Simultaneous estimation of recently FDA approved co-formulated ophthalmic solution benoxinate and fluorescein: Application to aqueous humor

[Display omitted] •For the first time, spectrophotometric approach was developed to simultaneously quantify benoxinate and fluorescein in eye drop.•The developed spectrophotometric methods are: direct UV, dual wavelength, first derivative, second derivative, ratio difference, first derivative ratio, ratio subtraction, absorption factor, and isoabsorptive point.•Water was used as a diluting solvent.•Application in aqueous humor. Nine simple, cost-effective, and sensitive spectrophotometric methods were developed to simultaneously assay benoxinate HCl and fluorescein sodium in their co-formulated eye drops without barring common excipients. A direct UV spectrophotometric method (method I) was developed for their FLU assay at 481 nm over the concentration range of 0.6–10.0 μg mL−1. Meanwhile, BEN was assayed over the concentration range of 1.0–25.0 μg mL−1 by different UV based methods, namely, conventional dual-wavelength method (DW) (method II), first derivative1D spectrophotometry (method III), second derivative2D spectrophotometry (method IV), ratio spectra difference spectrophotometry (method V), the first derivative of ratio spectra (method VI), ratio subtraction method (method VII), isosbestic point method (ISP) (method VIII) and absorption factor method (method IX). The performance of the proposed methods was assessed relying on the correlation coefficients, relative standard deviations, and limits of detection and quantitation. The variance ratio F-test and Student t-test showed no significant differences between the obtained results of the developed methods and those of reference methods. The proposed methods were also applied to determine studied drugs in commercial eye drops and aqueous humor.