Scientific and technical

Investigation into the temporal stability of aqueous standard solutions of psilocin and psilocybin using high performance liquid chromatography

Although it has been published during the last 15 years that hallucinogens derived from new psychoactive substances are increasingly abused, the old psychedelic agents are still used. Among these vegetal compounds, magic mushrooms are frequently mentioned. The major ingredient of the Psilocybe species is psilocybin, which is rapidly converted into psilocin in the human body. Therefore, psilocin represents the key target to document magic mushrooms abuse. The aim of the present work was to develop a specific method to identify psilocin in human hair by LC-MS/MS and review potential pitfalls. The method involves incubation of 10 mg of cut hair in 1 mL of acid methanol in presence of psilocin-d₁₀ for 2 h at 4 °C. The chromatographic separation was performed using a reverse phase column HSS C18 with a gradient elution of 8 min. Linearity was verified from 1 to 200 pg/mg (r² = 0.9994), after spiking blank hair with the corresponding amounts of psilocin. The limit of detection was estimated at 0.4 pg/mg. The precision was lower than 20 % and there was no interference with any extractable material present in hair. Psilocin was identified in the hair of a repetitive magic mushrooms consumer at 2.5 pg/mg (segment 0–1 cm), 4.4 pg/mg (segment 1–2 cm) and 5.4 pg/mg (segment 2–3 cm). Cold conditions during hair preparation and protection from the light were found mandatory to avoid psilocin degradation.

In nature, there are >200 species of fungi with hallucinogenic properties. These fungi are classified as Psilocybe, Gymnopilus, and Panaeolus which contain active principles with hallucinogenic properties such as ibotenic acid, psilocybin, psilocin, or baeocystin. In Chile, fungi seizures are mainly of mature specimens or spores. However, clandestine laboratories have been found that process fungus samples at the mycelium stage. In this transient stage of growth (mycelium), traditional taxonomic identification is not feasible, making it necessary to develop a new method of study.

Currently, DNA analysis is the only reliable method that can be used as an identification tool for the purposes of supporting evidence, due to the high variability of DNA between species. One way to identify the species of a distinctive DNA fragment is to study PCR products analyzed by real time PCR and sequencing. One of the most popular sequencing methods of forensic interest at the generic and intra-generic levels in plants is internal transcribed spacer (ITS). With real time PCR it is possible to distinguish PCR products by differential analysis of their melting temperature (Tm) curves.

This paper describes morphological, chemical, and genetic analysis of mycelia of psychedelic fungi collected from a clandestine laboratory. The fungus species were identified using scanning electron microscopy (SEM), mass spectrometry, HRM analysis, and ITS sequencing. The sporological studies showed a generally smooth surface and oval shape, with maximum length 10.1 μm and width 6.4 μm. The alkaloid Psilocyn was identified by mass spectrometry, while HRM analysis and ITS sequencing identified the species as Psilocybe cubensis. A genetic match was confirmed between the HRM curves obtained from the mycelia (evidence) and biological tissue extracted from the fruiting bodies. Mycelia recovered from the evidence and fruiting bodies (control) were genetically indistinguishable.

A two-step synthesis of psilocin glucuronide (PCG), the main metabolite of psilocin, with methyl 2,3,4-tri-O-isobutyryl-1-O-trichloroacetimidoyl-α-d-glucopyranuronate is reported. With the synthesized PCG, hydrolysis conditions in serum and urine were optimized. Escherichia coli proved to be a better enzyme source for β-glucuronidase than Helix pomatia. It was essential to add ascorbic acid to serum samples to protect psilocin during incubation.

Furthermore the stability of PCG and psilocin was compared as stability data are the basis for forensic interpretation of measurements. PCG showed a greater long-term stability after six months in deep frozen serum and urine samples than psilocin. The short-term stability of PCG for one week in whole blood at room temperature and in deep frozen samples was also better than that of psilocin. Therefore, PCG can be considered to be more stable than the labile psilocin and should always be included if psilocin is analyzed in samples.

Psilocybin, a psychoactive alkaloid contained in hallucinogenic mushrooms, is nowadays given a lot of attention in the scientific community as a research tool for modeling psychosis as well as due to its potential therapeutic effects. However, it is also a very popular and frequently abused natural hallucinogen. This review summarizes all the past and recent knowledge on psilocybin. It briefly deals with its history, discusses the pharmacokinetics and pharmacodynamics, and compares its action in humans and animals. It attempts to describe the mechanism of psychedelic effects and objectify its action using modern imaging and psychometric methods. Finally, it describes its therapeutic and abuse potential.

Many N,N-dialkylated tryptamine derivatives can induce altered states of consciousness in humans. The term “hallucinogen” is often used when describing the effect on the human mind, and the extent of psychoactivity depends on the nature of substituents attached to the ethylamine side chain and the indole ring. Naturally-occurring tryptamines (e.g., N,N-dimethyltryptamine and psilocybin) have increasingly been investigated in human clinical studies, which increased interest within a number of scientific communities and the public. Many of these derivatives are controlled substances and an increasing number of previously unreported and structurally modified N,N-dialkylated “designer” tryptamines with unknown bioactive profiles have become available.

This review provides an overview of analytical methodologies published in recent years on detection and characterization of 40 N,N-dialkylated derivatives. The majority of literature available utilized reversed-phase high-performance liquid chromatography, gas chromatography and/or capillary electrophoresis. Derivatization was not normally required for sufficient separation and detection.

Bioanalytical applications and characterization of natural products have not been included due to space limitations. The majority of analytical data described in the literature indicated the dominance of N,N-dialkylated derivatives carrying a methoxy group or hydrogen at the 5-position of the indole ring. A consistent body of data has been produced and should set the scene for the detection of derivatives in order to inform healthcare providers, clinicians, forensic scientists and potential consumers.