• Development of DA colorimetric detection by bio-based NBP validated assay • DoE ensured efficient optimized condition for the DA assay with minimal experiments • Optimized NBP method achieved improved performances reducing solvent consumption • The NBP-based assay assured selectivity for DA over other clinically relevant adrenergic drugs . The drive towards sustainability in analytical chemistry has prompted the replacement of hazardous solvents with greener alternatives, in line with circular chemistry principles. We here report a rapid, low-cost, and sustainable colorimetric assay for dopamine (DA) detection in pharmaceutical formulations. Building on our previously developed dimethyl sulfoxide (DMSO)-based melanochrome (MN) assay, three bio-based solvents—Cyrene™, γ-valerolactone (GVL), and N-butylpyrrolidone (NBP)—were evaluated as DMSO substitutes. Screening indicated that DMSO and NBP supported effective MN formation, whereas GVL and Cyrene were unsuitable. NBP was selected for optimization using a Design of Experiments (DoE) strategy to assess pH, solvent fraction, and temperature effects. Temporal contributions and interactions of parameters were analyzed through dynamic DoE modeling, with curvature effects evaluated via triplicate center-point measurements. Model predictivity was verified at the center and at the predicted maximum absorbance region, achieving the highest signal at 30 min. The optimized assay (NBP:buffer 1:4 v/v, pH 10.0, 45 °C) exhibited excellent linearity (R² = 0.9998) across 11–80 μg/mL, a low detection limit (LOD 1.35 μg/mL), and quantification limit (LOQ 4.59 μg/mL). Compared to the reference DMSO system, the NBP assay showed improved sensitivity and reduced solvent usage, minimizing environmental impact. Selectivity was confirmed against adrenergic drugs (adrenaline, noradrenaline, ephedrine, etilefrine, dobutamine), none of which produced MN chromophore. This results method robustness in clinically relevant contexts. Overall, the proposed NBP-based assay integrates eco-friendly solvent selection with DoE-guided optimization, delivering sensitivity, selectivity, and practicality for pharmaceutical quality control. Beyond simple solvent substitution, this study presents a systematic green re-evaluation of a validated analytical protocol, effectively balancing sustainability, functionality, and analytical performance. .
Cuffaro et al. (Thu,) studied this question.