Sustainable spectrophotometric fingerprinting resolution technique for concurrent quantification of ofloxacin and bromfenac in rabbit aqueous humor with ocular pharmacokinetic application.

The simultaneous quantification of multiple drugs in biological matrices is crucial for therapeutic drug monitoring and pharmacokinetic studies. A sustainable spectrophotometric fingerprinting resolution technique was developed for the simultaneous determination of ofloxacin (OFL) and bromfenac (BMF) in rabbit aqueous humor. The technique manipulates spectral data to extract the original zero-order absorption spectrum (D 0) of each analyte from overlapping signals in the mixture. This approach relies on the ratio subtraction method (RSM) combined with one or more additional methods, including extended ratio subtraction (ERS), unified constant subtraction (UCS), constant multiplication (CM), or spectrum subtraction (SS). Validation per ICH-Q2R1 showed a linearity range of 1-16 µg mL-1 for both drugs, with LOD/LOQ values between 0.16 and 0.63 µg mL-1. Following a simple protein-precipitation step, the technique was successfully applied to spiked rabbit aqueous humor samples, with recoveries between 97.8% and 102.1%. Matrix effect assessment confirmed negligible interference, with ME% values of -1.12% (OFL) and -0.98% (BMF). The validated technique was then used in an ocular pharmacokinetic study in rabbits following a single topical instillation of a combined OFL-BMF ophthalmic solution. The concentration-time profiles of both drugs in aqueous humor were effectively characterized, and key pharmacokinetic parameters were determined. Sustainability assessment indicated excellent trichromatic scores, confirming their suitability for ocular matrix analysis. This technique offers a sustainable platform for analyzing OFL and BMF in biological fluids, making it highly valuable for ocular bioavailability and pharmacokinetic studies.