A Simple and Validated Stability Indicating HPLC Method for
Simultaneous Quantification of Brimonidine, Timolol and
Benzalkonium Chloride in Anti-Glaucoma Ophthalmic Formulations

The combined dosage form of brimonidine, timolol and benzalkonium chloride is prescribed for
lowering intraocular pressure in patients with ocular hypertension. As no analytical HPLC method
reported for the estimation of brimonidine, timolol and benzalkonium chloride together in ophthalmic
formulations, the present study was aimed to fulfill the gap identified in literature. The separation of
analytes was achieved on Cosmosil C18 (250 × 4.6 mm; 5 μ id) as stationary phase, pH 4.3 acetate
buffer and methanol in 30:70 (v/v) as mobile phase at 1.0 mL/min and UV detection at 227 nm. In this
condition, well resolved, retained peaks were identified at 3.11 min for brimonidine, 4.86 min for
timolol and 5.57 min for benzalkonium chloride. The method reports 0.5, 1.25 and 0.0125 μg/mL,
respectively for brimonidine, timolol and benzalkonium chloride as LOD, which proves that the method
have enough sensitivity levels for the detection analytes in samples. The method passes all the validation
parameters as per the guidelines proved that the method was valid. The method shows 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 analyzing the drug content in Combigan®
ophthalmic drops and method reports the assay of 98.92%, 99.36% and 98.46% for brimonidine,
timolol and benzalkonium chloride, respectively. Based on the results, it can be concluded that the
method can adequately suitable for the separation and quantification of brimonidine, timolol and
benzalkonium chloride and hence can be applicable for the routine analysis of brimonidine, timolol
and benzalkonium chloride in ophthalmic formulations.