Analytical Challenges in the Separation and Identification of Ten Substituted Cathinone Isomers (C12H17NO) Using EI-GC-MS and ESI-LC-MS/MS

Synthetic cathinones are among the most frequently encountered classes of new psychoactive substances, and many occur as structural isomers sharing identical molecular formulas and highly similar mass-spectral features. Among them, substituted cathinones with the molecular formula C12H17NO (MW 191 Da) present particular analytical challenges because of their similar chromatographic behavior and overlapping ionization patterns. This study evaluated a combined EI-GC-MS and ESI-LC-MS/MS workflow, incorporating derivatization with trifluoroacetic anhydride (TFAA) and acetic anhydride (AA), for the differentiation of ten MW 191 Da isomers. TFAA-derivatized GC-MS enabled preliminary classification of the isomers, although several EMC and MEC analogs remained only partially resolved. AA derivatization improved the separation of unresolved isomers under slower oven temperature conditions, demonstrating the value of alternative acylation for enhancing chromatographic discrimination. LC-MS/MS provided complementary confirmation for several analytes, but some isomers remained difficult to distinguish because of shared product ions and peak fusion in mixed-standard analysis. Overall, this study establishes a practical analytical workflow for distinguishing MW 191 Da substituted cathinone isomers and highlights both the strengths and limitations of combining derivatization-based GC-MS with LC-MS/MS confirmation in routine forensic or clinical laboratories.