A stability-indicating RP-HPLC method for the simultaneous quantification of brimonidine tartrate and timolol maleate in bulk drug substances and pharmaceutical dosage forms.

A robust, rapid, and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous quantification of Brimonidine Tartrate and Timolol Maleate (Combigan) in ophthalmic dosage forms. Chromatographic separation was achieved on a Supelco Discovery C18 column (25 cm × 4.6 mm, 5 μm) maintained at ambient temperature, employing isocratic elution with two mobile phases: phase A (buffer at pH 7.0 containing 30 mM triethylamine) and phase B (acetonitrile) in a ratio of 80:20. The flow rate was set at 1.0 mL/min, and detection was performed at 245 nm and 295 nm using a diode array detector (DAD). Method validation, conducted in accordance with ICH Q2(R1), USP, and FDA guidelines, confirmed excellent linearity over the ranges of 0.24-500 ppm for Brimonidine Tartrate and 0.60-1250 ppm for Timolol Maleate. Accuracy results ranged from 99.42% to 99.82% for Brimonidine Tartrate and from 98.71% to 101.10% for Timolol Maleate. Relative standard deviations (RSDs) for precision, specificity, and robustness were all below 2%, demonstrating the method's consistency and reliability. Additionally, the limits of detection (LOD) were determined to be 0.08 ppm for Brimonidine Tartrate and 0.20 ppm for Timolol Maleate, while the limits of quantification (LOQ) were 0.24 ppm and 0.60 ppm, respectively. Forced degradation studies under various stress conditions, including acid and base hydrolysis and hydrogen peroxide oxidation, demonstrated that the method successfully separated Brimonidine Tartrate and Timolol Maleate from their degradation products, confirming its stability-indicating capability. Notably, both drugs remained stable under thermal and photolytic stress; however, Timolol Maleate was significantly more prone to degradation under strong hydrolytic and oxidative conditions, underscoring the need for stringent control during formulation and storage. Additionally, the three complementary green analytical chemistry (GAC) metrics were evaluated. The method achieved an Eco-Scale score of approximately 75. The GAPI pictogram for this method showed a mixture of green and yellow zones. The AGREE evaluation yielded a score of 0.57 (out of 1.0), indicating moderate greenness. Overall, this method effectively quantified both active pharmaceutical ingredients without interference from excipients or degradation products, supporting its suitability for routine quality control and stability testing of combined ophthalmic formulations.