Int J App Pharm, Vol 13, Issue 4, 2021, 265-271Original Article

STABILITY INDICATIVE AND COST EFFECTIVE ANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF FAVIPIRAVIR AND PERAMIVIR IN BULK AND PHARMACEUTICAL DOSAGE FORM BY USING RP-HPLC

SRINIVAS LINGABATHULA*, NEELU JAIN

Sri Satya Sai University of Technology and Medical Sciences, Sehore, (M. P.), India
*Email: lsrinivas19888@gmail.com

Received: 17 Dec 2020, Revised and Accepted: 29 May 2021

ABSTRACT

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

INTRODUCTION

Peramivir (trade name Rapivab) is an antiviral medication developed for the treatment of influenza by BioCryst pharmaceuticals [1]. 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 [2]. 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 [3]. 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 [7]. 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 [11]. Favipiravir, sold under the brand name Avigan among others, is an antiviral medication used to treat influenza in Japan [12]. It is also being studied to treat a number of other viral infections [13]. 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.

A B

Fig. 1: Structure of (A) Favipiravir and (B) Peramivir

MATERIALS AND METHODS

Chemicals

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.

The instrumentation

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 [16].

Method optimization

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 [20].

Validation procedure

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.

Chromatographic conditions

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.

Diluent

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.

RESULTS AND DISCUSSION

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.

System suitability

In System, suitability injecting standard solution and reported USP tailing and plate count values are tabulated in table 1.

Specificity

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
Favipiravir Peramivir
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
1 10 208946 10 188941
2 25 509623 25 460832
3 50 1044728 50 944706
4 75 1554351 75 1405538
5 100 2089456 100 1889419
6 125 2599079 125 2350244
7 150 3134184 150 2834118
Correl coef 0.99998 0.9999
Slope 20879.909 18880.871
intercept 3797.945 3434.331

A

B

Fig. 4: Calibration plots of (A) Favipiravir (B) Peramivir

Linearity

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.

Accuracy

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.

Precision

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
1 50 100.154 99.512
2 100 100.624 99.861
3 150 100.417 99.981
mean 100.534 99.78467
SD 0.106108 0.24364

Mean+SD (n=3)

Table 4: Intraday precision results of favipiravir and peramivir

Favipiravir Peramivir
S. No. Conc.(µg/ml) Area counts % assay as is Conc.(µg/ml) Area counts
1 100 2089456 100.561 100 1889419
2 2089451 100.478 1889426
3 2089444 100.362 1889435
4 2089441 100.287 1889441
5 2089420 100.514 1889422
6 2089450 100.136 1889434
% RSD 1.06 0.918
mean 100.3897 100.3575
SD 0.160168 0.119252

Mean+SD (n=6)

Fig. 5: Chromatogram of sample

Intermediate precision

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.

Intraday precision

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.

Interday precision

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 [16].

Table 5: Inter-day outcomes of accuracy of favipiravir and peramivir

Favipiravir Peramivir
S. No. Conc.(µg/ml) Area counts % assay as is Conc.(µg/ml) Area count
1 100 2089451 100.241 100 1889414
2 2089461 100.054 1889406
3 2089312 100.354 1889396
4 2089366 100.687 1889354
5 2089414 100.471 1889341
6 2089457 100.289 1889411
%RSD 0.89 1.04
Mean 2089410 100.4993
SD 60.078 0.07611

Mean+SD (n=6)

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 [30].

Robustness

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

Favipiravir Peramivir
LOD LOQ LOD LOQ
Concentration s/n Concentration s/n concentration s/n Concentration s/n
1.818µg/ml 8 6.248µg/ml 23 1.818µg/ml 6 6.248µg/ml 28

A

B

Fig. 6: Chromatogram of (A) LOD and (B) LOQ

Table 7: Robustness data of favipiravir and peramivir

Parameter name % RSD
Favipiravir Peramivir
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

Stability

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 [31]. 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

Stability Favipiravir Peramivir
Purity % of deviation Purity % of deviation
Initial 99.86 0.14 99.91 0.09
6 H 99.74 0.12 99.84 0.07
12 H 99.62 0.24 99.73 0.18
18 H 99.51 0.35 99.61 0.30
24 H 99.46 0.40 99.54 0.37

Degradation studies

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.

Acid degradation

In acid degradation was done at 1N HCl and degradation was formed 12.7% for Favipiravir and 13.5% for Peramivir.

Alkali degradation

In alkali degradation was done at 1N NaOH and degradation was formed 13.7% for Favipiravir and 14.7% for Peramivir.

Peroxide degradation

In peroxide degradation was peroformed at 20% hydrogen peroxide at 15.14% Favipiravir at 15.14% and 15.15% for Peramivir.

Reduction degradation

In reduction degradation, they formed 13.5% Favipiravir and Peramivir.

Table 9: Forced degradation results of favipiravir and peramivir

Degradation condition Favipiravir Peramivir
% Assay % Deg % Assay % Deg
Acid degradation 87.246 12.754 86.421 13.579
Alkali degradation 86.271 13.729 85.284 14.716
Peroxide degradation 84.854 15.146 84.841 15.159
Reduction degradation 86.412 13.588 86.472 13.528
Thermal degradation 99.231 0.769 99.547 0.453
Hydrolysis degradation 99.457 0.543 99.145 0.855

Thermal degradation

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.

CONCLUSION

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.

ACKNOWLEDGEMENT

The authors thankful to management of Sri Satya Sai University of Technology and Medical Sciences for their support to complete this research work.

FUNDING

Nil

AUTHORS CONTRIBUTIONS

All the authors have contributed equally.

CONFLICTS OF INTERESTS

Declared none

REFERENCES

  1. Drug approval package: Rapivab (peramivir) injection U. S. Food and Drug Administration (FDA); 2015.

  2. Life-saving H1N1 Drug unavailable to most, CBS Evening News, Atlanta, GA, USA: CBS interactive; 2009.

  3. Emergency use authorization granted for Biocryst’s Peramivir; 2009.

  4. Kohno S, Kida H, Mizuguchi M, Shimada J. Efficacy and safety of intravenous peramivir for treatment of seasonal influenza virus infection. Antimicrob Age Chemother 2010;54:4568-74.

  5. Shobugawa Y, Saito R, Sato I, Kawashima T, Dapat C, Dapat IC. Clinical effectiveness of neuraminidase inhibitors–oseltamivir, zanamivir, Laninamivir, and peramivir-for treatment of influenza A(H3N2) and A(H1N1)pdm09 infection: an observational study in the influenza season in Japan. J Infect Chemother 2012;18:858-64.

  6. Tanaka A, Seki M, Kosai K, Kawano S. Peramivir (in Japanese). Japan J Clin Exp Med 2010;87:1702-6.

  7. McLaughlin MM, Skoglund EW, Ison MG. Peramivir: an intravenous neuraminidase inhibitor. Expert Opin Pharmacother 2015;16:1889-90.

  8. Kohno S, Kida H, Mizuguchi M, Shimada J. Efficacy and safety of intravenous peramivir for treatment of seasonal influenza virus infection. Antimicrob Agents Chemother 2010;54:4568-74.

  9. Kohno S, Yen MY, Cheong HJ. Phase III randomized, double-blind study comparing single-dose intravenous peramivir with oral oseltamivir in patients with seasonal influenza virus infection. Antimicrob Agents Chemother 2011;55:5267-76.

  10. Kohno S, Kida H, Mizuguchi M. Intravenous peramivir for treatment of influenza A and B virus infection in high-risk patients. Antimicrob Agents Chemother 2011;55:2803-12.

  11. drugs.com [Last accessed on 10 Nov 2019]

  12. https://www.business-standard.com/article/companies/glenmark-launches-covid-19-drug-fabiflu. [Last accessed on 10 Nov 2019]

  13. Du YX, Chen XP. Favipiravir: pharmacokinetics and concerns about clinical trials for 2019-nCov infection. Clin Pharmacol Ther 2020;108:242-7.

  14. Shiraki K, Daikoku T. Favipiravir, an anti-influenza drug against life-threatening RNA virus infections. Pharmacol Ther 2020;209:107512.

  15. Murugan S, Upendra Janardhan CH, Niranjan Babu M. RP-HPLC method for simultaneous estimation of albendazole and niclosamide in oral suspension for veterinary use. Res J Pharm Tech 2016;9:27-32.

  16. Bhavani P, Prasada Rao K, Mohan S. Novel validated reversed-phase high-performance liquid chromatography method for determination of glucosamine, diacerein, and methyl sulfonyl methane in micro sample rat plasma and its application to pharmacokinetic and dissolution studies. Asian J Pharm Clin Res 2020;13:50-63.

  17. Naresh Kumar DS, Patel D. Stability indicating chromatographic method development and validation for the simultaneous estimation of escitalopram oxalate and flupentixol in its pharmaceutical dosage form by HPLC. WJPR 2017;6:549-66.

  18. Supriya T, Naresh D, Vijaya Kumar G, Haneer MA. Stability indicating RP-HPLC method development and validation for simultaneous estimation of escitalopram and flupentixol pure and marketed formulation. Asian J Pharm Res 2018;8:4-10.

  19. Shivani CP, Maheshwari DG. Development and validation of UV spectrometric and HPLC method for estimation of escitalopram oxalate and flupentixol dihydrochloride in combined dosage form. AJPTI 2016;4:59-70.

  20. Malathi S, Devakumar D. Development and validation of rp-hplc method for the estimation of escitalopram oxalate and flupentixol dihydrochloride in combined dosage form and plasma. Int J Pharm Pharm Sci 2021;13:61-6.

  21. Mukta D Naykode, Durgacharan A Bhagwat, Swapnil D Jadhav, Harinath N. Analytical and bio analytical method for quantification of pure azilsartan, not its salt by RP-HPLC. Res J Pharm Tech 2017;10:708-14.

  22. Mayanka Singh, Manoj Charde, Rajesh Shukla, Rita MC. Determination of calcipotriene in calcipotriene cream 0.05% w/w by RP-HPLC method development and validation. Res J Pharm Tech 2011;4:1219-23.

  23. Malathi S, Arunadevi N. Development and validation of stability-indicating simultaneous estimation of metformin and alogliptin in tablets by high-performance thin layer chromatography. Int J Pharm Pharm Sci 2020;12:68-73.

  24. Senthil Rajan D, Muruganathan G, Shivkumar K, Ganesh T. Development and validation of hplc method for simultaneous quantification of vasicine, glycyrrhizin and piperine in poly herbal cough syrup. Int J Curr Pharm Res 2020;12:15-9.

  25. Palani Shanmugasundaram, Kamarapu SK. RP-HPLC method for the simultaneous estimation and validation of amlodipine besylate and atenolol in bulk and tablet dosage form in biorelevant dissolution medium (Fassif). Res J Pharm Tech 2017;10:3379-85.

  26. Gomathy S, Narenderan ST, Meyyanathan SN, Gowramma B. Development and validation of hplc method for the simultaneous estimation of apigenin and luteolin in commercial formulation. J Crit Rev 2020;7:4785-90.

  27. Ashutosh Kumar S, Manidipa Debnath, Seshagiri Rao JVLN, Gowri Sankar D. Development and validation of a sensitive RP-HPLC method for simultaneous estimation of rosuvastatin and fenofibrate in tablet dosage form by using PDA detector in gradient mode. Res J Pharm Tech 2016;9:549-54.

  28. Malak Y, Al-Bathish AA, Gazy MK, El-Jamal. Rp-hplc and chemometric methods for the determination of two anti-diabetic mixtures; metformin hydrochloride-canagliflozin and metformin hydrochloride-gliclazide in their pharmaceutical formulation. Int J Pharm Pharm Sci 2020;12:83-94.

  29. Gadhvi MP, Bhandari A, Suhagia BN, Desai UH. Development and validation of RP-HPLC method for simultaneous estimation of atazanavir and ritonavir in their combined tablet dosage form. Res J Pharm Tech 2013;6:200-3.

  30. Swati K, Abhishek P, Sushank S, Bothiraja C, Atmaram P. High-performance liquid chromatography for the simultaneous estimation of cefoperazone and sulbactam in rat plasma and its importance in therapeutic drug monitoring. Int J Pharm Pharm Sci 2020;12:92-7.

  31. Vijayakumari M, Balasekhar Reddy Ch. Stability indicating validated hplc method for the determination of zanubrutinib in bulk and pharmaceutical dosage form. Asian J Pharm Clin Res 2020;13:159-62.