A sustainable AQbD-based UHPLC–MS/MS method for the determination of hallucinogenic phenethylamines in urine

A systematic Analytical Quality by Design (AQbD) approach was applied to the development and validation of a UHPLC–MS/MS method for the determination of hallucinogenic phenethylamine-derived novel psychoactive substances in urine. Owing to the continuous emergence of structurally related compounds on the illicit drug market, AQbD enables rapid and knowledge-based development of robust analytical methods. An Analytical Target Profile was defined to ensure adequate separation, sensitivity, robustness, and compliance with white analytical chemistry principles. Method development followed a structured AQbD workflow, including chromatographic scouting of five stationary phases and different mobile-phase compositions, followed by multivariate screening and response surface optimization of critical method parameters. Optimal performance was achieved on a biphenyl column using an initial mobile-phase composition of 20 % phase B and a column temperature of 40 °C. The method operable design region was defined for a flow rate of 0.375–0.435 mL min−1 and a formic acid concentration of 0.06–0.085 %. The method was fully validated, demonstrating excellent linearity (R2 ≥ 0.99), satisfactory precision and accuracy, and limits of detection of 0.1–1 ng mL−1. The White Analytical Chemistry evaluation yielded an overall score of 91.9, indicating balanced analytical performance and sustainability for routine forensic and clinical toxicological analyses.