Emerging arylcyclohexylamine new psychoactive substances: Comprehensive characterization and differentiation by NMR, GC-MS, LC-QTOF-MS and LC-MS/MS.

Fluorinated ketamine analogues and structurally related arylcyclohexylamines have increasingly emerged on the illicit drug market as new psychoactive substances (NPS), posing significant challenges for forensic identification due to their high structural similarity and the presence of numerous positional isomers and homologous derivatives. Reliable analytical differentiation of these compounds is therefore essential for forensic drug identification and regulatory monitoring. In this study, nine arylcyclohexylamine analogues were systematically characterized and differentiated using nuclear magnetic resonance (NMR) spectroscopy, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The investigated compounds included 2-fluoro-2-oxo-PCM (2-fluoro-deschloroketamine) and its positional isomers (3-fluoro-2-oxo-PCM and 4-fluoro-2-oxo-PCM), N-alkyl substituted derivatives (2-fluoro-2-oxo-PCE, 2-fluoro-2-oxo-PCPr, and 2-fluoro-2-oxo-PCiPr), the non-fluorinated analogue 2-oxo-PCPr, the phencyclidine-based analogue 2-oxo-PCP, and the methoxy-substituted derivative 3-MeO-2-oxo-PCP. Comprehensive NMR analysis enabled unambiguous structural elucidation of all compounds and revealed diagnostic spectral differences associated with fluorine substitution positions and N-alkyl chain variations. GC-MS analysis under electron ionization conditions produced characteristic fragmentation patterns that allowed differentiation of positional isomers and homologous analogues based on diagnostic ions and relative fragment intensities. High-resolution LC-QTOF-MS further supported compound identification through accurate mass measurements and product ion spectra, while LC-MS/MS provided reproducible fragmentation patterns suitable for routine forensic screening. The combined analytical approach demonstrated here provides a robust strategy for distinguishing structurally similar arylcyclohexylamine derivatives that are difficult to differentiate using a single analytical method. The analytical data generated in this study provide useful reference information for forensic laboratories dealing with emerging ketamine- and phencyclidine-related NPS.