A versatile LC method for the simultaneous quantification of latanoprost, timolol and benzalkonium chloride and related substances in the presence of their degradation products in ophthalmic solution

Abstract

A challenging and multipurpose stability-indicating HPLC method is developed and validated for the quantification of latanoprost, timolol, benzalkonium chloride (BKC) and related substances in the presence of their degradation products in ophthalmic solution. Various stress conditions, e.g. acid, base, oxidation, heat, UV radiation and heat were employed to assess the stability-indicating nature of the method. A strategic experimental approach was implemented for the method development. The desired chromatographic separation was achieved on a reverse phase cyano column {Hypersil BDS CN (250 × 4.6 mm), 5 μm} under gradient elution conditions. A mixture of phosphate buffer of pH 3.2, acetonitrile and methanol was used as the mobile phase. The determination was carried out at 295 nm and 210 nm. Due to the very low content of latanoprost (0.005%) in the formulation, the injection volume was optimized as 80 μl in order to achieve an optimum response. In totality, peaks of latanoprost and its two known impurities, four BKC homologs, timolol and its known impurities were obtained with a minimum resolution of 3.0. This sensitive method was found to be precise and linear through out the range with a lowest quantification of 0.068 μg ml⁻¹ for 15-keto latanoprost, 0.030 μg ml⁻¹ for latanoprost acid, 0.018 μg ml⁻¹ for latanoprost (unknown impurity), 0.011 μg ml⁻¹ for timolol impurity I and 0.013 μg ml⁻¹ for timolol (unknown impurity). The method was validated according to ICH guidelines. This versatile method can be used as a combined assay and related substance method for said ophthalmic solution formulation.