LC-HRMS suspect screening for expanded detection of (new) psychoactive substances in oral fluid: a practical approach...

Liquid chromatography tandem mass spectrometry (LC-MS/MS) methods are routinely used in forensic toxicology to confirm illicit drugs in oral fluid (OF). However, they are inherently limited to predefined analytes and may fail to detect newly emerging psychoactive substances (NPS) or compounds not included in routine panels. This study evaluates the applicability of a liquid chromatography-high resolution mass spectrometry (LC-HRMS) suspect screening workflow for the reanalysis of oral fluid extracts previously processed using a routine targeted LC-MS/MS method, with focus on its implementation in forensic casework. OF samples (0.25 mL) were extracted using a solid-phase extraction procedure routinely employed for the confirmation of traditional illicit drugs and psychopharmaceuticals. The same extracts were reanalyzed by LC-HRMS operating in data-dependent acquisition mode. Data processing was performed using a Personal Compound Database Library (PCDL) containing 2376 entries (1693 with MS/MS spectra), generated from HighResNPS and expanded with in-house acquired spectra and retention times. Qualitative validation was performed for 105 available reference standards, with limits of detection ≤ 10 ng/mL for 64.8% of them. The applicability of the workflow was demonstrated through the analysis of 25 authentic OF samples from drivers (n = 15), chemsex users (n = 6) and emergency unit patients (n = 4). LC-HRMS screening confirmed compounds previously detected by targeted LC-MS/MS, while enabling the identification of additional psychoactive substances not included in routine panels. This strategy provides a complementary approach for forensic toxicology laboratories, expanding screening capabilities from existing sample extracts and supporting the improved monitoring of emerging NPS in routine forensic casework.