Enantioselective analysis of synthetic cathinones and amphetamine-type stimulants in biological fluids via LC-MS/MS.

Synthetic cathinones are the second largest group of new psychoactive substances (NPS), with 123 new compounds reported in Europe between 2014 and 2023. Many possess a chiral center and are sold as racemic mixtures. Since enantiomers and other structural isomers may differ in pharmacological and pharmacokinetic properties, developing analytical methods capable of their separation and quantitative analysis is essential, particularly in forensic and clinical toxicology, where accurate determination of therapeutic and toxic concentrations is critical. This study developed a liquid chromatography-tandem mass spectrometry method for separating cathinone-type NPS, including structural isomers and enantiomers. Chromatographic separation was performed at 10 °C using a Lux 3 μm chiral column (Cellulose-2 phase). The method enables both qualitative and quantitative analysis, improving differentiation of structurally similar compounds and supporting toxicological interpretation. The method achieved successful chromatographic separation of the target cathinone enantiomers and numerous structural isomers within a 15-min runtime after a simple liquid-liquid extraction step. Validation demonstrated excellent analytical performance: calibration models were linear (R2 ≥ 0.99), accuracy and precision exceeded predefined acceptance criteria across the tested range, limits of detection and quantification at 1 ng/mL produced signal-to-noise values well above thresholds, and no significant carry-over or cross-interference was observed. Matrix effects were present in the range of -72% to +57%, and stability testing indicated superior preservation at higher concentrations and under frozen or cooled autosampler conditions. Therefore, the developed method constitutes a significant advancement in the analysis of synthetic cathinones, providing precise and reliable tools for forensic and clinical applications.