Green, Validated HPLC Method Coupled With Fluorescence Detection for the Analysis of Tafluprost in its Pure Form and Ophthalmic Formulation: Application to Aqueous Humor and Content Uniformity Testing

Abstract

A green, simple and sensitive HPLC method coupled with fluorescence detection was implemented for the quantitative determination of the anti-glaucoma drug tafluprost (TFL). Liquid chromatography was performed on HyperClone™ ODS (C18) column of dimensions; 150 × 4.6 mm i.d. and 5 μm particle size using a green eluent; ethanol:0.01 M phosphate buffer (60:40 v/v, pH 4.5) delivered at 1 mL min-1. Fluorescence detection was accomplished at 220 nm (excitation) and 292 nm (emission). Bimatoprost (BIM) was used as an internal standard (I.S.). In this method, TFL was eluted after 6.70 minutes. The method satisfied International Council for Harmonization (ICH) validation guidelines, as proved by good linearity (r = 0.9999, over the range 0.05-2 μg mL-1), accuracy (recovery average 100.13 ± 1.27%), precision, robustness and specificity. The limit of detection and limit of quantification were found to be 0.016 and 0.048 μg mL-1, respectively. The proposed method has been successfully applied for the estimation of TFL in eye drops and aqueous humor. For the first time, the approach was applied with acceptable results for the evaluation of the uniformity of TFL eye drops content. Furthermore, Green Analytical Procedure Index (GAPI) and analytical Eco-scale were used to prove that the proposed HPLC method is environmentally friendly.