Research and Development for the Creation of Validated Protocols for the Forensic Detection and Quantification of Psilocybin and Psilocin in Complex Edible Matrices

Psilocybin, a phosphorylated tryptamine natural product that is present in over 200 species of Psilocybe mushrooms (aka “magic mushrooms”), and its hallucinogenic dephosphorylated form psilocin, are Schedule I drugs in the U.S. However, these drugs are being increasingly legalized or decriminalization at the state level, due in part to the results of medical research showing its potential efficacy as a treatment for mental health disorders. This has contributed to a spike in availability, production, and retail sales of food and drink products containing psilocybin mushrooms or their psychoactive small-molecule components, raising public health concerns. While crime laboratories have well-established protocols for the detection by GC- and LC-MS of psilocybin and psilocin, methods for the quantification of these psychedelic substances are very limited. Moreover, reports of forensic methods for the detection and quantification of psilocybin and psilocin contained within food and drink products are sparse. Efficient extraction protocols for maximum recoveries from magic mushrooms and various other complex matrices must be created in order to reliably quantify their content and ensure product safety. A major challenge forensic scientists face when extracting and analyzing psilocybin and psilocin is the instability of psilocybin when exposed to heat and/or light, conditions which lead to the dephosphorylation of psilocybin to psilocin. Proposed here is the development of an optimized protocol for the rapid detection and differentiation of psilocybin and psilocin in edible matrices by direct analysis in real time-high resolution mass spectrometry (DART-HRMS), with no sample pretreatment steps (Specific Aim I). Furthermore, a rapid, universal, and efficient extraction approach for psilocybin and psilocin, independent of the complexity of the matrix (Specific Aim II) will be developed. With the optimized extraction protocol, quantification of psilocybin and psilocin by several techniques including GC-MS, DART-HRMS, and LC-MS (Specific Aim III) will be developed. Finally, investigations into the stability of psilocybin and psilocin in food and drink products as a function of the temperature and pH of the environment (Specific Aim IV) will be conducted. The anticipated outcomes of this work include development of validated protocols for the extraction, detection, and quantification of psilocybin, psilocin and their related derivatives that are infused within food and drink matrices by DART-HRMS, GC-MS, and LC-MS. Semi-annual and final progress and financial reports as well as scholarly products (e.g. peer reviewed articles) will be produced. CA/NCF