Fragmentation pathways of dihydro-metabolites of synthetic cathinones by high-resolution mass spectrometry on Orbitrap and quadrupole time-of-flight (qTOF) mass spectrometers

Cathinones represent one of the most prevalent classes of new psychoactive substances (NPS), largely due to the extensive variety of structural analogues. However, in forensic toxicology, their instability in post-mortem biological matrices such as blood and urine complicates their detection, making sample preservation at −20 °C essential prior to analysis. To overcome this limitation, dihydro (DH) metabolites—formed via reduction of the ketone group—have been proposed as reliable biomarkers of cathinone intake, owing to their enhanced stability. In this study, 28 DH-metabolites were synthesised through selective reduction of commercial cathinones. Their fragmentation behaviour was carefully studied using isotopically labelled analogues and analysed on different LC-HRMS platforms (Orbitrap and qTOF). A predictive fragmentation model was developed, allowing for the identification of anticipated MS 2 fragments and the characterization of specific diagnostic ions. These ions aid in the detection of DH-metabolites during untargeted LC-HRMS analyses. In line with previously described fragmentation pathways for synthetic cathinones, DH-metabolites can produce as well odd-electron ions from even-electron precursor ions during electrospray ionization (ESI). However, compared to their parent compounds, DH-metabolites exhibit some differences in fragmentation pathways. Understanding the fragmentation mechanisms of a compound family is essential when applying non-targeted analysis methods using LC-HRMS techniques. The development of a predictive fragmentation model can significantly improve the detection of novel, unstable emerging cathinones in post-mortem samples or in wastewater monitoring, simply by targeting their DH-metabolites. • Dihydro-metabolites of cathinones proposed as biomarkers of exposure. • Fragmentation patterns rationalised on Orbitrap and qTOF platforms. • Isotope-labelled synthesis used to confirm fragmentation pathways. • Odd-electron vs even-electron fragmentation. • Characteristic fragments proposed to identify DH-metabolite structural cores.