Distinguishing 4- vs 5-Hydroxy- N , N -Dimethyltryptamine (Psilocin vs Bufotenine) Using Hydrogen–Deuterium Back-Exchange

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.

Study at a glance

Abstract

Distinguishing metabolite isomers often relies on comparing relative data, such as relative chromatographic retention times and ion mobility arrival time orders, or relative product ion abundances. These approaches necessitate the need for quality reference data and/or chemical standards. An ideal method for differentiating isomers would leverage one of the absolute physiochemical properties of the isomers, and would have no reliance on instrument vendor, chromatographic column chemistry, or external reference data. For example, the pKa of an aromatic hydroxy hydrogen changes according to ring position across isomers (e.g., 4- vs 5-hydroxyindole). Herein, we leverage the difference in pKa to resolve 4- and 5-hydroxy positional isomers of hydroxy-N,N-dimethyltryptamine (psilocin and bufotenine), the structural moiety of compounds with profound effects on the serotonergic system. We first use hydrogen-deuterium exchange (HDX) to rapidly exchange the indole amine hydrogen and gradually exchange the indole hydroxy hydrogen atoms to deuterium atoms. We then back-exchange the indole amine deuterium atom back to a hydrogen atom on the LC column and monitor the kinetic exchange rates of the retained aromatic hydroxy deuterium atom using high resolution mass spectrometry (HRMS). HDX kinetics allow for facile differentiation of the two isomers, with only 4-hydroxy-N,N-dimethyltryptamine exchanging at an appreciable amount within hours. These results could ultimately be used to characterize a variety of unknown structural isomers.