Analytical quality-by-design-assisted RP-HPLC method for simultaneous determination of cinnamaldehyde and chrysin in a lipid-based delivery system and its greenness assessment.

An analytical quality-by-design (AQbD)-assisted reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous determination of cinnamaldehyde (CN) and chrysin (CR) in a lipid-based delivery system. The CN : CR ratio was initially selected based on glucose utilisation studies in insulin-resistant HepG2 cells and Chou-Talalay combination index analysis, which identified a 1 : 1 ratio as most favourable. Chromatographic conditions were systematically optimised using a central composite design (CCD), with wavelength, aqueous-phase composition, pH, flow rate, and column temperature as critical method parameters. The optimised method employed methanol:acetonitrile : 0.1% orthophosphoric acid (40 : 40 : 20, v/v/v; pH 3.0; flow rate 0.8 mL min-1; column temperature of 23 °C) and achieved baseline separation of CN and CR, with retention times of 5.602 and 7.683 min, respectively, and a resolution of 5.363. The method demonstrated satisfactory linearity, accuracy, precision, and sensitivity over the investigated concentration range and was successfully applied to quantify CN and CR in the optimised self-nanoemulsifying drug delivery system (SNEDDS), as well as in dissolution and in vitro lipolysis studies. The optimised SNEDDS exhibited a droplet size of 57.49 nm, a zeta potential of -21.6 mV, 97.1% transmittance, and an emulsification time of 4.26 s. At 240 min, the SNEDDS showed 61.77% CN and 90.91% CR release, compared with 37.42% and 68.71%, respectively, for the pure drugs. The environmental profile of the developed method was evaluated using AGREE and ComplexMoGAPI, yielding scores of 0.65 and 81. The developed AQbD-assisted RP-HPLC method provides a reliable analytical approach for the simultaneous quantification of CN and CR in a complex lipid-based formulation and for evaluating its performance.