Application of reactive dyes in Validation of favipiravir from pharmaceutical dosages

 

Rele Rajan V.*, Tiwatane Prathamesh P.

Central research laboratory, D.G. Ruparel college Mahim Mumbai 400 016.

*Corresponding Author E-mail: drvinraj@gmail.com

 

 

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 nowadays 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 corona virus 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 noro-viruses^{5, 6} as well as being active against the influenza virus. In a pre-pilot trial by University of Wuhan, it was observed that a good recovery rate in corona virus patients in the favipiravir compared to the other drugs⁷, favipiravir is considered as better for as a potential drug for this disease.

 

According to the literature search, there are published work on high performance liquid chromatography (HPLC) methods for determining favipiravir assay and impurities in active pharmaceutical ingredients^8-12, colorimetric^13,14 and non-aqueous titration¹⁵. In HPLC method, 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 spectrophotometric methods with reactive dyes are 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. The proposed methods involve formation of ion pair complexes of favipiravir with reactive dyes like Congo red, eriochrome black T, methyl orange in acidic medium.

 

Structure of Favipiravir:

 

MATERIALS AND METHODS:

A SHIMADZU -160 A double beam UV-VISIBLE recording spectrophotometer with pair of 10mm matched quartz cell was used to measure absorbance of solutions. A SHIMADZU analytical balance was used.

Congo red, eriochrome black T, methyl orange, hydrochloric acid, potassium hydrogen phthalate and chloroform of A.R. grade were used in the study.

 

Preparation of standard solution and reagents:

Stock solution of favipiravir, (100 μg/ml) was prepared in ethanol. From this stock solution working standard (10 μg/ml) was prepared by diluting 10 ml stock solution to 100 m lwith ethanol. A 0.05 % w/v Congo red, 0.25 % eriochrome black T and 0.02 % methyl orange solutions were prepared in distilled water respectively.

Potassium hydrogen phthalate buffer solution of pH 4.01was prepared in distilled water. Dilute hydrochloric acid was used to adjust desired pH of buffer solution.

 

EXPERIMENTAL:

Method 1 (with Congo red):

Into a series of separating funnels appropriate amount of the working standard drug solutions were pipetted out. To each funnel 1.0 ml of buffer (pH= 3.7) and 5.0 ml of 0.05%w/v congo red were added. 10 ml of chloroform was added to each funnel. The solutions were shaken for thorough mixing of the two phases and were allowed to stand for clear separation of the layers.

The absorbance values of the chloroform layers were measured against their respective reagent blank at the wavelength of the maximum absorbance (λ max =490 nm.)

 

Method 2(with eriochrome black T):

Into a series of separating funnels appropriate amount of the working standard drug solutions were pipetted out. To each funnel 4.0 ml of buffer (pH = 3.7) and 3.5 ml of 0.25% w/v eriochrome black T were added. 10ml of chloroform was added to each funnel. The solutions were shaken for thorough mixing of the two phases and were allowed to stand for clear separation of the layers. The absorbance values of the chloroform layers were measured against their respective reagent blank at the wavelength of the maximum absorbance (λ max= 500 nm).

 

Method 3(with methyl orange):

Into a series of separating funnels appropriate amount of the working standard drug solutions were pipetted out. To each funnel 1.0ml of buffer (pH= 3.7) and 5.5 ml of 0.02% w/v methyl orange were added. 10ml of chloroform was added to each funnel. The solutions were shaken for thorough mixing of the two phases and were allowed to stand for clear separation of the layers. The absorbance values of the chloroform layers were measured against their respective reagent blank at the wavelength of the maximum absorbance (λ max =430 nm).

 

Estimation from tablets:

Twenty tablets of labelled claim 200 mg of favipiravir were weighed accurately. Average weight of each tablet was determined. Tablets were crushed into fine powder. An accurately weighed quantity of powder equivalent to 10 mg of favipiravir was transferred into a beaker and it was shaken with 50 ml of ethanol and filtered. The filtrate and the washing were collected in a 100.0 ml volumetric flask. This filtrate and the washing were diluted up to the mark with ethanol to obtain final concentration as 100 μg/ml. This solution was further diluted to give 10 μg/ml. Such solution was used for methods respectively.

 

Appropriate aliquots of drug solution were taken and the individual assay procedures were followed for the estimation of drug contents in tablets. The concentration of the drug in the tablets was calculated using calibration curve. The recovery experiment was carried out by standard addition method. Results of analysis of optical and regression of drug are given in table 1.

 

Table 1: Values of results of optical and regression of drug

 

RESULTS:

The extractive spectrophotometric methods are popular due to their sensitivity in assay of the drug and hence ion pair extractive spectrophotometric methods have gain considerable attention for quantitative determination of many pharmaceutical preparations. These proposed methods are extractive spectrophotometric methods for the determination of favipiravir, by using chloroform as solvent from its formulations. The colour ion pair complexes formed are very stable. The working conditions of these methods were established by varying one parameter at time and keeping the other parameters fixed by observing the effect produced on the absorbance of the colour species. The various parameters involved for maximum colour development for these methods were optimized. The proposed methods were validated statistically and by recovery studies. The molar absorptivity values showed the sensitivity of methods while the precision was confirmed by %RSD (relative standard deviation).The optical characteristics such as absorption maxima (nm), co-relation coefficient (r) were calculated and are also summarized in table 1. Assay results of recovery studies are given in table 2 A,B,C.

 

 

Table no 2:A (Congo red)

 

 

Table no 2:B (Eriochrome black T  )

 

 

Table no 2:C (methyl orange)

 

 

Results are in good in agreement with labelled value.

 

DISCUSSION:

The percent recovery obtained indicates non-interference from the common excipients used in the formulation. The reproducibility, repeatability and accuracy of these methods were found to be good, which is evidenced by low standard deviation. The proposed methods are simple, sensitive, accurate, precise and reproducible. They are directly applied to drug to form chromogen. Hence they can be successfully applied for the routine estimation of favipiravir, in bulk and pharmaceutical dosage form even at very low concentration and determination of stability of drug in formulation. The strong recommendation is made here for the proposed methods for determination of favipiravir, from its formulation.

 

ACKNOWLEDGMENTS:

Authors express sincere thanks to the Principal, of D.G. Ruparel college, for providing necessary facility for research work.

 

REFERENCES:

1.      Sohrabi, C., Alsafi, Z.;O’Neill, N.; Khan, M.; Kerwan, A.; Al-Jabir, A.;et al. World Health Organization declares global emergency: A review of the 2019 novel coronavirus (COVID-19), Int. J. Surg., 76;2020:, 71–76.

2.      Zhu, R. F.; Gao, R. I.; Robert, S. H.; Gao, J. P.; Yang, S. G.; Zhu, C. Systematic review of the registered clinical trials of coronavirus diseases 2019 (COVID-19), J. Transl. Med. 18; 2020: 274–279.

3.      Prajapat, M.; Sarma, P.; Shekhar, N.; Avti, P.; Sinha, S.; Kaur, H.; et al. Drug targets for corona virus: A systematic review. Indian J.Pharm. 52(1); 2020: 56–63.

4.      Dong, L.; Hu, S.; Gao, J. Discovering drugs to treat coronavirus disease 2019 (COVID-19). Drug Discov. Ther. 14(1);2020: 58–60.

5.      De Clercq, E. New nucleoside analogues for the treatment of hemorrhagic fever virus infections. Chem.–Asian Journal. 14(22);2019: 3962–3968.

6.      Delang, L.; Abdelnabi, R.; Neyts, J. Favipiravir as a potential countermeasure against neglected and emerging RNA viruses. Antiviral Research. 153;2018: 85–94.

7.      Chen, C.; Huang, J.; Cheng, Z.; Wu, J.; Chen, S.; Zhang, Y.; et al. Favipiravir versus arbidol for COVID-19: A randomized clinical trial, medRxiv. 3; 2020: 17–20.

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.    Journal.pntd.0005389 Thi Huyen Tram Nguyen and et.al. Favipiravir pharmacokinetics in Ebola-Infected patients of the JIKI trial reveals concentrations lower than targeted, 23; 2017: https://doi.org/10.1371/journal.pntd.0005389.

13.    Rele Rajan V. Tiwatane Prathamesh P. Determination of favipiravir from pharmaceutical dosage form By Extractive ion pair complex colorimetric method. Asian J. Research Chem 14(5); 2021:321-323. DOI: 10.52711/0974-4150.2021.00054.

14.    Rele Rajan V. Tiwatane Prathamesh P. Simple Extractive Spectrophotometric Method for Determination of Favipiravir from Pharmaceutical Formulation. Asian J. Research Chem. 15(4); 2022):299-302. DOI: 10.52711/0974-4150.2022.00053.

15.    Rele Rajan V. Tiwatane Prathamesh A Validated Potentiometric Titration Method for Quantitative Determination of Favipiravir from Pharmaceutical Preparation. Asian J. Research Chem.15(1);2022):49-51. DOI: 10.52711/0974-4150.2022.00007.

 

 

Received on 26.09.2022                    Modified on 31.10.2022

Accepted on 29.11.2022                   ©AJRC All right reserved