Eco-friendly Indirect Spectrophotometric Method for Estimation of Amiloride Hydrochloride in its Pharmaceutical Preparations Via Eosin Y
DOI:
https://doi.org/10.56286/t1f5hk02Keywords:
Amiloride hydrochloride, Eosin Y dye, Calcium hypochlorite, Indirect Spectrophotometry, GAPIAbstract
A simple, sensitive, and precise indirect spectrophotometric method has been developed for quantifying amiloride hydrochloride (AMD. HCl), both as a pure compound and in its dosage forms (tablets). This work is based on indirect spectrophotometric method of the oxidation of (AMD. HCl) by adding a known amount of oxidizing agent calcium hypochlorite (Ca(OCl)2) in acidic medium. The unreacted amount of Ca(OCl)2 then reacted with a known quantity of the dye eosin Y to shorten its color, and then the absorption of the remaining amount of the dye to form an orange-colored product that can be measured at a wavelength of 518 nm. A thorough investigation was conducted into the optimization of method conditions. Beer's law was followed under ideal circumstances in the concentration range of 2–12 µg/mL, with a relative standard deviation of 2.62%, a recovery of 100.03%, and a determination coefficient (R2) of 0.9992. The limit of quantification (LOQ) was 0.0932 µg/mL, while the limit of detection (LOD) was 0.028 µg/mL. The molar absorptivity coefficient was 1.8856×104 L/mol. cm, which corresponds to Sandall’s sensitivity of 0.0138 µg/cm2. The method was applied to the assay amiloride hydrochloride (AMD. HCl) in tablets, yielding acceptable analytical results. Additionally, a new tool for assessing analytical protocols related to green analytical chemistry attributes had been used in order to assess the environmental impact of the proposed technique. The new instrument is known as the Green Analytical Procedure Index, or GAPI.
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References
British Pharmacopoeia. (2020). Amiloride hydrochloride. The Stationery Office, London. (Vol. I), (pp. 1–143).
Sabarwal, N., Jain, S., & Agarwal, D. D. (2021). Spectrophotometric Method for Simultaneous Determination of Amiloride and Hydrochlorothiazide in Combined Dosage Form. Journal of Advanced Scientific Research, 12(03), 170–174.
Suraj, K., & Bishnu, U. (2024). Formulation and Evaluation of Gastroretentive Floating Microspheres of Amiloride Hydrochloride. Asian Pacific Journal of Health Sciences, 11(3), 14–24. https://doi.org/10.21276/apjhs.2024.11.3.04
Sun, Q., & Sever, P. (2020). Amiloride: A review. JRAAS - Journal of the Renin-Angiotensin-Aldosterone System, 21(4).
Shah, J. V., Shah, P. A., Sanyal, M., & Shrivastav, P. S. (2017). Simultaneous quantification of amiloride and hydrochlorothiazide in human plasma by liquid chromatography–tandem mass spectrometry. Journal of Pharmaceutical Analysis, 7(5), 288–296.
Gong, M., Chang, Y., Shao, J., Li, S., Zhang, Y., & Chang, H. (2019). Microbial Pathogenesis Antiviral effect of amiloride on replication of foot and mouth disease virus in cell culture. Microbial Pthogenesis, 135(1), 103638. https://doi.org/10.1016/j.micpath.2019.103638
Yellepeddi, V., Sayre, C., Burrows, A., Watt, K., Davies, S., Strauss, J., & Battaglia, M. (2020). Stability of extemporaneously compounded amiloride nasal spray. PLoS ONE, 15(7), 1–11. https://doi.org/10.1371/journal.pone.0232435
Nagori, B. P., & Solanki, R. (2010). RP-HPLC method for simultaneous estimation of frusemide and amiloride hydrochloride in tablet formulation. Indian Journal of Pharmaceutical Sciences, 72(3), 384–387. https://doi.org/10.4103/0250-474X.70491
Mahgoub, S. (2016). Novel Stability-Indicating RP-HPLC Method for Determination of Hydrochlorothiazide, Amiloride Hydrochloride and Timolol Maleate in Tablet Dosage Form. American Journal of Modern Chromatography, 3(1), 23–32.
Kumar, R. A., Babu, G. R., M., S., & M., R. (2021). Validated RP-HPLC method for the estimation of Amiloride and hydrochlorothiazide in combined tablet dosage form. Asian Journal of Pharmaceutical Analysis, 11(3), 207–211. https://doi.org/10.52711/2231-5675.2021.00037
Shaikh, J. S., & Rao, N. N. (2018). Simultaneous estimation and forced degradation studies of amiloride hydrochloride and furosemide in a pharmaceutical dosage form using reverse-phase high-performance liquid chromatography method. Asian Journal of Pharmaceutical and Clinical Research, 11(7), 215–221. https://doi.org/10.22159/ajpcr.2018.v11i7.25783
Sabarwal, N., Jain, S., & Agarwal, D. D. (2023). Stability Indicating Reverse Phase Ultra Performance Liquid Chromatography Method Development and Validation for Amiloride and Hydrochlorothiazide Tablet in the Presence of Degradation Products and Application to Green Analytical Principles. International Journal of Pharmaceutical Quality Assurance, 14(4), 1058–1066. https://doi.org/10.25258/ijpqa.14.4.38
Lakshmi, A. (2015). Spectrophotometric method for estimation of amiloride in bulk and tablet dosage form. Drug Development and Therapeutics, 6(2), 62. https://doi.org/10.4103/2394-6555.162443
Lotfy, H. M., & Saleh, S. S. (2016). Recent development in ultraviolet spectrophotometry through the last decade (2006–2016): A review. International Journal of Pharmacy and Pharmaceutical Sciences, 8(10), 40–56. https://doi.org/10.22159/ijpps.2016v8i10.13537
Turkey, N.S., & Alamri, M.H (2014). A new mode for an on-line determination of amiloride in pure and pharmaceutical preparation using CFIA with homemade Ayah 3SBGRx3-3D solar cell microphotometer analyzer. Iraqi Journal of Science, 55, 1137–1152.
Wedian, F., Lataifeh, A., & Mohammed, M. S. (2020). Simultaneous spectrofluorometric analysis of tablets containing hydrochlorothiazide combined with timolol maleate or amiloride hydrochloride. Acta Pharmaceutica, 70(3), 373–385. https://doi.org/10.2478/acph-2020-0027
Turkey, N. S., & Nafea, H. M. (2020). Determination of amiloride via quenched continuous fluorescence of azo dye using low-pressure mercury lamp tube (UV-light) and multi solar cells at 2 x 90° as detectors. International Journal of Applied Pharmaceutics, 12(5), 211–218. https://doi.org/10.22159/ijap.2020v12i5.38268
Nascimento, T. O., Leite, F. R. F., Mourão, H. A. J. L., & Malagutti, A. R. (2020). Development of an electroanalytical methodology using differential pulse voltammetry for amiloride determination. Journal of Solid-State Electrochemistry, 24(8), 1735–1741. https://doi.org/10.1007/S10008-020-04559-5/METRICS
Zayed, S. I. M., & Arida, H. A. M. (2013). Preparation of carbon paste electrodes and it using in voltametric determination of amiloride hydrochloride using in the treatment of high blood pressure. International Journal of Electrochemical Science, 8(1), 1340–1348. https://doi.org/10.1016/s1452-3981(23)14102-2
Mohammadzadeh, S., Bavali, A., & Mokhtari, F. (2024). Laser induced fluorescence spectroscopy of aqueous eosin Y solution. Dyes and Pigments, 222, 111900. https://doi.org/10.1016/J.DYEPIG.2023.111900
Bandoh, C. K., Haruna, M., Adu-Poku, D., Danu, B. Y., Toku, M., Ampong, F. K., Nkum, R. K., & Agorku, E. S. (2025). Gadolinium doped-zirconium oxide-graphitic carbon nitride heterostructures for photocatalytic degradation of eosin yellow dye in water. Next Materials, 6(November 2024). https://doi.org/10.1016/j.nxmate.2024.100424
Rahman, H. (2017). Utilization of Eosin Dye as an Ion Pairing Agent for Determination of Pharmaceuticals: A Brief Review. International Journal of Pharmacy and Pharmaceutical Sciences, 9(12), 1. https://doi.org/10.22159/ijpps.2017v9i12.21220
Barghash, S., Abd El-Razeq, S., El -Awady, M., & Belal, F. (2021). Validated Spectrophotometric method for analysis of Dipyridamole and Lamivudine using eosin Y. Azhar International Journal of Pharmaceutical and Medical Sciences, 1(1), 87–96. https://doi.org/10.21608/aijpms.2021.52761.1016
Khalid W, Al-Behadili H, Jawad Y, Whaab W. Effect of Solvent on Intensity of Absorption and Fluorescence of Eosin Y Dye and Spectral Properties of Eosin Y Dye. 2023 Feb 13;6:322–34.
Yim, J. H., Song, K. Y., Kim, H., Bae, D., Chon, J. W., & Seo, K. H. (2021). Effectiveness of calcium hypochlorite, quaternary ammonium compounds, and sodium hypochlorite in eliminating vegetative cells and spores of Bacillus anthracis surrogate. Journal of Veterinary Science, 22(1), 1–7. https://doi.org/10.4142/jvs.2021.22.e11
Pauletto, G., Brum, N. F., Carlotto, I. B., da Rosa, L. S., & Bier, C. A. S. (2025). Effect of sodium and calcium hypochlorite with or without surfactant on the adhesion of epoxy resin-based endodontic sealer. Brazilian Journal of Oral Sciences, 24, 1–11. https://doi.org/10.20396/BJOS.V24I00.8674080
Coaguila-Llerena, H., Ramos, J. D. S. T. A. P., & Faria, G. (2022). Physicochemical properties and penetration into dentinal tubules of calcium hypochlorite with surfactants. Brazilian Dental Journal, 33(2), 1–11. https://doi.org/10.1590/0103-6440202204567
Abdel-Hay, M. H., Ragab, M. A., Ahmed, H. M., & Mohyeldin, S. M. (2019). The use of Arrhenius kinetics to evaluate different hydrolytic stability of amiloride hydrochloride and cyclopenthiazide using chromatographic methods. Microchemical Journal, 147, 682-690.
Christian GD, Dasgupta PK, Schug K. Analytical chemistry. Seventh edition. Hoboken, NJ: John Wiley and Sons, Inc.; 2014. ISBN: 978-1-118-80527-5.
Hanamshetty, P. C., Siddappa, K., Chavan, P., & Nagabhushana, M. M. (2023). Spectrophotometric Assay Methods for Determination of Amiloride: A Charge-Transfer Reaction Approach with 2,3-Dichloro-5,6-Dicyano-1,4-Benzoquinone and p-Chloranilic Acid Reagents. In Indian Journal of Pharmaceutical Sciences (Vol. 85, Issue 6). https://doi.org/10.36468/pharmaceutical-sciences.1221
Saleem, K.A., Fakhre, N.A.,and Qader. H. A. (2024). Simultaneous Determination of Chlorhexidine and Triclosan in Some Pharmaceutical Personal Care Products Using Q-Absorbance Ratio Spectra and H-point Standard Addition Methods, Advanced Journal of Chemistry, Section A, 7(6), 740-757. https://doi.org/10.48309/AJCA.2024.456004.1518.
Nowak, P.M., Wietecha-Pos?uszny, R. and Pawliszyn, J. (2021). White analytical chemistry: An approach to reconcile the principles of green analytical chemistry and functionality, TrAC Trends in Analytical Chemistry, 138, 116223. https://doi.org/10.1016/j.trac.2021.116223.
P?otka-Wasylka,J. (2018). A new tool for the evaluation of the analytical procedure: Green analytical procedure index, Talanta, 181, 204-209.
El-Masry, A.A., El-Wasseef, D.R., Eid, M., Shehata, I.A., and Zeid, A.M. (2022). Development of three ecological spectroscopic methods for analysis of betrixaban either alone or in mixture with lercanidipine: Greenness assessment, Royal Society Open Science, 9, 211457. https://doi.org/10.1098/rsos.211457.
Evangelin, M.P., Priyanka, G., Babu S., M. (2018). Development of new RP-HPLC method for simultaneous estimation of amiloride and hydrochlorothiazide, Indo American Journal of Pharmaceutical Sciences, 05 (05), 3590-3604. http://doi.org/10.5281/zenodo.1244653
Pena-Pereira,F., Wojnowski,W. , Tobiszewski, M., (2020). Agree-analytical greenness metric approach and software, Analytical chemistry, 92, 14, 10076-10082. https://doi.org/10.1021/acs.analchem.0c01887.
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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

