HPLC Analysis of Hallucinogenic Mushroom Alkaloids (Psilocin and Psilocybin) Applying Hydrophilic Interaction Chromatography (HILIC)

Abstract

Hallucinogenic mushrooms containing psilocin and psilocybin psychoactive compounds are permanent offers on the black market palette. Reliable quantifications of psilocin and psilocybin are especially important task of forensic analysis because their results have significant effect on the hardness of judgement to be punished by the court. For quantification of psilocybin mainly HPLC procedures are applied because the compound is thermally labile and converts into psilocin by dephosphorylation at gas chromatographic conditions. The majority of HPLC procedures available in the scientific literature is based on reversed phase separation which is not advantageous particularly for the psilocybin because of its insufficient retention caused by the high polarity of the compound. The elution at the unretained section may even hinder the mass spectrometric detection of psilocybin because of the quenching of formed ions by other unretained co-eluting matrix components. To overcome drawbacks outlined previously the application of hydrophilic interaction chromatography (HILIC) is promising which allows analysis of polar compounds with appropriate retention and allowing mass spectrometric detection. The aim of the present study was to develop a procedure for quantification of psilocin and psilocybin in hallucinogen mushrooms applying HPLC separation and subsequent UV-photometric and mass spectrometric detection. For this reason, an isocratic hydrophilic interaction chromatographic (HILIC) phase system consisting of a zwitterionic stationary phase and a mixture of acetonitrile and formate buffer as mobile phase was applied. The developed system assures sufficient retention for both psilocin and psilocybin, baseline separation of the compounds from each other and from matrix components, too. Unique feature of the applied system that psilocybin elutes with a retention factor of approximately two times greater than that of psilocin which is a scientific novelty. The applicability of the proposed procedure is demonstrated via results obtained by analysis of mushrooms seized by police.