Ecofriendly Stability Indicating RP-UHPLC Method for Simultaneous Estimation of Amiloride and Hydrochlorothiazide in Bulk and pharmaceutical dosage form using AGREE and GAPI Tools
Abstract
An easy, quick and reliable RP-UHPLC technique for the quantification of Amiloride (AML) and Hydrochlorothiazide (HCTZ) is established as well as validated in the present article using the green analytical chemistry (GAC) tools. The mobile phase consisted of a mixture of 0.1% formic acid with ethanol in a 55:45 % v/v ratio delivered at a flow rate of 0.5 mL/min through a 120x4.6mm Hypersil gold ODS endcapped reverse phase column having a particle size of 3 micron. AML and HCTZ were detected at 265 nm with respective retention periods of 2.982 and 5.654 min. This methodology was rigorously validated in alignment with ICH guidelines. The calibration plots exhibited linearity over the concentration ranges of 2.5–25 μg/mL for AML and 25-150 μg/mL for HCTZ. The detection limits for AML and HCTZ were 0.69 and 0.61 μg/mL whereas quantification limits were 2.10 and 1.84 μg/mL. At three different levels of a standard addition, the percentage recovery was determined to be between 99.37 and 100.7%. The precision of the method for both intra as well as inter-day fell under the acceptable range (RSD < 2). Both the bulk medication as well as its formulation were subjected to a forced degradation trial and the results indicate that the technique is stability indicating and can differentiate between the peaks of the active analytes and the degradants. The evaluation of the method’s environmental impact utilized green analytical chemistry tools such as GAPI and AGREE revealing a greenness rate exceeding 75%, which is excellent. Hence, the established chromatographic approach was straightforward, highly sensitive, quick, precise, cost effective as well as eco-friendly than the earlier reported methods making it suitable for routine quality control testing in industrial sector.
Keywords:
Amiloride, Hydrochlorothiazide, ICH, UHPLC, Forced degradation studies, Method validationDOI
https://doi.org/10.25004/IJPSDR.2025.170202References
Van Mil JWF, Sweetman SC (E). The Martindale- The complete drug reference. London: Pharmaceutical press; 2009. Available from: http://dx.doi.org/10.1007/s11096-011-9543-9 .
Carlucci G, Palumbo G, Mazzeo P, Quaglia MG. Simultaneous determination of losartan and hydrochlorothiazide in tablets by high-performance liquid chromatography. Journal of pharmaceutical and biomedical analysis. 2000;23(1):185-9. Available from: https://doi.org/10.1016/s0731-7085(00)00268-5 .
Kartal M, Erk N. Simultaneous determination of hydrochlorothiazide and amiloride hydrochloride by ratio spectra derivative spectrophotometry and high-performance liquid chromatography. Journal of pharmaceutical and biomedical analysis. 1999;19(3-4):477-85. Available from: https://doi.org/10.1016/s0731-7085(98)00241-6
Al-Saidi KH, Abdlaziz S, Semer S. Simultaneous determination of amiloride hydrochloride and hydrochlorothiazide in pharmaceuticals by derivative spectrophotometry. Journal of Al-Nahrain University Science. 2010;13(4):52-61. Available from: https://doi.org/10.22401/jnus.13.4.07
Tang J, Li X, Feng Y, Liang B. Simultaneous determination of amiloride and hydrochlorothiazide in a compound tablet by diffuse reflectance spectroscopy and chemometrics. Journal of Applied Spectroscopy. 2016;83(4):710-716. Available from: https://doi.org/10.1007/s10812-016-0352-8
Kopuri G, Anusha K, Srinivasa Rao D. Development of a new HPLC method for simultaneous estimation of Amiloride and Hydrochlorothiazide. Journal of chemical and Pharmaceutical Sciences. 2015;8(4): 661- 663.
Kumar RA, Babu GR, Sowjanya M, Ramayyappa M. Validated RP-HPLC method for the estimation of Amiloride and hydrochlorothiazide in combined tablet dosage form. Asian Journal of Pharmaceutical Analysis. 2021;11(3):207-211. Available from: https://doi.org/10.52711/2231-5675.2021.00037
De Croo F, Van den Bossche W, De Moerloose P. Simultaneous quantitative determination of amiloride hydrochloride and hydrochlorothiazide in tablets by high-performance liquid chromatography. Chromatographia. 1985;20(8):477-481. Available from: https://doi.org/10.1007/bf02344789
Zecevic M, Zivanovic LJ, Agatonovic-Kustrin S, Ivanovic D, Maksimovic M. Statistical optimization of a reversed-phase liquid chromatographic method for the analysis of amiloride and hydrochlorothiazide in tablets. Journal of pharmaceutical and biomedical analysis. 2000;22(1):1-6. Available from: https://doi.org/10.1016/s0731-7085(99)00253-8
Naguib IA, Abdelaleem EA, Abdallah FF, Emam AA. Development and validation of two chromatographic methods for simultaneous determination and quantification of amiloride hydrochloride, hydrochlorothiazide, and their related substances, in pure and tablet forms. Journal of AOAC International. 2020;103(3):747-754. Available from: https://doi.org/10.1093/jaocint/qsz015
Shah JV, Shah PA, Sanyal M, Shrivastav PS. Simultaneous quantification of amiloride and hydrochlorothiazide in human plasma by liquid chromatography–tandem mass spectrometry. Journal of pharmaceutical analysis. 2017;7(5):288-296. Available from: https://doi.org/10.1016/j.jpha.2017.03.007
Song M, Hang T, Zhao H, Wang L, Ge P, Ma P. Simultaneous determination of amiloride and hydrochlorothiazide in human plasma by liquid chromatography/tandem mass spectrometry with positive/negative ion‐switching electrospray ionisation. Rapid Communications in Mass Spectrometry: An International Journal Devoted to the Rapid Dissemination of Up‐to‐the‐Minute Research in Mass Spectrometry. 2007;21(21):3427-3434. Available from: https://doi.org/10.1002/rcm.3235
Van Der Meer MJ, Brown LW. Simultaneous determination of amiloride and hydrochlorothiazide in plasma by reversed-phase high-performance liquid chromatography. Journal of Chromatography B: Biomedical Sciences and Applications. 1987;423:351-357. Available from: https://doi.org/10.1016/0378-4347(87)80363-8
Jangid AG, Tale RH, Vaidya VV. A single, selective and simple validated method for simultaneous estimation of amiloride and hydrochlorothiazide in human plasma by liquid chromatography–tandem mass spectrometry. Biomedical Chromatography. 2012;26(1):95-100. Available from: https://doi.org/10.1002/bmc.1632
Martın ME, Hernandez OM, Jimenez AI, Arias JJ, Jimenez F. Partial least-squares method in analysis by differential pulse polarography. Simultaneous determination of amiloride and hydrochlorothiazide in pharmaceutical preparations. Analytica chimica acta. 1999;381(2-3):247-256. Available from: https://doi.org/10.1016/s0003-2670(98)00732-6
Li YN, Moore DE, Tattam BN. Photodegradation of amiloride in aqueous solution. International journal of pharmaceutics. 1999;183(2):109-116. Available from: https://doi.org/10.1016/s0378-5173(99)00035-6
Mohammed NS, Mohammed AJ. Development and validation of RP‐HPLC method for the determination of hydrochlorothiazide in bulk drug and pharmaceutical dosage form. Chromatography Research International. 2016;2016(1):1-7. Available from: https://doi.org/10.1155/2016/1693024
Gotardo MA, Pezza L, Pezza HR. Determination of hydrochlorothiazide in pharmaceutical formulations by diffuse reflectance spectroscopy. Eclética Química. 2005;30:17-24. Available from: https://doi.org/10.1590/s0100-46702005000200002
Hapse SA, Wagh VS, Kadaskar PT, Dokhe MD, Shirsath AS. Spectrophotometric estimation and validation of hydrochlorothiazide in tablet dosage forms by using different solvents. Der Pharma Chemica. 2012;4(1):10–14.
Rapolu K, Boggula N, Aleti RR, Patlolla P, Tadikonda RR. Development and Validation of UV Spectrophotometric Method for the Determination of Hydrochlorothiazide in Pure and Formulations. International Journal of Pharmacy and Pharmaceutical Research. 2024;30(4): 304-312.
Shaikh JS, Rao NN. Simultaneous estimation and forced degradation studies of amiloride hydrochloride and hydrochlorothiazide in a pharmaceutical dosage form using RPHPLC method. International Journal of Research in Pharmaceutical and Nano Sciences. 2017; 6(6):300-309.
Anastas P, Eghbali N. Green chemistry: principles and practice. Chemical Society Reviews. 2010;39(1):301-312. Available from: https://doi.org/10.1039/b918763b
Tobiszewski M, Namieśnik J. Greener organic solvents in analytical chemistry. Current Opinion in Green and Sustainable Chemistry. 2017;5:1-4. Available from: https://doi.org/10.1016/j.cogsc.2017.03.002
Ivanković A, Dronjić A, Bevanda AM, Talić S. Review of 12 principles of green chemistry in practice. International Journal of Sustainable and Green Energy. 2017;6(3):39-48. Available from: https://doi.org/10.11648/j.ijrse.20170603.12
Borman P, Elder D. Q2 (R1) Validation of Analytical Procedure: Text and methodology. ICH Quality guidelines [Interent]. 2017;127-66. Available from: http://dx.doi.org/10.1002/9781118971147.ch5
Bakshi M, Singh S. Development of validated stability indicating assay methods-critical review. J Pharm Biomed Anal. 2002;28(6):1011-1040. Available from: https://doi.org/10.1016/s0731-7085(02)00047-x
Maggio RM, Vignaduzzo SE, Kaufman TS. Practical and regulatory considerations for stability-indicating methods for the assay of bulk drugs and drug formulations. TrAC Trends in Analytical Chemistry. 2013;49:57-70. Available from: https://doi.org/10.1016/j.trac.2013.05.008
Gałuszka A, Migaszewski ZM, Konieczka P, Namieśnik J. Analytical Eco-Scale for assessing the greenness of analytical procedures. TrAC Trends in Analytical Chemistry. 2012;37:61-72. Available from: https://doi.org/10.1016/j.trac.2012.03.013
Płotka-Wasylka J. A new tool for the evaluation of the analytical procedure: Green Analytical Procedure Index. Talanta. 2018;181:204-209. Available from: https://doi.org/10.1016/j.talanta.2018.01.013
Pena-Pereira F, Wojnowski W, Tobiszewski M. AGREE—Analytical GREEnness metric approach and software. Analytical chemistry. 2020;92(14):10076-10082. Available from: https://doi.org/10.1021/acs.analchem.0c01887
Published
Abstract Display: 122 
PDF Downloads: 45 How to Cite
Issue
Section
Copyright (c) 2025 Neetu Sabarwal, Suman Jain, D. D. Agarwal
This work is licensed under a Creative Commons Attribution 4.0 International License.
