Abstract
Introduction:
The simultaneous quantification of Brimonidine Tartrate and Timolol Maleate in ophthalmic formulations is essential for ensuring product quality, efficacy, and stability. However, few methods provide adequate selectivity, sensitivity, and environmental sustainability. This study aimed to develop and validate a robust, rapid, and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC) method with stability-indicating and green chemistry attributes.
Methods:
Chromatographic separation was performed on a Supelco Discovery C18 column (25 cm × 4.6 mm, 5 μm) using isocratic elution with phase A (30 mM triethylamine buffer, pH 7.0) and phase B (acetonitrile) in a ratio of 80:20. The flow rate was 1.0 mL/min, and analytes were detected at 245 nm and 295 nm using a diode array detector. Method validation followed ICH Q2 (R1), USP, and FDA guidelines, evaluating linearity, accuracy, precision, specificity, robustness, and sensitivity. Forced degradation under acid, base, oxidative, thermal, and photolytic conditions was conducted to assess stability-indicating capability. Green analytical chemistry (GAC) metrics were calculated using Eco-Scale, GAPI, and AGREE tools.
Results:
The method exhibited excellent linearity over 100-500 ppm for Brimonidine Tartrate and 250-1,250 ppm for Timolol Maleate. Accuracy ranged from 99.42% to 99.82% for Brimonidine Tartrate and 98.71% to 101.10% for Timolol Maleate. Precision, specificity, and robustness results showed relative standard deviations below 2%. The LODs were 0.08 ppm for Brimonidine Tartrate and 0.20 ppm for Timolol Maleate, while LOQs were 0.24 ppm and 0.60 ppm, respectively. Forced degradation confirmed the method's ability to separate both drugs from their degradation products. Brimonidine Tartrate remained stable under all stress conditions, whereas Timolol Maleate was susceptible to hydrolytic and oxidative degradation. The method demonstrated moderate greenness with an Eco-Scale score of ~75, a GAPI pictogram with mixed green/yellow zones, and an AGREE score of 0.57.
Discussion:
The validated RP-HPLC method proved accurate, precise, sensitive, and stability-indicating for the simultaneous determination of Brimonidine Tartrate and Timolol Maleate. Its moderate GAC performance supports a balance between analytical rigor and sustainability. These findings establish the method as suitable for routine quality control and stability testing of ophthalmic formulations containing both drugs.
Keywords:
accuracy; brimonidine tartrate; high-performance liquid chromatography (HPLC); limit of detection (LOD); linearity; precision; specificity; timolol maleate.