Development and validation of an LC-MS/MS method for the bioanalysis of psilocybin’s main metabolites, psilocin and 4-hydroxyindole-3-acetic acid, in human plasma

A rapid LC-MS/MS method was developed and validated to quantify psilocin, the active metabolite of psilocybin, and its metabolite 4-hydroxyindole-3-acetic acid (4-HIAA) in human plasma. Plasma samples were processed by protein precipitation with methanol. The method achieved inter-assay accuracy of 100-109% and precision ≤8.7%, with recovery ≥94.7% and consistent across concentration levels and plasma batches (CV% ≤4.1%). Plasma matrix caused negligible ion suppression, and endogenous interferences were separated. Samples could undergo three freeze-thaw cycles, remain at room temperature for 8 hours, or be stored at -20°C for 1 month without degradation (≤10%). The linear range (R ≥ 0.998) covered concentrations observed after a 25 mg oral dose of psilocybin, enabling pharmacokinetic assessment.

Study at a glance

Abstract

Psilocin is the active metabolite of psilocybin, a serotonergic psychedelic substance. It is used recreationally and investigated in substance-assisted psychotherapy. The pharmacokinetic properties of psilocin are only partially characterized. Therefore, we developed and validated a rapid LC-MS/MS method to quantify psilocin and its metabolite 4-hydroxyindole-3-acetic acid (4-HIAA) in human plasma. Plasma samples were processed by protein precipitation using methanol. The injected sample was mixed with water in front of a C18 analytical column to increase retention of the analytes. Psilocin and 4-HIAA were detected by multiple reaction monitoring (MRM) in positive and negative electrospray ionisation mode, respectively. An inter-assay accuracy of 100-109% and precision of ≤8.7% was recorded over three validation runs. The recovery was near to complete (≥94.7%) and importantly, consistent over different concentration levels and plasma batches (CV%: ≤4.1%). The plasma matrix caused negligible ion suppression and endogenous interferences could be separated from the analytes. Psilocin and 4-HIAA plasma samples could be thawed and re-frozen for three cycles, kept at room temperature for 8 h or 1 month at -20 °C without showing degradation (≤10%). The linear range (R ≥ 0.998) of the method covered plasma concentrations observed in humans following a common therapeutic oral dose of 25 mg psilocybin and was therefore able to assess the pharmacokinetics of psilocin and 4-HIAA. The LC-MS/MS method was convenient and reliable for measuring psilocin and 4-HIAA in plasma and will facilitate the clinical development of psilocybin.