pKa Estimation of Ambrisentan by Reversed-Phase High-Performance Liquid Chromatography

Erkan Tütüncü; Dilara Başat Dereli

doi:10.17798/bitlisfen.1917355

pKa Estimation of Ambrisentan by Reversed-Phase High-Performance Liquid Chromatography

Abstract

The characterization of ionization behavior in pharmaceutical active ingredients is fundamental to understanding their bioavailability, pharmacokinetics, and formulation development. Ionization constants (pKa) are critical parameters that directly influence the solubility, stability, and membrane permeability of drugs, especially those with weakly acidic or basic characters. Ambrisentan, a selective endothelin receptor antagonist used in the treatment of pulmonary arterial hypertension, is a lipophilic compound whose ionization behavior requires detailed investigation. In this study, a reversed-phase high-performance liquid chromatography (RP-HPLC) method was employed to determine the pKa value of Ambrisentan using a C18 column at 37 °C across a mobile phase pH range of 2.5–7.5. To examine the effect of solvent composition on ionization, pKa determinations were performed in 40%, 45%, and 50% (v/v) acetonitrile-water binary mixtures. The experimental data were evaluated using the dielectric constant method and mole fraction (XACN– pKa) extrapolation methods. The calculated pKa value of Ambrisentan in aqueous medium was successfully estimated as 4.145±0.156 and 4.134±0.156 for the respective models. The results demonstrate that the developed chromatographic approach provides a reliable and reproducible tool for the pKa determination of drugs with limited aqueous solubility, which is strongly supported by the excellent agreement between the two extrapolation methods and high linear correlation coefficients (r > 0.999). This study contributes to the literature by providing essential physicochemical characterization data for Ambrisentan, which is vital for future pharmaceutical applications and analytical method development.

Keywords

Supporting Institution

Scientific Research Projects Coordination Office of Bitlis Eren University

Project Number

BEBAP 2018.05

Ethical Statement

The study is complied with research and publication ethics.

Thanks

This study was derived from a master's thesis and was supported by the Scientific Research Projects Coordination Office of Bitlis Eren University under project number BEBAP 2024.07. The authors would like to thank the Scientific Research Projects Coordination Office of Bitlis Eren University for their financial support.

References

N. Galié, R. N. Channick, R. P. Frantz, E. Grünig, Z. C. Jing, O. Moiseeva, I. R. Preston, T. Pulido, Z. Safdar, Y. Tamura, and V. V. McLaughlin, “Risk stratification and medical therapy of pulmonary arterial hypertension,” *European Respiratory Journal*, vol. 53, no. 1, 2019, doi: 10.1183/13993003.01889-2018.
R. L. Benza, M. K. Kanwar, A. Raina, J. V. Scott, C. L. Zhao, M. Selej, C. G. Elliott, and H. W. Farber, “Development and validation of an abridged version of the REVEAL 2.0 Risk Score Calculator, REVEAL Lite 2, for use in patients with pulmonary arterial hypertension,” *Chest*, vol. 159.
T. Thenappan, S. J. Shah, S. Rich, and M. Gomberg-Maitland, “Survival in pulmonary arterial hypertension: A comparison of the REVEAL registry and historical cohorts,” *Chest*, vol. 132, no. 2, pp. 410–417, 2007.
M. M. Hoeper *et al*., “Incidence and prevalence of pulmonary arterial hypertension: A systematic review,” *European Respiratory Journal*, vol. 30, no. 6, pp. 1047–1057, 2006.
N. Galié *et al*., “Initial use of ambrisentan plus tadalafil in pulmonary arterial hypertension,” *New England Journal of Medicine*, vol. 373, no. 9, pp. 834–844, 2015.
M. Beghetti *et al*., “Safety and efficacy of the endothelin receptor antagonist macitentan in pediatric pulmonary hypertension,” *Pediatric Pulmonology*, vol. 55, no. 12, pp. 3406–3414, 2020.
M. Humbert, G. Kovacs, M. M. Hoeper, R. Badagliacca, R. M. F. Berger, M. Brida, *et al*., “2022 ESC/ERS guidelines for the diagnosis and treatment of pulmonary hypertension,” *European Heart Journal*, vol. 43, no. 38, pp. 3618–3731, 2022.
N. Galié *et al*., “Ambrisentan for the treatment of pulmonary arterial hypertension: Results of the ARIES studies,” *Circulation*, vol. 117, no. 23, pp. 3010–3019, 2008.

M. Wiese and M. Gutschow, “Liquid chromatographic methods for the determination of pKa values,” *Journal of Pharmaceutical and Biomedical Analysis*, vol. 27, no. 5, pp. 867–873, 2002.
A. Avdeef, *Absorption and Drug Development: Solubility, Permeability, and Charge State*. Hoboken, NJ, USA: Wiley, 2012.
Z. Üstün and E. Ç. Demiralay, “Determination of the dissociation constants of six pharmacologically active imidazole antifungal agents by RPLC method,” *Microchemical Journal*, vol. 219, Art. no. 116019, 2025.
D. B. Dereli and A. G. Alsancak, “Determination of the ionization constants of sulfonylureas in THF–water media by potentiometric titration and RPLC methods,” *Journal of the Iranian Chemical Society*, vol. 19, pp. 1889–1898, 2022.
D. B. Dereli, S. Akkoc, and E. Ç. Demiralay, “Protonation constant determination of some benzimidazole-based drug candidates by reverse phase liquid chromatography method and cytotoxic activity studies,” *ChemistrySelect*, vol. 8, Art. no. e202301192, 2023.
M. Balakrishna, A. Unnisa, P. Reddy, and C. H. Suma, “RP-HPLC-PDA method for the analysis of ambrisentan in bulk drug and pharmaceutical dosage forms,” *International Journal of Chemical and Pharmaceutical Sciences*, vol. 4, no. 4, pp. 45–50, 2013.
P. R. Ravi, S. Ganga, and R. N. Saha, “Development and validation of stability indicating RP-HPLC method for estimation of ambrisentan in bulk and pharmaceutical formulation,” *International Journal of Pharmacy and Pharmaceutical Sciences*, vol. 5, no. 4, pp. 674–678, 2013.
M. Iglarz, C. Binkert, K. Morrison, W. Fischli, J. Gatfield, A. Treiber, T. Weller, M. H. Bolli, C. Boss, S. Buchmann, B. Capeleto, P. Hess, C. Qiu, and M. Clozel, “Pharmacology of macitentan, an orally active tissue-targeting dual endothelin receptor antagonist,” *Journal of Pharmacology and Experimental Therapeutics*, vol. 327, no. 3, pp. 736–745, 2008.
J. A. Angus, P. F. Soeding, R. J. A. Hughes, and C. E. Wright, “Functional estimation of endothelin-1 receptor antagonism by bosentan, macitentan and ambrisentan in human pulmonary and radial arteries in vitro,” *European Journal of Pharmacology*, vol. 804, pp. 111–116, 2017.
P. H. Sherrod, *NLREG: Nonlinear Regression Analysis Program*. Nashville, TN, USA: Phillip H. Sherrod, 2015. [Online]. Available: https://philsherrod.com/NLREGweb/. [Accessed: Mar. 27, 2025].
Swiss Institute of Bioinformatics, “SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules.” [Online]. Available: http://www.swissadme.ch. [Accessed: Mar. 12, 2025].
J. Barbosa, I. Toro, and V. Sanz-Nebot, “Acid-base behaviour of tripeptides in solvents used in liquid chromatography: Correlation between pK values and solvatochromic parameters of acetonitrile-water mixtures,” *Analytica Chimica Acta*, vol. 347, no. 3, pp. 295–304, 1997.

Details

Primary Language

English

Subjects

Instrumental Methods

Journal Section

Research Article

Authors

Erkan Tütüncü
0009-0007-6014-7943
Türkiye

Dilara Başat Dereli ^*
0000-0003-1328-077X
Türkiye

Publication Date

September 30, 2026

Submission Date

March 27, 2026

Acceptance Date

June 26, 2026

Published in Issue

Year 2026 Volume: 15 Number: 3

DOI

https://doi.org/10.17798/bitlisfen.1917355

IZ

https://izlik.org/JA35HB24ME

Cite

RIS / Bibtex

APA

Tütüncü, E., & Başat Dereli, D. (2026). pKa Estimation of Ambrisentan by Reversed-Phase High-Performance Liquid Chromatography. Bitlis Eren Üniversitesi Fen Bilimleri Dergisi, 15(3), 1417-1425. https://doi.org/10.17798/bitlisfen.1917355

AMA

1.Tütüncü E, Başat Dereli D. pKa Estimation of Ambrisentan by Reversed-Phase High-Performance Liquid Chromatography. Bitlis Eren Üniversitesi Fen Bilimleri Dergisi. 2026;15(3):1417-1425. doi:10.17798/bitlisfen.1917355

Chicago

Tütüncü, Erkan, and Dilara Başat Dereli. 2026. “PKa Estimation of Ambrisentan by Reversed-Phase High-Performance Liquid Chromatography”. Bitlis Eren Üniversitesi Fen Bilimleri Dergisi 15 (3): 1417-25. https://doi.org/10.17798/bitlisfen.1917355.

EndNote

Tütüncü E, Başat Dereli D (September 1, 2026) pKa Estimation of Ambrisentan by Reversed-Phase High-Performance Liquid Chromatography. Bitlis Eren Üniversitesi Fen Bilimleri Dergisi 15 3 1417–1425.

IEEE

[1]E. Tütüncü and D. Başat Dereli, “pKa Estimation of Ambrisentan by Reversed-Phase High-Performance Liquid Chromatography”, Bitlis Eren Üniversitesi Fen Bilimleri Dergisi, vol. 15, no. 3, pp. 1417–1425, Sept. 2026, doi: 10.17798/bitlisfen.1917355.

ISNAD

Tütüncü, Erkan - Başat Dereli, Dilara. “PKa Estimation of Ambrisentan by Reversed-Phase High-Performance Liquid Chromatography”. Bitlis Eren Üniversitesi Fen Bilimleri Dergisi 15/3 (September 1, 2026): 1417-1425. https://doi.org/10.17798/bitlisfen.1917355.

JAMA

1.Tütüncü E, Başat Dereli D. pKa Estimation of Ambrisentan by Reversed-Phase High-Performance Liquid Chromatography. Bitlis Eren Üniversitesi Fen Bilimleri Dergisi. 2026;15:1417–1425.

MLA

Tütüncü, Erkan, and Dilara Başat Dereli. “PKa Estimation of Ambrisentan by Reversed-Phase High-Performance Liquid Chromatography”. Bitlis Eren Üniversitesi Fen Bilimleri Dergisi, vol. 15, no. 3, Sept. 2026, pp. 1417-25, doi:10.17798/bitlisfen.1917355.

Vancouver

1.Erkan Tütüncü, Dilara Başat Dereli. pKa Estimation of Ambrisentan by Reversed-Phase High-Performance Liquid Chromatography. Bitlis Eren Üniversitesi Fen Bilimleri Dergisi. 2026 Sep. 1;15(3):1417-25. doi:10.17798/bitlisfen.1917355