First enantioselective determination of N-ethylpentedrone and its major phase-1 metabolites using supercritical fluid chromatography coupled with tandem mass spectrometry and its application to human oral fluid and urine samples.

The enantioselective bioanalysis of synthetic cathinones and their metabolites remains analytically challenging. Due to the basic nature of these compounds, mobile phases with elevated pH are preferable for their analysis in high-performance liquid chromatography (HPLC). On the other hand, as it has been recently reported, cathinone derivatives are chemically and stereochemically instable, especially at high pH. As the alternative to HPLC, a supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) approach was developed in this study, to enable the chiral separation and detection of N-ethylpentedrone (NEP) and its major phase-1 metabolites. Careful selection of the chiral column and mobile phase compositions provided efficient retention and stereoselective resolution of both the parent compound and its polar metabolites. The applicability of the method was confirmed through the analysis of authentic human oral fluid (OF) and urine samples. The results of this study underscore the expanded analytical potential of SFC-MS/MS for chiral metabolite profiling and demonstrate its value as a complementary analytical tool to HPLC in forensic toxicology, particularly for new psychoactive substances.