SAGE-Hindawi Access to Research Chromatography Research International Volume 2011, Article ID 929876, 9 pages doi:10.4061/2011/929876 Research Article Stability-Indicating Gradient RP-LC Method for the Determination of Process and Degradation Impurities in Bosentan Monohydrate: An Endothelin Receptor Antagonist Sanjay A. Jadhav, 1 Shashikant B. Landge, 1 Sonali L. Jadhav, 1 Navanath C. Niphade, 1 Saroj R. Bembalkar, 2 and Vijayavitthal T. Mathad 1 1 Research and Development, Megafine Pharma (P) Ltd., Nashik 422 202, India 2 Department of Chemistry, Deogiri College, Aurangabad 431004, India Correspondence should be addressed to Vijayavitthal T. Mathad, drvtmathad@yahoo.co.in Received 16 June 2011; Revised 29 July 2011; Accepted 2 August 2011 Academic Editor: Cristina Minguill ´ on Copyright © 2011 Sanjay A. Jadhav et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Described is a simple, rapid, selective, and stability-indicating RP-LC method for the determination of process and degradation- related impurities of bosentan monohydrate. Chromatographic separation was achieved on Zorbax SB-Phenyl column thermostated at 35 ◦ C under gradient elution by a binary mixture of solvent A (60% phosphate buffer, pH 2.5, and 40% methanol) and solvent B (acetonitrile) at a flow rate of 1.0 mL/min. Forced degradation was carried out under acidic, alkaline, oxidative, photolytic, and thermal conditions. Significant degradation is observed in acid and alkali stress conditions. Process- and degradation-related impurities were characterized by mass spectrometry, 1 H NMR, and FT-IR spectral data. Validation of the developed method was carried out as per ICH requirements. Regression analysis shows an “r ” value (correlation coefficient) of greater than 0.999 for bosentan and five potential impurities. This method was capable to detect the five impurities at 0.01% of the test concentration of 1.0 mg mL −1 . 1. Introduction Bosentan monohydrate (4-tert-butyl-N-[6-(2-hydroxye- thoxy)-5-(2-methoxyphenoxy)-2-(pyrimidin-2-yl) pyrim- idin-4-yl]benzene-1-sulfonamide monohydrate), a dual endothelin receptor antagonist (ERA) has molecular for- mula of C 27 H 29 N 5 O 6 S·H 2 O with relative molecular mass of 569.64. It is the first orally active drug approved by United States Food and Drug Administrative as Tracleer (65mg and 125 mg) for the successful treatment of pulmonary arterial hypertension (PAH). Tracleer improves the exercise ability and decreases the rate of clinical worsening in patients with WHO Class III or IV symptoms of PAH, by blocking the binding of endothelin to its receptors, thereby negating endothelin’s deleterious effects [1–8]. Further Tracleer has been demonstrated to be effective in remodeling the pulmonary vascular tree through several mechanisms including vasodilatation, antifibrotic and antithrombotic actions [9]. An extensive literature survey revealed that there are several bioanalytical HPLC methods for the determination of bosentan monohydrate and its metabolite in blood plasma, whereas, there are few other literatures disclosed only for the quantitative determination of bosentan in biological and formulation samples [10–13]. The reported HPLC method [13] was not capable to separate the peaks of impurities and bosentan. The literature survey also revealed that there was no stability-indicating RP-LC method for the determination of process and degradation-related impurities formed under the stress conditions in bosentan monohydrate. In this paper we describe development and validation of related substances method for accurate quantification of five potential process impurities in bosentan monohydrate samples as per International Conference on Harmonization (ICH) recommendations. Intensive stress studies are carried out on bosentan monohydrate; accordingly a stability- indicating method is developed, which could separate vari- ous degradation products. The present active pharmaceutical