Distinguishing 4- vs 5-Hydroxy- N , N -Dimethyltryptamine (Psilocin vs Bufotenine) Using Hydrogen–Deuterium Back-Exchange
Journal of the American Society for Mass Spectrometry December 30, 2025 Michael W. Christopher, Boone M. Prentice, Timothy J. Garrett
Distinguishing between structural isomers of metabolites typically requires reference standards or comparative data like retention times. A new method uses hydrogen-deuterium exchange (HDX) to differentiate 4- and 5-hydroxy positional isomers of hydroxy-N,N-dimethyltryptamine (psilocin and bufotenine) based on their absolute pKa differences. The indole amine hydrogen exchanges quickly, while the aromatic hydroxy hydrogen exchanges at a rate dependent on ring position. After back-exchange on the LC column, only the 4-hydroxy isomer shows appreciable deuterium retention within hours, enabling clear differentiation via high-resolution mass spectrometry. This approach could characterize unknown structural isomers without relying on external standards or instrument-specific data.