Development of N,P-doped carbon quantum dots as a green fluorescent probe for fexofenadine determination: mechanistic studies, Box–Behnken optimization, and pharmacokinetic application†

A sensitive and selective fluorescence quenching method based on nitrogen and phosphorus co-doped carbon quantum dots (N,P CQDs) was developed for the quantitative determination of the antihistamine drug fexofenadine. The optical and morphological properties of the N,P CQDs were extensively characterized using dynamic light scattering, transmission electron microscopy, UV-vis absorption, and fluorescence spectroscopy. A blue emitting N,P CQDs with excitation and emission maxima at 324 and 425 nm, respectively, exhibited strong fluorescence quenching upon interaction with fexofenadine. The sensing mechanism was investigated through Stern–Volmer analysis and thermodynamic studies, revealing a static quenching process. Additionally, the experimental parameters affecting the quenching efficiency, such as pH, N,P CQDs volume, and incubation time, were optimized using a Box–Behnken design to achieve maximum sensitivity. A significant quadratic model was developed to maximize the fluorescence quenching efficiency of these N,P CQDs upon interaction with fexofenadine which was further employed in the optimization process. The proposed method showed excellent linearity (r² = 0.9998) over the dynamic range of 0.02–1.5 μg mL⁻¹, with a limit of detection of 0.006 μg mL⁻¹. The analytical performance of the method was validated according to ICH M10 guidelines, demonstrating high accuracy, precision, and selectivity. The applicability of the method was demonstrated by determining fexofenadine in pharmaceutical formulations with no significant difference from the reported HPLC-UV method. Furthermore, the method was successfully applied for pharmacokinetic profiling in rabbits following oral administration (10 mg kg⁻¹), revealing characteristic parameters including C_max of 344 ± 84 ng mL⁻¹ at 3.6 h, elimination half-life of 12.5 h, and AUC_0→∞ of 6495 ng·h mL⁻¹, which aligned well with previously reported pharmacokinetic behavior. The greenness and practicality of the proposed method were evaluated using the AGREE, MOGAPI and BAGI tools, indicating its superior environmental sustainability and acceptable practical applicability compared to conventional HPLC-UV and UPLC-MS/MS techniques and presenting its suitability for routine pharmaceutical and bioanalytical applications.