INTRODUCTION:

Favipiravir, is chemically, 6-fluoro-3-oxo-3,4-dihydropyrazine-2-carboxamide, Formula, C₅H₄FN₃O₂ (molecular weight 157.1 g/mol). It becomes a new influenza drug. favipiravir is a modified pyrazine analog that was initially approved for therapeutic use in resistant cases of influenza. The antiviral targets RNA-dependent RNA polymerase (RdRp) enzymes, which are necessary for the transcription and replication of viral genomes. Favipiravir inhibit replication of influenza A and B, but the drug has shown promise in the treatment of avian influenza, and may be an alternative option for influenza strains that are resistant to neuramidase inhibitors. Favipiravir has been investigated for the treatment of life-threatening pathogens such as Ebola virus, Lassa virus, and now COVID-19.

Active pharmaceutical dosages are urgently required for rising COVID-19 pandemic conditions on global health¹.

Pharmaceutical dosages such as remdesivir, chloroquine, and favipiravir are currently undergoing clinical test in different countries for treating coronavirus disease ^2,3. So far, there is no any specific drug available for the treatment of corona virus due to there is not enough evidence⁴.

It exhibit antiviral activity against alpha-, filo-, bunya-, arena-, flavi-, and noroviruses ^{5, 6} as well as being active against the influenza virus.

In a pilot trial by Wuhan University, it was observed a good recovery rate in corona virus patients in the favipiravir compared to the other drugs ⁷, favipiravir is considered as better for as a potential candidate drug for this disease.

According to the literature search, there are two published high performance liquid chromatography (HPLC) methods for determining favipiravir assay and impurities in active pharmaceutical ingredients^8-11andpotentiometric titration¹². In both of these methods, a gradient HPLC mode was used for chromatographic separation and the run time was 60 min. favipiravir is not officially available in any pharmacopoeia and there is still a need for validated HPLC and other methods to determine favipiravir in pharmaceutical dosages.

Simple, rapid and reliable non aqueous titration method is developed for the determination of favipiravir. This method can be used for the routine analysis. In the proposed methods optimization and validation of this method are reported.

Structure of Favipiravir:

EXPERIMENTAL:

Instrumentation:

An potentiometric titrator was used (Lab- India-Auto titrator) for assay method development and validation. A Shimadzu analytical balance with 0.01 mg was used.

Reagents and chemical:

Reference standard of favipiravir was obtained from reputed firm with certificate of analysis.

Potassium hydrogen phthalate, perchloric acid, mercury acetate and glacial acetic acid of A. R. grade were used.

General procedure:

Standardization of 0.1 N perchloric acid:

About 0.350 mg of potassium hydrogen phthalate (previously powdered lightly, dried at 120^oC for 2 hours) was weighed accurately into clean and dry titration jar. It was dissolved in 50 ml of glacial acetic acid. It was titrated with 0.1 N perchloric acid by using auto titrator. Blank determination was performed out for necessary correction. The titration was performed in duplicate.

One ml of 0.1 N HClO₄ is equivalent to 0.2042 g. of potassium hydrogen phthalate (C₈H₅KO₄)

Normality of perchloric acid = ---------------

B.R. x 0.2042

Where, W is weight of potassium hydrogen phthalate in g. B.R. is burette reading in ml.

Quantitative determination of Favipiravir:

About 0.200 g. of favipiravir test sample was weighted accurately into a clean and dried titration jar. It was dissolved in 60 ml. of anhydrous glacial acetic acid. It was heated to dissolve favipiravir.

It was titrated with 0.1 N perchloric acid potentiometrically.

Blank determination was also carried out for necessary correction.

One ml of 1 N perchloric acid is equivalent to 0.00523 g. of favipiravir.

% (Percentage) Favipiravir on the dried basis was calculated as below.

B.R. x N x 0.00523 x 100 x dilution factor

% assay = _____________________________

Where, B.R. is burette reading in ml at the potentiometric end point.

N is actual normality of 0.1 N perchloric acid.

W is weight of the sample taken in g.

RESULT AND DISCUSSION:

Determination of Favipiravir:

The objective of this work was to determine accurately the content of favipiravir sulphate. The assay of favipiravir (on the dried basis) of various batches of test sample was analyzed using the above method. It was in the range of 99.33 to 101.32 %.

Analytical method validation:

The method precision was checked after analyzing six different preparations of homogeneous test sample of favipiravir. The % RSD of results obtained was found to be 0.7634. It confirms good precision of the method. The results are presented in table 1.

Table No. 1: Method of precision

Sr. No	Weight of favipiravir	Burette reading in ml	Normality of perchloric acid	% assay
1	0.2	3.8	0.1007	100.06
2	0.202	3.81	0.1007	99.33
3	0.199	3.79	0.1007	100.30
4	0.197	3.79	0.1007	101.32
5	0.202	3.81	0.1007	99.33
6	0.201	3.8	0.1007	99.56
			Mean	99.98
			Std. Deviation	0.7633
			RSD	0.7634

Linearity:

For the establishment of method linearity, five different weights of favipiravir test samples corresponding to 20 %, 40%, 60%, 80%, 100 % and 120 % of the about weight (0.500 g.) were taken and analyzed for % (percentage) of favipiravir content. The results are in table 2.

Table No.2: Linearity

Sr. No.	Weight of Favipiravir in gm	Burette reading	Normality of Perchloric acid	% Assay
1	0.1	1.9	0.1007	100.0656
2	0.2	3.8	0.1007	100.0656
3	0.3	5.71	0.1007	100.2411
4	0.4	7.59	0.1007	99.93392
5	0.5	9.51	0.1007	100.1709
	0.6	11.4	0.1007	100.0656
			Std. Deviation	0.117043
			RSD	0.116966

The potentiometric titration was conducted once at each level. Linearity curve Figure no .1 was drawn by plotting test sample weight in gram on x axis and titre values on y axis.

Fig. no.1: Linearity curve

The values of correlation coefficient, slope and intercept are given in table 3.

Table No.3: Regression values

Parameter	Values
Slope	19.000
Intercept	4E-15
Coefficient of co-relation	0.9999

Accuracy and recovery

Accuracy was determined at five different levels i.e., 20 %, 40%, 60%, 80% and 100 % of the nominal concentration. (0.500 g.) The titration was conducted in triplicate at each level and the titre value was recorded. The tire value obtained in linearity study was considered as true value during the calculation of percentage (%) recovery. The percentage recovery is calculated using following equation.

Titre value x 100

Percentage recovery = ---------------------------

True titre value

The percentage range recovery of Favipiravir was in 99.65 to 100.08 %. It confirms the accuracy of the proposed method. (Table 4).

Table No 4: accuracy and precision

Level no.	Weight of Favipiravir added	Weight of Favipiravir found	% Assay	Mean % assay
1	0.1	0.100066	100.0656	100.0847
	0.101	0.100592	99.59629
	0.1	0.100592	100.5923
2	0.2	0.200131	100.0656	99.6563
	0.202	0.200658	99.33556
	0.201	0.200131	99.56775
3	0.3	0.300197	100.0656	99.7922
	0.301	0.300723	99.90812
	0.302	0.300197	99.4029
4	0.4	0.400789	100.1973	99.94791
	0.402	0.400262	99.56775
	0.401	0.401316	100.0787
5	0.5	0.500328	100.0656	99.90116
	0.501	0.500855	99.97098
	0.502	0.500328	99.66692

Ruggedness:

The ruggedness of the method is defined as degree of reproducibility of results obtained by analysis of favipiravir sample under variety of normal test conditions such as different laboratories, different analysts and different lots of reagents. Quantitative determination of favipiravir was conducted potentiometrically on one laboratory. It was again tested in another laboratory using different instrument by different analyst. The assays obtained in two different laboratories were well in agreement. It proved ruggedness of the proposed method.

CONCLUSION:

The proposed method of non-aqueous potentiometric titration was found to be precise, accurate and rugged. The values of percentage recovery and standard deviation showed sensitivity. The method was completely validated. It showed satisfactory data for all the parameters of validation. Hence it can be applied for routine quality control application.

REFERENCES:

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8. Ibrahim Bulduk, HPLC-UV method for quantification of favipiravir in pharmaceutical Formulations, Acta Chromatographica 2020:1-7 DOI: 10.1556/1326.2020.00828.

9. R. Suzuki, Y. Osaka, Quantitative Analysis of Favipiravir Spiked in Plasma Using by HPLC, Shimadzu excellence in science, application news no L 570, http://www.shimadzu.com/about/trademarks/index.html.

10. China patent. A kind of Favipiravir has the HPLC assay method of related substance. 21.09.2016. (CN104914185B)

11. China patent HPLC method for measuring related substances in Favipiravir. 16.09.2015. (CN104914185A).

12. Rele Rajan V. Tiwatane Prathamesh P. Determination of Favipiravir from Pharmaceutical Dosage Form by Extractive Ion Pair Complex Colorimetric Method, Asian Journal of Chemistry, 14(5);2021:321-323

Received on 12.10.2021 Modified on 20.11.2021

Asian J. Research Chem. 2022; 15(1):49-51.

DOI: 10.52711/0974-4150.2022.00007