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

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 the 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.