Application of quality-by-design for adopting an environmentally green fluorogenic determination of benoxinate hydrochloride in eye drops and artificial aqueous humour

Design and strategy for assay development

The proposed reaction mechanism shown in Scheme 1 illustrates how BEN-HCl reacts with the reagent via its primary aliphatic amino group, stimulating the reagent’s fluorescence^{35,36,37,38,39}. The obtained fluorophore emits light at a specific wavelength of 483 nm after its excitation at 393 nm (Fig. 2).

Excitation/Emission spectra of the reaction product of BEN-HCl (0.4 µg/mL), 0.1 M borate buffer (pH = 8.2, 1.5 mL), and fluorescamine (0.04%, w/v…