Investigating fluorogenic labelling of amine coupled with first-order derivative spectrofluorimetry as a versatile analytical methodology: Application to estimation of benoxinate in its eye drops

In this research, fluorogenic labelling followed by applying first-order derivative spectrofluorimetry for the developed fluorophore is discussed as an alternative, sensitive and selective analytical approach. Benoxinate, containing a primary amine and fluorescamine reagent were selected for the study. Then the proposed methodology relies on the reaction between the primary amine in benoxinate with fluorescamine that selectively reacts with primary amines to develop highly fluorescent products. The fluorescamine-benoxinate developed fluorophore is identified by its sharp first-order derivative peak at 465 nm following excitation at 386 nm in borate buffer, pH 8. The optimum reaction conditions were ascertained. Following ICH validation guidelines, the first order derivative of the relative fluorescence intensity for the developed fluorophore was linearly related to benoxinate concentration and ranged from 20.0 to 200.0 ng/mL with a detection limit of 3.36 ng/mL and a quantitation limit of 10.19 ng/mL, moreover, satisfying accuracy and precision values were obtained upon statistical analysis of results. The offered analytical method was successfully applied to quantify benoxinate in raw material and Benox® eye drops as a direct application to a commercial formulation.