Psilocybin is a prodrug for psilocin, which produces psychedelic effects by activating serotonin 5-HT2A receptors. This study examined three naturally occurring compounds from psilocybin-containing mushrooms—psilocybin, baeocystin, and aeruginascin—along with their synthetic 4-acetoxy and 4-hydroxy analogues. In cell-based assays, secondary and tertiary tryptamines with 4-acetoxy or 4-hydroxy substitutions showed nanomolar affinity for several human serotonin receptor subtypes, including 5-HT2A and 5-HT1A. In mice, only the tertiary amines psilocin, psilocybin, and psilacetin induced head twitch responses (ED50 0.11–0.29 mg/kg), indicating psychedelic-like activity, which was blocked by a 5-HT2A antagonist.
A liquid chromatography–tandem mass spectrometry method was developed and validated to detect and quantify six 4-position ring-substituted tryptamines in plasma, including psilocybin, psilacetin, 4-Pro-DMT, and their metabolites psilocin and 4-HO-DPT. The method showed linearity from 0.5 to 100 ng/mL for most analytes (psilocybin from 5 to 100 ng/mL), with acceptable bias and imprecision. Matrix effects were minimal except for ion enhancement of psilocin and psilocybin. Extraction efficiency was about 50%. Applied to plasma from male rats given psilacetin, psilacetin was not detected, and psilocin concentrations ranged up to 32.7 ng/mL. The method provides a robust tool for future research and clinical applications.