Simultaneous determination of 40 novel psychoactive stimulants in urine by liquid chromatography–high resolution mass spectrometry and library matching

•Simultaneous novel psychoactive substances (NPS) analysis is highly challenging.•NPS constantly emerge with similar or completely new chemical structures.•The method quantifies 40 urinary NPS and 4 metabolites by LC–HRMS.•Screening and confirmation data acquired in full scan and data dependent MS2.•100 μL urine volume achieved 2.5–5 μg/L limit of quantification (LOQ).

The emergence of novel psychoactive substances is an ongoing challenge for analytical toxicologists. Different analogs are continuously introduced in the market to circumvent legislation and to enhance their pharmacological activity. Although detection of drugs in blood indicates recent exposure and link intoxication to the causative agent, urine is still the most preferred testing matrix in clinical and forensic settings. We developed a method for the simultaneous quantification of 8 piperazines, 4 designer amphetamines and 28 synthetic cathinones and 4 metabolites, in urine by liquid chromatography coupled to high-resolution mass spectrometry (LC–HRMS). Data were acquired in full scan and data dependent MS2 mode. Compounds were quantified by precursor ion exact mass, and confirmed by product ion spectra library matching, taking into account product ions’ exact mass and intensities. One-hundred μL urine was subjected to solid phase cation exchange extraction (SOLA SCX). The chromatographic reverse-phase separation was achieved with gradient mobile phase of 0.1% formic acid in water and in acetonitrile in 20 min. The assay was linear from 2.5 or 5 to 500 μg/L. Imprecision (n = 15) was <15.4%, and accuracy (n = 15) 84.2–118.5%. Extraction efficiency was 51.2–111.2%, process efficiency 57.7–104.9% and matrix effect ranged from −41.9% to 238.5% (CV < 23.3%, except MDBZP CV < 34%). Authentic urine specimens (n = 62) were analyzed with the method that provides a comprehensive confirmation for 40 new stimulant drugs with specificity and sensitivity.