Analytical quality-by-design-guided green RP-HPLC method to quantify cyclosporin A and luteolin simultaneously in a combination of two drug solutions and two drug-loaded next-generation emulsions or nanocapsules.

The US FDA-approved oral finasteride and topical minoxidil for alopecia have adverse effects and fewer therapeutic outcomes, highlighting the need for alternative topical delivery approaches. This study aimed to develop and validate a robust and sustainable reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous quantification of cyclosporin A (CsA) and luteolin (Lu) in combined drug solutions and two compositionally distinct nanoformulations, namely nanosized emulsions and nanocapsules. The novelty of the study lies in integrating AQbD-assisted simultaneous CsA and Lu quantification with its application to two compositionally distinct nanoformulations, and a comprehensive assessment of analytical greenness and blueness. The AQbD approach was employed to systematically optimise chromatographic conditions. The optimised method employed a Phenomenex Luna C18 column (250 × 4 mm), acetonitrile-0.1% formic acid in water (pH 2.9), 206 nm wavelength, a flow rate of 1.2 ml/min, and a column temperature of 59 °C. CsA and Lu exhibited retention times of 9.14 and 6.04 min, respectively. The method demonstrated excellent linearity (R2 = 0.9994 and 0.9996 for CsA and Lu, respectively), precision (%RSD <2%), and robustness, and was validated in accordance with ICH Q2(R2). Drug content and entrapment efficiency ranged from 69.80 to 86.17% and 57.66 to72.38%, respectively. Regardless of the two compositionally distinct nanoformulations, the Lu release was higher than the CsA release (57.42 ± 5.98% vs 12.24 ± 1.40%). Analytical sustainability was supported by an Eco-Scale score of 84, AGREE score of 0.63, GAPI assessment, and BAGI score of 75.