Quantitative Analysis of Psilocybin and Psilocin in Psilocybe cubensis Mushrooms: From Sample Preparation to LC-MS and UV Quantitation

Background: Psilocybin and psilocin, are naturally occurring psychoactive compounds found in magic mushrooms including P. cubensis. These mushrooms have garnered increased attention as psychoplastogens for the treatment of brain-related disorders. While many methodologies are based off standards and food grade mushroom matrices, we present quantitation based on actual psilocybin mushrooms for an authentic and meaningful analysis. Problem Statement: There is still much confusion on what is necessary to overcome challenges such as low sensitivity and sample degradation. There is a critical need for standardized, simple methodologies for reliable and efficient quantitation. Methods: We developed LC-MS and LC-UV methods for the accurate quantitation of psilocybin and psilocin. Sample preparation approaches including drying, milling, and extraction were explored to make homogenous samples with reproducible extractions for introduction into HPLC coupled to either MS or UV. The selectivity of MS as well as the use of multiple UV wavelengths to perform an Intelligent Peak Deconvolution Analysis (i-PDeA) reduced matrix interference. Results: Key results include a suitable sample preparation protocol as well as fully resolved analytes for quantitation. Furthermore, key preparation steps critical for achieving optimum sensitivity, accuracy, and precision, such as sample dilution to allow for mobile phase matching while maintaining sample concentration within limits of detection, will be discussed. Conclusions: Our HPLC-UV/MS methods represents a turn-key approach for both routine analysis and as a springboard for additional, in-depth interrogation. Future work should focus on standardizing methodologies for easier comparison across testing labs. Expanding the application of this method to analyze other psychedelic molecules may be of research interest.