A Sustainable Microextraction of Hallucinogenic New Psychoactive Substances for Clinical and Forensic Applications

The application of Green Analytical Chemistry (GAC) principles in method development aims to reduce waste and replace hazardous solvents with environmentally friendly alternatives. Natural Deep Eutectic Solvents (NADESs) have recently emerged as sustainable replacements for traditional organic solvents. In this study, hydrophobic NADESs were used in dispersive liquid–liquid microextraction (DLLME) to extract four synthetic hallucinogenic phenethylamines (2C-B, 25B-NBOMe, 25C-NBOMe, and 25I-NBOMe) in urine samples. Nine NADESs were formed using menthol and different organic acids, with menthol–decanoic acid (1:1 molar ratio) providing the best extraction efficiency. A fractional factorial design identified pH, vortex speed, and vortex time as key factors, which were then optimized using a Box–Behnken design. The statistical model showed strong validity and high predictive power, and the optimal conditions (pH 12, vortex time 20 s, vortex speed 30,000 rpm, centrifugation at 14,000 rpm for 3 min) resulted in the highest recoveries. Greenness and operational sustainability, evaluated using ComplexGAPI, AGREEprep, BAGI, and SPRS tools, revealed clear advantages over existing extraction approaches. Overall, the proposed method represents a sustainable, white-chemistry–driven microextraction strategy suitable for clinical and forensic toxicological applications.