The first validated HPLC method with UV detection for concurrent assay of lidocaine and fluorescein in their co-formulated ophthalmic solution

A simple, fast, and eco-friendly high-performance liquid chromatography (RP-HPLC) method has been developed for the concurrent quantification of lidocaine and fluorescein, employing ultraviolet detection at a wavelength of 220 nm. The chromatographic separation was applied under isocratic conditions on an Inertsil© C18 column (250 mm × 4.6 mm, 3.5 μm particle size), utilizing a mobile phase consisting of acetonitrile and water containing 0.3% triethylamine (adjusted to pH 3.5) in a volumetric ratio of 75:25%v/v. The analysis was performed at a constant flow rate of 1.0 mL/min. Method validation was conducted in accordance with the guidelines of International Council for Harmonisation (ICH), confirming that the procedure is accurate, precise, robust, and exhibits linearity within the concentration ranges of 20–280 µg mL− 1 for lidocaine and 1–20 µg mL− 1 for fluorescein. The detection limits were determined to be 5.27 µg mL− 1 for lidocaine and 0.29 µg mL− 1 for fluorescein, while the quantitation limits were 15.97 µg mL− 1 and 0.88 µg mL− 1, respectively. This analytical method was effectively applied to quantify both compounds in a combined ophthalmic formulation, yielding mean recovery values of 100.80 ± 0.67% for lidocaine and 100.13 ± 0.99% for fluorescein, thereby demonstrating its suitability for routine quality control purposes. The environmental impact of the proposed method was evaluated using Analytical Eco-Scale, GAPI, and AGREE tools, yielding an Eco-Scale score 78 and an AGREE score of 0.63, indicating acceptable greenness based on quantitative assessment.