Simple Extractive Spectrophotometric Method for Determination of Favipiravir from Pharmaceutical Formulation
Rele Rajan V.*, Tiwatane Prathamesh P.
Central Research Laboratory, D. G. Ruparel College, Mahim, Mumbai - 400 016.
*Corresponding Author E-mail: drvinraj@gmail.com
ABSTRACT:
Favipiravir, is chemically, 6-fluoro-3-oxo-3,4-dihydropyrazine-2-carboxamide formula C5H4FN3O2, (molecular weight 157.1g/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 disease2,3. 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-viruses5,6 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 high performance liquid chromatography (HPLC) methods for determining favipiravir assay and impurities in active pharmaceutical ingredients8-12, Colorimetric13, non-aqueous titration14 and miscellaneous15-16. 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 spectrophotometric 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.
The proposed methods involve formation of ion pair complexes of favipiravir with bromophenol blue, solochrome dark blue and bromocresol green in acidic medium.
Structure of Favipiravir:
Materials and methods:
A Shimadzu-160 A double beam UV-Visible recording spectrophotometer with pair of 10mmmatched quartz cell was used to measure absorbance of solutions. A Shimadzu analytical balance was used.
Bromophenol blue, solochrome dark blue, bromocresol green, 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 (100μg/ml) was prepared in ethanol. From this stock solution working standard (10 μg/ml) was prepared by diluting 10ml stock solution to 100mlwith ethanol. 0.6%w/v solution of bromophenol blue, solochrome dark blue and bromocresol green 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 bromophenol blue)
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= 4.2) and 5.8ml of 0.6% w/v bromophenol blue 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 (λmax 420nm).
Method 2 (with solochrome dark blue):
Into a series of separating funnels appropriate amount of the working standard drug solutions were pipetted out. To each funnel 4.0ml of buffer (pH=1.10) and 4.1ml of 0.25% w/v solochrome dark blue 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 (λmax=495 nm).
Method 3 (with bromocresol green):
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 5.2ml of 0.02% w/v bromocresol green 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 (λmax = 430 nm).
Estimation from tablets:
Twenty tablets of labelled claim 200mg 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 10mg of favipiravir was transferred into a beaker and it was shaken with 50ml of ethanol and filtered. The filtrate and the washing were collected in a 100.0ml volumetric flask. This filtrate and the washing were diluted up to the mark with ethanol to obtain final concentration as 100ìg/ml. This solution was further diluted to give 10ìg/ml. Such solution was used for methods (1-3) 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 are given in table 1.
Table 1: Values of results of optical and regression of drug
|
Parameter |
Bromophenol blue |
Solochrome dark blue |
Bromocresol green |
|
Detection Wavelength (nm) |
420 |
495 |
430 |
|
Beer Law Limits (µg/ml) |
1-12 |
2-20 |
1-16 |
|
Correlation coefficient (r2) |
0.9999 |
0.9999 |
0.9999 |
|
Regression equation (y=b+ac) |
|
|
|
|
Slope (a) |
0.0001 |
0.0010 |
0.0305 |
|
Intercept (b) |
0.050 |
0.020 |
-.00008 |
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 i.e. tablets. 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 show the sensitivity of methods while the precision was confirmed by %RSD (relative standard deviation). The optical characteristics such as absorption maxima (nm), molar absorptivity (l -mole-1 cm-1), co-relation coefficient (r) were calculated and are also summarized. Assay results of recovery studies are given in table 2 (A, B, C).
Table No 2: A (Bromophenol blue)
|
Amount of Sample Added in (µg/ml) |
Amount of Standard Added in (µg/ml) |
Total amount recovered |
Percentage recovery (%) |
Standard deviation |
Percentage of relative standard deviation (C.O.V.) |
|
1 |
0 |
1.005602 |
100.5602 |
0.02458 |
2.44428 |
|
2 |
1 |
1.991597 |
99.57983 |
0.019135 |
0.960802 |
|
3 |
2 |
2.994398 |
99.81326 |
0.021818 |
0.728614 |
|
4 |
3 |
3.994398 |
99.85994 |
0.027061 |
0.677484 |
|
|
|
|
|
Mean= 0.023149 |
Mean= 1.202795 |
Table No 2: B (Solochrome dark blue)
|
Amount of Sample Added in (µg/ml) |
Amount of Standard Added in (µg/ml) |
Total amount recovered |
Percentage recovery (%) |
Standard deviation |
Percentage of relative standard deviation (C.O.V.) |
|
2 |
0 |
1.97619 |
98.80952 |
0.115011 |
5.81983 |
|
2 |
2 |
3.97619 |
99.40476 |
0.178174 |
4.481027 |
|
2 |
4 |
6.119048 |
101.9841 |
0.18545 |
3.030693 |
|
2 |
6 |
8.047619 |
100.5952 |
0.125988 |
1.565533 |
|
|
|
|
|
Mean= 0.151156 |
Mean= 3.724271 |
Table No 2: C (Bromocresol green)
|
Amount of Sample Added in (µg/ml) |
Amount of Standard Added in (µg/ml) |
Total amount recovered |
Percentage recovery (%) |
Standard deviation |
Percentage of relative standard deviation (C.O.V.) |
|
2 |
0 |
2.014052 |
100.7026 |
0.031997 |
1.588675 |
|
2 |
2 |
4.009368 |
100.2342 |
0.05588 |
1.393733 |
|
2 |
4 |
5.990632 |
99.84387 |
0.064788 |
1.081496 |
|
2 |
6 |
7.995316 |
99.94145 |
0.039836 |
0.498237 |
|
|
|
|
|
Mean= 0.048125 |
Mean= 1.140535 |
Results are in good in agreement with labelled value.
DISCUSSION:
The percent recovery obtained indicates noninterference 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 such as tablets. 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.
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Received on 02.04.2022 Modified on 01.05.2022
Accepted on 20.05.2022 ©AJRC All right reserved
Asian J. Research Chem. 2022; 15(4):299-302.