Determination of psychoactive alkaloids in Psilocybe subaeruginosa grown in Victoria by HPLC-PDA
Psilocin, psilocybin and the associated tryptamines are psychoactive alkaloids which are naturally synthesised by Psilocybe (Fr.) P.Kumm. fungi, commonly referred to as magic mushrooms. These psychoactive fungi were and may still be used in traditional healing rituals, particularly by certain ethnic groups for mental healing, influencing modern psychedelic studies. Since these substances are intermittently determined in analytical laboratories, validated methods for fast, accurate and reliable analysis are in demand.
Therapeutic potentials of these psychoactive compounds has encouraged academic and commercial activities to further explore the chemistry of these molecules as well as finding advanced methods for consistent production.
In this study, a method for clinical potency determination of psychoactive alkaloids in hallucinogenic mushroom, Psilocybe subaeruginosa, was developed using high-performance liquid chromatography coupled with a photodiode array detector (HPLC-PDA). Mushroom samples were obtained from parks around Melbourne, grown from different natural substrates. An extraction method was developed for maximum recovery and no significant matrix effects was observed.
The sample-to-solvent ratio was considered to be 10 mg to 1 mL. The major stages of the extraction process include vortex, ultrasonic bath, centrifuge and filtration. Different drying methods were employed to evaluate its effect on the quantity of psychoactive substances. For the separation of molecules of interest, reverse-phase chromatography was used by adopting an Ultimate 3000 HPLC-PDA instrument. For quantifying the target alkaloids, the external standard calibration method was used.
20 ug/mL stock solution was made by mixing six reference compounds with an original concentration of 1,000 ug/mL. Six dilutions with various concentrations, including 0.01 ug/mL, 0.1 ug/mL, 1 ug/mL, 5 ug/mL, 10 ug/mL and 15 ug/mL were made. Assessment of the accuracy and precision of the method indicated Relative Standard Deviation (RSD) was <3.3 % at all concentration levels except for psilocin with RSD of 8.5%. In this study, norbaeocystin, baeocystin, psilocybin and psilocin were detected and quantified in fresh and dry samples. The quantity of detected tryptamines are shown in mg/g of dry mushrooms.
