Development and application of a simplified, quantitative method for 30+ New Psychoactive Substances in hair

The rapid emergence of novel psychoactive substances (NPS) poses a significant challenge for forensic toxicology and public health. While blood and urine enable rapid detection for early warning systems, long-term monitoring is essential for retrospective exposure assessment and temporal trend analysis. In this context, hair represents a valuable biological matrix; however, multi-class NPS determination remains difficult due to their chemical diversity and typically low concentrations. In this work, we targeted 12 synthetic opioids, 8 synthetic cathinones, 3 dissociatives, 3 designer benzodiazepines, 3 hallucinogens, and 2 synthetic cannabinoids. Sample preparation included a three-step wash procedure (water, methanol, and ethyl acetate), followed by simultaneous pulverization and methanolic extraction of 20 mg of hair, using an Omni Ruptor bead-mill homogenizer (10 cycles, 40 s at 4.5 m/s with dwell periods; total time ∼43 min). After centrifugation (5000 rpm, 10 min), extracts were evaporated under nitrogen stream at 40°C (20 psi) and reconstituted in 100 µl of 90:10 (v/v) water/acetonitrile, both containing 0.1% formic acid, followed by filtration. Chromatographic separation was achieved by liquid chromatography on a Kinetex C18 column (100 × 2.1 mm, 1.7 µm) using gradient elution with a total runtime of 10 min. Identification and quantitation were performed by tandem mass spectrometry in positive electrospray ionization using multiple reaction monitoring with quantifier and qualifier transitions. The method was validated according to ANSI/ASB Standard 036. Linearity was demonstrated from 5 to 500 pg/mg. Intra- and inter-day precision were <20% and bias within ±20% for most analytes, with no significant carryover or interferences observed. Matrix effects were observed but consistent across analytes, allowing reliable quantitation. Application to authentic hair samples from electronic music festival attendees in Europe and Brazil confirmed the presence of emerging NPS. This integrated pulverization-extraction approach enables rapid, robust multi-class NPS detection in hair for biomonitoring and forensic applications.