Direct Quantitation of Psilocybin and Psilocin by 1H and 31P qNMR in Wild Psilocybe from Greece

Abstract The genus Psilocybe (Basidiomycota) comprises over 300 mushroom-forming species, many of which produce the secondary metabolite psilocybin, a prodrug of its dephosphorylated active metabolite psilocin. Psilocybin has recently attracted significant interest, with numerous active, recruiting, and completed clinical trials registered on ClinicalTrials.gov, primarily investigating its therapeutic potential in central nervous system disorders. Nonetheless, psilocybin remains a controlled substance in many jurisdictions, posing regulatory and legal challenges, not only for clinical research but also for forensic analysis, standardization, and quality control, since it is required as a reference material in chromatographic analyses. To this end, qNMR-based methods for rapid quantitation of psilocybin and psilocin were developed using wild-origin Psilocybe fruitbodies, preserved in the Dried Specimen Collection of the Mycetotheca ATHUM (NKUA). A dephosphorylation step was employed to convert psilocybin to psilocin, followed by GC-MS analysis, confirming its presence in mycelium of the isolated strains. Both native and dephosphorylated extracts were analyzed by 1H and 31P qNMR in D2O using calcium formate and dimethyl methylphosphonate as internal standards, respectively. To the best of our knowledge, this represents the first officially documented occurrence of a psychoactive Psilocybe species from Greece in the peer-reviewed scientific literature as well as the application of reference-standard-free qNMR methods for the quantitation of a psilocybin- and psilocin-containing fungus.