Development of HPTLC method for simultaneous estimation of Gatifloxacin and Flurbiprofen Sodium using 32 factorial design.

INTRODUCTION: This study aimed to develop and validate an Analytical Quality by Design (AQbD)-based HPTLC method for simultaneous estimation of gatifloxacin and flurbiprofen sodium in ophthalmic formulations. METHODS: Separation was achieved on silica gel 60 F254 precoated HPTLC plates using n-propanol: ammonia (8:2, v/v) as the mobile phase, with densitometric detection at 254 nm. A 32 full factorial design was employed to evaluate the effects of mobile phase composition and chamber saturation time on the Rf values of both drugs. The optimized method was validated according to ICH Q2(R1) guidelines for specificity, linearity, accuracy, precision, robustness, LOD, and LOQ. Sustainability and practicality were assessed using AGREE, GAPI, Analytical Eco-Scale, ComplexGAPI, BAGI, RAPI, and RGB12 multi-colour assessment. RESULTS: The method produced sharp, well-resolved peaks with Rf values of 0.32 ± 0.02 and 0.65 ± 0.02 for gatifloxacin and flurbiprofen sodium, respectively. Excellent linearity was observed over 600-2100 ng/band for gatifloxacin and 60-210 ng/band for flurbiprofen sodium, with correlation coefficients >0.999. Recoveries ranged from 98.30% to 99.96%, with %RSD <2%, confirming accuracy and precision. The method was successfully applied to FLUBIGAT® eye drops without interference from excipients. RGB12 multi-colour assessment yielded a Whiteness score of 80.5%. CONCLUSION: A simple, accurate, robust, and AQbD-based HPTLC method was successfully developed and validated for simultaneous determination of gatifloxacin and flurbiprofen sodium in ophthalmic formulations.