Addressing a major interference in the quantification of psilocin in mouse plasma: Development of a validated liquid chromatography tandem mass spectrometry method

Psilocin, the active metabolite of the psychedelic compound psilocybin, is being studied for treating anxiety, depression, and PTSD. Evaluating its pharmacokinetics is essential for drug development. During reversed-phase liquid chromatography analysis of mouse plasma, a previously unreported interference was encountered. The interference was identified as tryptophan using exact mass measurement and MS/MS analysis. A fast and reliable hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) method was developed and validated that separates psilocin from tryptophan, achieving a lower limit of quantification of 0.5 ng/ml. The method was successfully applied to a pharmacokinetic study in C57BL/6 mice, determining psilocin concentrations in all analyzed plasma samples.

Study at a glance

Abstract

Psilocybin is a psychedelic compound found in some hallucinogenic "magic mushrooms". Psilocin is the active metabolite of Psilocybin, and it is the subject of several studies for the treatment of psychological disorders, such as anxiety, depression, and post-traumatic stress disorder. As such, the pharmacokinetic properties of psilocin should be evaluated to ensure its safety and efficacy as part of the drug development process. Based on the previously published studies, reversed-phase liquid chromatography (LC) was tested for psilocin quantification. The analysis, however, showed a major interference in mouse plasma that was not, to the best of our knowledge, reported previously. We, therefore, aimed to identify and separate the interference, using various chromatographic columns, mobile phase conditions, and mass spectrometers (MS) instruments. Chromatographic separation was achieved on an ultra high performance liquid chromatography (UHPLC) system, and a quadrupole-linear ion trap equipped with an electrospray ionization (ESI) source was used in positive ion mode with multiple reaction monitoring (MRM). Several chromatographic conditions and column chemistries, including C-18 and Phenyl-hexyl were initially tested, and failed to separate the interference. Exact mass measurement and MS/MS analysis were used to determine the structure of the interfering compound, which was confirmed to be tryptophan. Using the identified structure of the interfering compound, a fast and reliable hydrophilic interaction liquid chromatography (HILIC)-MS/MS method was developed and validated, that was capable of separating psilocin from the interference while achieving a 0.5 ng/ml lower limit of quantification (LLOQ). The validated method was successfully applied to a pharmacokinetic study where psilocin was orally administered to C57BL/6 mouse subjects. Psilocin concentration in all the analyzed mouse plasma samples was successfully determined.