DEVELOPMENT AND VALIDATION OF A STABILITY-INDICATING HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR THE QUANTITATIVE DETERMINATION OF NITAZOXANIDE AND IT’S RELATED SUBSTANCES IN ACTIVE PHARMACEUTICAL INGREDIENTS

TATA SANTOSH; PRAFULLA KUMAR SAHU

doi:10.22159/ajpcr.2025v18i3.53968

Authors

TATA SANTOSH Department of Pharmaceutical Analysis, School of Pharmacy, Centurion University of Technology and Management, Odisha, India
PRAFULLA KUMAR SAHU Department of Pharmaceutical Analysis, School of Pharmacy, Centurion University of Technology and Management, Odisha, India

DOI:

https://doi.org/10.22159/ajpcr.2025v18i3.53968

Keywords:

Nitazoxanide, Aspirin, Salicylic acid, Tizoxanide, 2-amino-5-nitro thiazole

Abstract

Objective: Nitazoxanide (NAT) is a veterinary antibiotic used for the treatment of protozoal infections in livestock and sheep. The estimation of NAT, its process impurities, and degradation products have not been reported till date. In this study, we aimed to develop and validate a chromatographic separation method for the determination of NAT, its process impurities, and related substances as per International Conference on Harmonization (ICH) guidelines.

Methods: Chromatographic separation of process impurities such as 2-amino-5-nitro thiazole also called NAT-related substance A, aspirin, and degradation impurities such as salicylic acid and tizoxanide (TIZ) were separated by reverse-phase high-performance liquid chromatography using gradient elution. The separation of NAT and TIZ was most critical as they are structurally similar. The mobile phase consisted of a gradient elution containing a composition of acetonitrile and 2% orthophosphoric acid solution adjusted to pH 2.5 at a flow rate of 1 mL/min. Separation was achieved on a YMC Pack C8 L7 column with a run time of 40 min. The detection was carried out using a photodiode array detector and quantification was carried out at 210 nm. Forced degradation study was also conducted to confirm the specificity. The robustness and ruggedness of the method were evaluated.

Results: The relative retention times (RRT) for aspirin, salicylic acid, NAT, and TIZ were 6.33, 6.52, 22.43, and 6.45, respectively, indicating good separation. The asymmetry factor for all the peaks is ranged from 1.1 to 1.2 indicating acceptable chromatography. The % recovery from spiked studies ranged from 90% to 110% for all the impurities when spiked in the range of 50–150% of their nominal concentrations. For all the known impurities, the limit of detection ranged from 0.06 to 0.20 parts per million (ppm) and the limit of quantification ranged from 0.19 to 0.61 ppm.

Conclusion: The method was validated as per ICH guidelines and further was successfully applied for the quality evaluation of NAT in bulk active pharmaceutical ingredients.

Downloads

Download data is not yet available.

References

Crouch SP. Veterinary use of nitazoxanide: A review. Vet Ther. 2001;2(3):156-6.

U.S. Food and Drug Administration (FDA). FDA Approves Alinia for the Treatment of Diarrhea Caused by Cryptosporidium Parvum and Giardia Lamblia. U.S. Food and Drug Administration; 2002. Available from: https://www.accessdata.fda.gov/drugsatfda_docs/ label/2002/21498_alinia_lbl.pdf

Hemphill A, Mueller J, Esposito M. Nitazoxanide, a broad-spectrum

thiazolide anti-infective agent for the treatment of gastrointestinal infections. Expert Opin Pharmacother. 2006;7(7):953-64. doi: 10.1517/14656566.7.7.953

Rossignol JF. Nitazoxanide: A first-in-class broad-spectrum antiviral agent. Antiviral Res. 2009;83(1):1-9. doi: 10.1016/j.antiviral.2009.02.008

Rossignol JF. Nitazoxanide, a broad-spectrum antiviral agent: Mechanisms of action and potential clinical applications. Clin Infect Dis. 2016;62(7):944-52.

Rocco PR, Silva PL, Cruz FF, Melo-Junior MA, Tierno PF, Moura MA, et al. Early use of nitazoxanide in mild COVID-19 disease: Randomized, placebo-controlled trial. Eur Respir J. 2020;58(1):2003725. doi: 10.1183/13993003.03725-2020

Martins-Filho PR, Do Nascimento-Júnior EM, Barreto-Alves JA, Fakhouri R, Ferreira LC. Efficacy and safety of nitazoxanide in treating SARS-CoV-2 infection: A systematic review and meta-analysis of blinded, placebo-controlled, randomized clinical trials. Eur J Clin Pharmacol. 2022;78(11):1813-21. doi: 10.1007/s00228-022-03380-5

Irabuena C, Scarone L, De Souza GE, Aguiar AC, Mendes GR, Guido RV, et al. Synthesis and antiplasmodial assessment of nitazoxanide and analogs as new antimalarial candidates. Med Chem Res. 2022;31(3):426-35. doi: 10.1007/s00044-021-02843-1

Ballard TE, Wang X, Olekhnovich I, Koerner T, Seymour C, Salamoun J, et al. Synthesis and antimicrobial evaluation of nitazoxanide-based analogues: Identification of selective and broad spectrum activity. ChemMedChem. 2011 Feb 7;6(2):362-77. doi: 10.1002/cmdc.201000475

Blossom DB, Kallen AJ, Patel PR, Elward A, Robinson L, Gao G, et al. Outbreak of adverse reactions associated with contaminated heparin. N Engl J Med. 2008;359(25):2674-84. doi: 10.1056/NEJMoa0806450

Wax PM. Elixirs, diluents, and the passage of the 1938 federal food, drug and cosmetic act. Ann Intern Med. 1995;122(6):456-61. doi: 10.7326/0003-4819-122-6-199503150-00009

Burman K, Hennessey J, Mcdermott M, Wartofsky L, Emerson C. The FDA revises requirements for levothyroxine products. Thyroid. 2008;18:487-90. doi: 10.1089/thy.2008.0109

International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). ICH Q3A(R2): Impurities in New Drug Substances; 2005. Available from: https://www.ich/website

U.S. Food and Drug Administration (FDA). Guidance for Industry: Q3B(R2) Impurities in New Drug Products. U.S. Food and Drug Administration; 2020. Available from: https://www.fda/website

European Medicines Agency (EMA). Guideline on the Limits of Genotoxic Impurities. European Medicines Agency; 2018. Available from: https://www.ema/website

Gad SC. Pharmaceutical Manufacturing Handbook: Production and Processes. Hoboken: John Wiley and Sons; 2008.

Carstensen JT, Rhodes CT. Drug Stability: Principles and Practices. 3rd ed. New York: Marcel Dekker; 2000.

Swarbrick J. Encyclopedia of Pharmaceutical Technology. Vol. 2. London: Informa Healthcare; 2006.

Sharma S, Bhandari A, Choudhary VR, Rajpurohit H, Khandelwal P. RP-HPLC method for simultaneous estimation of nitazoxanide and ofloxacin in tablets. Indian J Pharm Sci. 2011;73(1):84-8. doi: 10.4103/0250-474X.89763

Hegazy M, Kessiba A, El Gindy AE, Abdelkawy M. Validated stability indicating RP-HPLC for quantitation of nitazoxanide in presence of its alkaline degradation products and their characterization by HPLC-tandem mass spectrometry. J Chromatogr Sci. 2014;52(9):1071-8. doi: 10.1093/chromsci/bmt163

Sakamoto T, Hiyama Y. Rapid determination of nitazoxanide in tablets using reversed-phase ultra-performance liquid chromatography (UPLC) and high-performance liquid chromatography. Pharmazie. 2008;63(7):503-7.

Malesuik MD, Paim CS, Schapoval EE, Steppe M. Development of a simple, rapid and validated spectrophotometric method for nitazoxanide in pharmaceutical formulations and comparison with HPLC. Quím Nova. 2010;33:739-42. doi: 10.1590/S0100-40422010000300045

Ali NW, Abbas SS, Zaazaa HE, Abdelrahman MM, Abdelkawy M. Validated stability indicating methods for determination of nitazoxanide in presence of its degradation products. J Pharm Anal. 2012 Apr;2(2):105-16. doi: 10.1016/j.jpha.2011.11.004

Gritti F, Guiochon G. Perspectives on the evolution of the column efficiency in liquid chromatography. Anal Chem. 2013 Mar 19;85(6):3017-35. doi: 10.1021/ac3033307

Altmaier S, Fridstrom A, Dube M. Simple Tips to Increase Sensitivity in U/HPLC Analysis; 2021 Mar. Available from: https://www. chromatographyonline.com/view/simple-tips-to-increase-sensitivity-in-uhplc-analysis

Wysocki J, Dong MW. Ultraviolet detectors: Perspectives, principles, and practices. LCGC North Am. 2019;37(10):750-9.

Reddy CS, Rao BT. Development and validation of a stability indicating related substances of trandolapril by rp-hplc and its degradation. Int J Appl Pharm. 2021;13(5):115-21. doi: 10.22159/ijap.2021v13i5.42113

Babu C, Devanna N, Reddy KV. Validated gradient stability indicating RP-HPLC method for the simultaneous quantification of 11 related substances in the combined dosage forms of lamivudine and tenofovir disopeoxil fumarate. Int J Appl Pharm. 2017;9(4):61-8. doi: 10.22159/ ijap.2017v9i4.19001