Development of Novel RP-HPLC Method for Separation and Estimation of Critical Geometric Isomer and Other Related Impurities of Tafluprost Drug Substance and Identification of Major Degradation Compounds by Using LC-MS.

Abstract

A novel, simple, sensitive and stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the quantitative determination of the geometric isomer (Trans) and other related substances in the active pharmaceutical ingredient (API) of Tafluprost (TFL), with their determination by an assay. A chromatographic separation of TFL and its impurities was achieved with a C18 analytical column, using gradient elution with mobile phase A consisting of a mixture of water, methanol and orthophosphoric acid (900:100:1, v/v) and mobile phase B consisting of a mixture of acetonitrile and water (900:100, v/v). The instrumental settings included a flow rate of 1.0 mL/min for related substances and 1.2 mL/min for the assay, a column temperature of 50°C and a detector wavelength of 210 nm, using a photodiode array detector. TFL was exposed to thermal, photolytic, hydrolytic and oxidative stress conditions and the stressed samples were analyzed by the proposed method. Peak homogeneity data of TFL were obtained by using a photodiode array detector in the stressed sample chromatograms, which demonstrated the specificity of the method for estimation in the presence of degradants. The developed method was validated for parameters such as precision, accuracy, linearity, limit of detection, limit of quantification, ruggedness and robustness as per ICH guidelines.