HPLC Sample Prep and Extraction SOP v1.3 for Fungi

A novel sample preparation and extraction technique for fungi uses microwave assisted extraction with 75% methanol and 25% water, plus ultrasonication and vortex mixing, to break down chitin in the cell wall and release bioactive compounds. Applied to magic mushroom fruit bodies, it extracts tryptamines (psilocybin and psilocin) for accurate chromatography measurements in two hours and forty-five minutes with minimal handling. The technique is superior to other methods in time and effectiveness, pulling all active compounds without degrading them. It can be adapted for other fungi like Cordyceps and Lion's mane. Sharing such methods reduces variability between testing labs and improves result consistency.

Study at a glance

Abstract

medicine, industry, and biotechnology. Fungi produce a wide range of bioactive compounds, such as alkaloids, antibiotics, antifungals, immunomodulators, anticancer agents, enzymes, and vitamins. However, these compounds are often locked inside the fungal cell wall, which is composed of chitin, a tough substance that is dif�icult to digest by humans1. Therefore, it is essential to have a good extraction technique that can break down the chitin and release the valuable compounds from the fungi, this is especially essential in the laboratory for accurate lab assays and potency determination during routine HPLC chromatography analysis. During licensure and/ or certi�ication any given lab will be required to take a pro�iciency test which gauges the lab’s pro�iciency at measuring a given matrices for accurate evaluation. They evaluate our abilities to run the gear and accurately measure the potency of what was extracted; however, at the time of this writing none existed for extraction of the fungal material itself, so this remains a variable between testing labs. It is important that we openly share our extraction techniques for evaluating fungi materials speci�ically for the clean extraction of active alkaloids for which potency can be measure via chromatography and/or spectrometry devices. In this way hopefully creating less variables between testing lab and more concise results. In this paper, we present a novel sample prep and extraction technique for fungi that uses speci�ic solvent composition in conjunction with M.A.E (microwave assisted extraction) in 75% methanol , 25% water which helps break the cell wall to release the compounds. Also used is an ultrasonication unit and vortex mixer. Our technique quickly releases all the available alkaloids for accurate chromatography measurements in just two hours, forty-�ive minutes with minimal handling. We demonstrate the effectiveness and ef�iciency of our technique by applying it to magic mushroom fruit bodies for the extraction of tryptamines namely psilocybin and its active derivative psilocin; however, this technique can be used for other species of fungi and compounds like Cordyceps/ cordycepin or Lions’ mane/ erinacines, etc.. We also compare our technique with other popular methods in terms of extraction techniques, digestion times and solvent compositions. Our results show that our technique is superior to the others in terms of time and effectiveness while pulling all the active compounds and not degrading them. Our extraction technique for fungi chromatography analysis offers a new and improved way to access the natural products of fungi and explore their potential for various biotechnological applications. We hope that our technique will inspire further research and innovation in the field of fungal extraction and natural product.