Development and Validation of Stability Indicting HPLC Method for the Separation and Simultaneous Analysis of Timolol, Dorzolamide and Latanoprost Inophthalmic formulations

Abstract

The present work is intended to establish a simple, precise and sensitive stability indicating HPLC method for the separation and simultaneous quantification of timolol, dorzolamide and latanoprost in pharmaceutical formulations. The separation of analytes was achieved on Spherisorb ods2 C18 (250mm × 4.6mm; 5µ)as stationary phase, methanol, acetonitrile and phosphate buffer (pH 5.2) in 55:45:05 (v/v) as mobile phase at 1.0 mL/min and UV detection at 239nm. In this condition, well resolved, retained peaks were identified at 3.45 min fortimolol, 2.66min for dorzolamideand 5.43min for latanoprost. The method reports 0.313µg/mL, 1.25µg/mL and 0.003µg/mL for timolol, dorzolamide and latanoprost respectivelyas LOD that proves that the method have enough sensitivity levels for the detectionanalytes in samples. The method passes all the validation parameters as per the guidelines proved that the method was valid. The method can shows very less % degradation in various stress studies such as acidic, base, peroxide, thermal and UV light conditions and can effectively separate various stress degradation compounds and confirms the stability indicating nature of the method. The method applicability was assessed by analysing the drug content in ophthalmic drops and reports the % assay of be 98.48, 99.37 and 98.32% for timolol, dorzolamide and latanoprost respectively. Based on the results, it can be concluded that the method can adequately suitable for the separation and quantification of timolol, dorzolamide and latanoprost and hence can be applicable for the routine analysis of timolol, dorzolamide and latanoprostin single or any combined ophthalmic formulations.