Comprehensive Analytical Method Development for Related Substance Determination in Dorzolamide Hydrochloride-Timolol Maleate Ophthalmic Solutions: Multi-Method Approach and Regulatory Compliance.

This study presents the development and validation of three selective chromatographic methods for the identification and quantification of related substances, including process-related impurities and degradation products, in combined ophthalmic formulations of dorzolamide hydrochloride (DH) and timolol maleate (TM). The methods were applied to ophthalmic solutions containing 2.0% DH and 0.5% TM (20 and 5 mg/mL, respectively) and were designed to comply with the requirements of the USP, B.P., and Ph. Eur. Both an in-house developed generic formulation and the reference listed drug formulations, COSOPT PF and COSOPT, were evaluated during method development and validation. The novelty of this work lies in the use of three complementary chromatographic approaches that significantly improve specificity, selectivity, and impurity profiling in complex ophthalmic matrices during the aqueous in-process of both phases (aqueous and polymer mixing phase). In addition, the study provides a comparative assessment between preservative-free and preserved formulations, supporting regulatory compliance, stability evaluation, and bioequivalence assessment of generic ophthalmic products.