The Determination of Psilocin and Psilocybin in Hallucinogenic Mushrooms by HPLC Utilizing a Dual Reagent Acidic Potassium Permanganate and Tris(2,2′‐bipyridyl)ruthenium(II) Chemiluminescence Detection System

A method using high-performance liquid chromatography with chemiluminescence detection was developed to measure psilocin and psilocybin in hallucinogenic mushrooms. Simple methanolic extraction proved most effective among tested methods. Separation was achieved on a C12 column with a methanol-ammonium formate mobile phase in five minutes. A dual chemiluminescence system using acidic potassium permanganate and tris(2,2′-bipyridyl)ruthenium(II) provided better detectability than UV absorption at 269 nm, with detection limits of 1.2 × 10⁻⁸ mol/L for psilocin and 3.5 × 10⁻⁹ mol/L for psilocybin. The procedure was applied to three Australian mushroom species.

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Abstract

ABSTRACT: This paper describes a procedure for the determination of psilocin and psilocybin in mushroom extracts using high‐performance liquid chromatography with postcolumn chemiluminescence detection. A number of extraction methods for psilocin and psilocybin in hallucinogenic mushrooms were investigated, with a simple methanolic extraction being found to be most effective. Psilocin and psilocybin were extracted from a variety of hallucinogenic mushrooms using methanol. The analytes were separated on a C 12 column using a (95:5% v/v) methanol:10 mM ammonium formate, pH 3.5 mobile phase with a run time of 5 min. Detection was realized through a dual reagent chemiluminescence detection system of acidic potassium permanganate and tris(2,2′‐bipyridyl)ruthenium(II). The chemiluminescence detection system gave improved detectability when compared with UV absorption at 269 nm, with detection limits of 1.2 × 10 −8 and 3.5 × 10 −9 mol/L being obtained for psilocin and psilocybin, respectively. The procedure was applied to the determination of psilocin and psilocybin in three Australian species of hallucinogenic mushroom.