A comprehensive HPLC-HRMS/MS targeted screening method to detect 90 new psychoactive substances in oral fluid samples

The continuous emergence of New Psychoactive Substances (NPS) poses a significant challenge to public health and forensic toxicology due to their unpredictable pharmacology and rapid turnover on the illicit market. This study describes the development and validation of a high-resolution screening method for the simultaneous detection of 90 NPS in oral fluid (OF), a matrix of choice for non-invasive sampling and roadside testing. The analytical workflow utilizes a “dilute-and-shoot” approach (1:2 v/v dilution) followed by ultra-high-performance liquid chromatography coupled with a quadrupole-Orbitrap hybrid mass spectrometer (UHPLC-HRMS/MS). Chromatographic separation was achieved in 11 min using a biphenyl column and a gradient elution. The method was validated according to ANSI/ASB Standard 036 guidelines, covering 90 substances including synthetic cannabinoids (e.g., HHC, MDMB-4en-PINACA), synthetic cathinones, and high-risk synthetic opioids such as nitazenes and fentanyl analogues. Results showed high sensitivity, with limits of identification (LOI) reaching 1 ng/mL for 44.4% of the analytes and 5 ng/mL for 37.8%, while the remaining compounds showed higher LOIs ranging from 10 to 100 ng/mL. No significant matrix interference or carryover was observed. The method was successfully applied to real samples from external quality control programs and forensic cases. This robust and versatile screening tool is suitable for clinical and forensic applications, supporting the monitoring of emerging NPS trends.