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

Michael W. Christopher, Boone M. Prentice, Timothy J. Garrett

Journal of the American Society for Mass Spectrometry December 30, 2025 DOI: 10.1021/jasms.5c00421 (opens in new tab)

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AI-extracted from the abstract
Characteristics Experimental study Peer reviewed
Topics 5-MeO-DMT DMT Psilocybin
Keywords Hydrogen–deuterium exchange Indole test Amine gas treating Moiety Structural isomer Hydrogen atom Tandem mass spectrometry Ion Analytical chemistry journal Stereochemistry Metabolite
Key findings Hydrogen-deuterium exchange kinetics, monitored by high-resolution mass spectrometry, differentiate 4- and 5-hydroxy-N,N-dimethyltryptamine isomers based on pKa differences, with only the 4-hydroxy isomer showing appreciable exchange within hours.

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.

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Unmasking of a neonatal somatovesical reflex in adult cats by the serotonin autoreceptor agonist 5-methoxy-N,N-dimethyltryptamine. Adult cats 1990 Experimental study n = 10
Studies on the Mechanism of Action of Monoamine Oxidase: Metabolism of N,N-Dimethyltryptamine and N,N-Dimethyltryptamine-N-Oxide 1962 Experimental study

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