboratories, different instruments and different analyst.

The result of the ruggedness for Favipiravir is shown in Table 4

Table 4: Ruggedness of Developed Method

S. No	S. No	Percentage Recovery	% RSD
1.	Analyst 1	99.68 ± 0.003	0.361
2.	Analyst 2	100 ± 0.003	0.434

Limit of Detection (LOD) and Limit of Quantification (LOQ):

From the linearity data the LOD and LOQ as calculated, using the formula LOD = 3.3 σ/S and LOQ = 10 σ/S, where σ = standard deviation of the y intercept of linearity equations and S =slope of the calibration curve of the analyte. The LOD and LOQ by UV- Spectroscopy were found to be 0.04 μg/ml and 0.13 μg/ml, respectively.

Estimation of Favipiravir in commercial formulation:

Favipiravir present in Commercial formulation (FABIFLU 400) was found to be 100.2%

DISCUSSION:

Ultraviolet spectroscopy is an absorption spectroscopy in which the molecule absorbs ultra violet light with a wavelength between 200 and 400nm. All molecules include either either n, π or σ or electrons. The radiation is absorbed by either bonded (and) or non-bonding (n) electrons, which then move from the ground state to the excited state¹². By the absorption peaks obtained for a particular molecule and the nature of the electron present in that particular molecule, the molecular structure of a molecule can be elucidated¹³.

The pharmaceutical sector relies extensively on analytical method validation to publish information on commercial batches and long-term stability. As a result, the data must be created in accordance with recognised scientific criteria. Hence, all analytical procedures should be thoroughly validated and documented in order to comply with regulatory authority regulations¹⁴. The method was validated according to ICH guidelines with respect to Linearity, precision, accuracy, ruggedness, limit of detection, limit of quantification, robustness and ruggedness. The calibration curve for the methods were linear over concentration range 0.5-25μg/ml for Favipiravir at 228nm. It was broad compared to earlier Methods¹⁵. The method was found to be precise and as the % RSD value for Intra and Inter day were found to be less than ±2%. The accepted limits of accuracy (recovery) were found to be 90% - 120% and all observed data are within required range which indicates good recovery value. The result of, robustness indicted the small change in the condition did not significantly affect the determination of Favipiravir. The LOD and LOQ were found to be 0.13µg/ml and 0. 40µg/ml. The method was also economic compared to earlier methods ¹⁶.

CONCLUSION:

A simple, accurate, precise and cost Effective UV-spectroscopic method has been developed for the estimation of Favipiravir. The proposed method is successfully applied for estimation of Favipiravir in any formulation. The method can be used for the routine quality control analysis of Favipiravir.

ACKNOWLEDGEMENT:

We are thankful to RBVRR Women’s College of Pharmacy for supporting and encouraging us to carry out the research.

CONFLICT OF INTEREST:

The authors declare that they don’t have any conflict of interest.

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Received on 11.03.2023 Modified on 12.10.2023

Asian J. Pharm. Ana. 2024; 14(2):65-68.

DOI: 10.52711/2231-5675.2024.00012