Sri Satya Sai University of Technology and Medical Sciences, Sehore, (M. P.), India
*Email: [email protected]
Received: 17 Dec 2020, Revised and Accepted: 29 May 2021
Objective: The current investigation was pointed at developing and progressively validating novel, simple, responsive and stable RP-HPLC method for the measurement of active pharmaceutical ingredients of Favipiravir and Peramivir and their related substances.
Methods: A simple, selective, validated and well-defined stability that shows gradient RP-HPLC methodology for the quantitative determination of Favipiravir and Peramivir. The chromatographic strategy utilized Inertsil ODS column of dimensions 250x4.6 mm, 5 micron, using isocratic elution with a mobile phase of acetonitrile and 0.1 percent orthophosphoric acid (70:30). A flow rate of 1 ml/min and a detector wavelength of 260 nm utilizing the PDA detector was given in the instrumental settings. Using the impurity-spiked solution, the chromatographic approach was streamlined.
Results: Validation of the proposed method was carried out according to an international conference on harmonization (ICH) guidelines. LOD and LOQ for the two active ingredients and their impurities were established with respect to test concentration. The calibration charts plotted were linear with a regression coefficient of R2>0.999, which means the linearity was within the limit. Recovery, specificity, linearity, accuracy, robustness, ruggedness was determined as a part of method validation and the results were found to be within the acceptable range.
Conclusion: The proposed method to be fast, simple, feasible and affordable in RS condition. During stability tests, it can be used for routine analysis of production samples and to verify the quality of drug samples during stability studies.
Keywords: Favipiravir, Peramivir, RP-HPLC, Development, Validation
© 2021 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijap.2021v13i4.40530. Journal homepage: https://innovareacademics.in/journals/index.php/ijap
Peramivir (trade name Rapivab) is an antiviral medication developed for the treatment of influenza by BioCryst pharmaceuticals . In October 2009, the experimental antiviral drug peramivir was confirmed to be “life saving” effective in the intravenous treatment of 8 extreme cases of swine flu . On October 23, the U. S. An emergency use authorization for peramivir was provided bt the Food and Drug Administration (FDA), authorizing the use of the drug in intravenous form for hospitalized parents only in cases where the other treatment options available are inadequate or unavailable . Two new anti-influenza medications were launched clinically in Japan in 2010. Rapiacta (peramivir), which is provided by intravenous drip injection, was one of them [4-6]. Peramivir was the first intravenous neuraminidase inhibitor (NAI) approved by the US food and drug administration (FDA) for the treatment of influenza . Data from randomized controlled trails have shown that peramivir is safe and well-tolerated in uncomplicated influenza, with similar or superior clinical efficacy to those of placebo and oseltamivir [8-10]. Signs of an allergic reaction hive, difficult breathing, swelling in face or throat, severe skin reactions . Favipiravir, sold under the brand name Avigan among others, is an antiviral medication used to treat influenza in Japan . It is also being studied to treat a number of other viral infections . There is evidence that use of during pregnancy may result in harm to the baby [14, 15]. Research in 2014, suggested that Favipiravir may have efficacy in humans, it went unaddressed.
Fig. 1: Structure of (A) Favipiravir and (B) Peramivir
Acetonitrile, HPLC-grade orthophosphoric acid, water, were purchased from Merck India Ltd, Mumbai, India. APIs of Favipiravir, Peramivir standards were procured from Glen mark, Mumbai.
Waters alliance liquid chromatography (model 2695) monitored with empower 2.0 data handling system and a detector of photodiode array (model 2998) was used for this study .
To optimize the chromatographic conditions, different ratios of phosphate buffer and the acetonitrile in the mobile phase with isocratic and gradient mode were tested. However, the mobile phase composition was modified at each trial to enhance the resolution and also to achieve acceptable retention times. Finally, 0.1% OPA buffer and acetonitrile with isocractic elution was selected because it results in a greater response of active pharmacy ingredient and their impurities. During the optimization of the method various stationary phases such as C8, C18 phenyl and amino, inertsil ODS columns were tested. From these trials the peak shapes were relatively good with a inertsil ODS column of 250 x 4.6 mm, 5 µ with a PDA detector. The mobile phase flow rate has been done at 254 nm in order to obtain enough sensitivity. By using above conditions we get retention times of Favipiravir and Peramivir were about 3.003 and 7.271 min with a tailing factor of 1.063 and 1.401. The number of theoretical plates for Favipiravir and Peramivir were 2089456,1889412 which indicate the column’s successful output the % RSD for six replicate injections was around 0.94% the proposed approach suggests that it is extremely precise. According to ICH guidelines, the method established was validated.
There are some HPLC methods [17-19] reported in the literature, but these methods are developed only for routine analysis of ESC and FLU in bulk and formulation studies. The developed HPLC method was utilized for the estimation of the combined drugs by in vitro method. Different extractions were tried using acetonitrile, methanol, and dimethylformamide .
The analytical parameters such as system suitability, precision, specificity, accuracy, linearity, robustness, LOD, LOQ, forced degradation and stability were validated according to ICH Q2 (R1) guidelines [21-29].
Preparation of buffer
1 ml of ortho phosphoric acid is dissolved in 1 lt of HPLC grade water and filter through 0.45 µ filter paper.
The HPLC analysis was performed on reverse phase HPLC system with isocratic elution mode using a mobile phase of acetonitrile and 0.1% OPA and Inertsil ODS column (250x4.6 mm, 5 μ) column with a flow rate of 1 ml/min.
Water and Acetonitrile in the ratio (50:50) is used as diluent.
Preparation of the standard stock solution
For standard stock solution preparation, add 70 ml of diluents to 100 mg of Favipiravir and 100 mg of Peramivir taken in a 100 ml volumetric flask and sonicate for 10 min to fully dissolve the contents and then make up to the mark with diluent.
Preparation of standard solution
1 ml of solution is drawn from the above normal stock solution into a 10 ml volumetric flask and diluted up to the level.
Preparation of sample solution
Take 130 mg of the sample drug Favipiravir and 100 mg of the sample drug Peramivir into a 100 ml volumetric flask and add 70 ml of diluents and sonicate for 10 min to fully dissolve the contents and then make up the mark with diluent. This solution is filtered into a device using a 0.45µ nylon syringe in a vial.
The main analytical challenge during the development of a new method was to separate active Pharma ingredients from their impurities. In order to provide a good performance, the chromatographic conditions were optimized.
In System, suitability injecting standard solution and reported USP tailing and plate count values are tabulated in table 1.
In this test method placebo, sample and standard solutions were analyzed individually to examine the interference. The below fig. shows that the active ingredients were well separated from blank and their excipients and there was no interference of placebo with the principal peak. Hence the method is specific.
Table 1: Results of system suitability
|System suitability parameter||Acceptance criteria||Drug name|
|USP Plate Count||NLT 2000||38417||3264|
|USP Tailing||NMT 2.0||1.07||1.04|
|USP Resolution||NLT 2.0||-||8.64|
|% RSD||NMT 2.0||0.71||0.89|
Fig. 2: Chromatogram of system suitability
Fig. 3: Chromatogram of blank
Table 2: Linearity of favipiravir and peramivir
|S. No.||Conc µg/ml||Favipiravir area count||Conc. µg/ml||Peramivir area count|
Fig. 4: Calibration plots of (A) Favipiravir (B) Peramivir
The area of the linearity peak versus different concentrations has been evaluated for Favipiravir, Peramivir, as 10,25,50,75,100,125,150 percent respectively. Linearity was performed in the range of 10-150µg/ml of Favipiravir and 10-150µg/ml of Peramivir. The correlation coefficients achieved greater than 0.999 for all.
In this method, Accuracy was conducted in triplicate by analyzing active pharma ingredient sample solution spiked with known amounts of all the impurities at three kinds of concentration levels of 50, 100 and 150% of each at a specified limit. For all impurities, percentage recoveries were measured and found to be within the limit. The accuracy and reliability of the developed method were established. The percentage recovery values were found to be in the range of 100.154-100.624% for Favipiravir and 99.512-99.918% for Peramivir. The results are given in table 3, 4 and 5.
In method precision study prepare six different samples in the concentration of Favipiravir (100 ppm) and Peramivir (100 ppm) are injected into HPLC system. Favipiravir %assay found to be in the range of 100.136-100.561 and Peramivir %assay found to be in range of 100.261-100.517. These results are given below table 4.
Table 3: Results of accuracy
|S. No.||% Level||Favipiravir % recovery||Peramivir % recovery|
Table 4: Intraday precision results of favipiravir and peramivir
|S. No.||Conc.(µg/ml)||Area counts||% assay as is||Conc.(µg/ml)||Area counts|
Fig. 5: Chromatogram of sample
Six replicates of the sample solution were studied by various researchers, and on separate days different instruments were tested. The peak regions used to determine mean percent RSD values have been calculated. The results are given in the following table.
Six replicates of a sample solution containing Favipiravir (100μg/ml) and Peramivir (100μg/ml) were analysed on the same day. Peak areas were calculated, which were used to calculate mean, SD and %RSD values.
Six replicates of a sample solution containing Favipiravir (100μg/ml) and Peramivir (100μg/ml) were analysed on a different day. Peak areas were calculated which were used to calculate mean, SD and %RSD values. The present method was found to be precise as the RSD values were less than 2% and also the percentage assay values were close to be 100%. The results are given in table 5 .
Table 5: Inter-day outcomes of accuracy of favipiravir and peramivir
|S. No.||Conc.(µg/ml)||Area counts||% assay as is||Conc.(µg/ml)||Area count|
LOD and LOQ
The LOD concentrations for Favipiravir are 1.818 µg/ml and s/n values is 8 and Peramivir 1.818 µg/ml and s/n value 6. The LOQ concentration for Favipiravir 6.248 µg/ml and their s/n values are 23 and Peramivir their 6.248 µg/ml and s/n value is 28. The method is validated as per the US FDA guidelines .
The conditions of the experiment were designed to test the robustness of the established system intentionally altered, such as flow rate, mobile phase in organic percentage in all these varied conditions. Robustness results for favipiravir and peramivir found to be within the limit and results are tabulated in table 7.
Table 6: LOD and LOQ for favipiravir and peramivir
Fig. 6: Chromatogram of (A) LOD and (B) LOQ
Table 7: Robustness data of favipiravir and peramivir
|Parameter name||% RSD|
|Flow minus (0.8 ml/min||0.62||0.70|
|Flow plus (1.2 ml/min)||0.34||0.66|
|Organic minus (-10%)||0.56||0.26|
|Organic plus (+10%)||0.51||0.61|
The standard and sample solution was kept at room temperature and at 2-8 °C up to 24 h. Then these solutions were pumped into the device and calculate the % of deviation from initial to 24 h . There was no significant deviation observed and confirmed that the solutions were stable up to 24 h percentage of the assay was not quite 2%. There is no effect in storage conditions for Favipiravir and Peramivir drugs. The results are given below table 8.
Table 8: Stability results of favipiravir and peramivir
|Purity||% of deviation||Purity||% of deviation|
The Peramivir and Favipiravir sample was subjected into various forced degradation conditions to effect partial degradation of the drug. Studies of forced degradation have been carried out to find out that the method is suitable for products of degradation. In addition, the studies provide details about the conditions during which the drug is unstable, in order that the measures are often taken during formulation to avoid potential instabilities.
In acid degradation was done at 1N HCl and degradation was formed 12.7% for Favipiravir and 13.5% for Peramivir.
In alkali degradation was done at 1N NaOH and degradation was formed 13.7% for Favipiravir and 14.7% for Peramivir.
In peroxide degradation was peroformed at 20% hydrogen peroxide at 15.14% Favipiravir at 15.14% and 15.15% for Peramivir.
In reduction degradation, they formed 13.5% Favipiravir and Peramivir.
Table 9: Forced degradation results of favipiravir and peramivir
|% Assay||% Deg||% Assay||% Deg|
In thermal degradation the sample was degradated 0.76% for Favipiravir and 0.45% for Peramivir.
Degradation of hydrolysis
In hydrolysis degradation the sample was degradated 0.54% for Favipiravir and 0.85% for Permaivir.
All degradation results are tabulated in table 9.
We present in this article simple, selective, validated and well-defined stability that shows gradient RP-HPLC methodology for the quantitative determination of Favipiravir and Peramivir. All the products of degradation formed during the stress conditions and the related active pharma ingredients are well separated and peaks were well resolved from each other and separate with an appropriate retention time indicating that the proposed method to be fast, simple, feasible and affordable in RS condition. Therefore the developed method during stability tests, it can be used for routine analysis of production samples and to verify the quality of drug samples during stability studies.
The authors thankful to management of Sri Satya Sai University of Technology and Medical Sciences for their support to complete this research work.
All the authors have contributed equally.
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