Psilocybe mushrooms: extraction and purification of psychedelic tryptamines

Abstract

"Magic mushrooms," belonging to the genus Psilocybe, contain the tryptamines psilocybin and psilocin, compounds with recognised therapeutic potential for mental health. Psilocybin is generally more abundant, with concentrations in dried mushrooms ranging from 0.5% to 1.5%. Their use for religious, recreational, and therapeutic purposes, along with their increased popularity and potential harm, has led to the development of specific regulatory frameworks, resulting in their increasing importance in the field of forensic sciences. The main goal of this work was to enhance the understanding of extraction and purification techniques applied to tryptamines present in Psilocybe cubensis mushrooms, aiming to guide future scale-up strategies by the pharmaceutical company Avextra. An analytical method was developed and validated using high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection, optimising parameters such as mobile phase, temperature, and detection wavelength. Validation according to ICH guidelines demonstrated excellent linearity for psilocybin and psilocin (R² > 0.999), with good precision (RSD < 3.4%) and accuracy (Bias < 9.6%). The extraction method was optimised by taking into account the known stability issues of tryptamines, specifically the presence of water, temperature, and exposure to light. The final analytical protocol involved pulverisation of mushrooms in a mortar and pestle on ice, followed by two 15-minute extractions using methanol containing 10% water (5 mL + 5 mL for 50.0 mg of mushroom) through dynamic maceration by stirring. Purification strategies included impurity precipitation with polar solvents and flash column chromatography, achieving psilocybin enrichment of the extract from 3% to 51% using isocratic 100% water elution with a C18 (Aq) column. Future studies should focus on further optimisation of purification strategies to achieve extracts with a higher percentage of psilocybin, such as the exploration of different chromatography stationary phases followed by recrystallisation. Furthermore, the developed and validated method for the extraction and quantification of psilocybin from mushrooms can be helpful for screening illicit mushroom samples, thereby contributing to the field of forensic sciences.