6:05pm - 7:05pm
Development and Validation of an LC‐MS/MS Method for the Bioanalysis of Psilocybin’s Metabolites, Psilocin and 4‐Hydroxyindole‐3‐Acetic Acid, in Human Plasma
Introduction: Psilocybin is a psychedelic substance being explored in substance‐assisted psychotherapy. However, its pharmacokinetics ﴾PK﴿ are only partially characterized. Thus, we developed and validated a liquid chromatography-tandem mass spectrometry method to quantify psilocybin’s metabolites psilocin and 4‐hydroxyindole‐3‐acetic acid ﴾4‐HIAA﴿ in human plasma, to further explore ﴾or investigate﴿ the PK properties of psilocybin.
Methods: Psilocin and 4‐HIAA were detected by multiple reaction monitoring ﴾MRM﴿ in positive and negative electrospray ionisation mode, respectively. The analytes were separated on a C18 analytical column using water + 0.1% formic acid and methanol +0.1% formic acid as mobile phase A and B, respectively. Plasma samples ﴾time course: 0 to 420 min after treatment﴿ from 3 healthy subjects receiving an oral dose of 25 mg psilocybin were quantified in the presence and absence of Escherichia coli β‐glucuronidase to determine the PK parameters of psilocin, psilocin glucuronide, and 4‐HIAA.
Results: Three independent validation runs exhibited an inter‐assay accuracy of 100‐109% and precision ≤8.7%. Both analytes could be recovered almost completely ﴾>94.7%﴿ and showed no degradation ﴾≤10%﴿ under various storage conditions. Endogenous plasma components did not impair the selectivity of the analysis. Furthermore, the method was linear ﴾R≥ 0.998﴿ and covered the entire concentration range of psilocin ﴾0.36 – 94.1 ng/ml﴿ and 4‐HIAA ﴾7.2 – 156.7 ng/ml﴿ observed in PK samples. The applicability of the method was assessed by analysing PK samples of three healthy volunteers treated with an oral dose of 25 mg psilocybin. Maximal plasma level of psilocin and 4‐HIAA were 19.2 ng/ml and 137.3 ng/ml, respectively. Tmax was reached after 120 to 140 min post treatment. The half‐life of psilocin and 4‐HIAA was around 130 –140 min. Moreover, 4‐HIAA was not glucuronidated, while the majority of psilocin underwent extensive O‐glucuronidation reaching maximal plasma levels of 78.3 ng/ml after approximately 220 min post‐treatment.
Conclusions: The developed method is reliable for the quantification of psilocin and 4‐HIAA in plasma and is capable of determining their pharmacokinetic properties. Overall, the developed analytical method may serve as an important tool in the clinical development of psilocybin as a therapeutic agent.