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.

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.

Species differences in mammalian renal function: 5-HIAA, a reported marker of tubular secretion in humans, is handled exclusively by filtration in the rat.

Renal clearance of endogenous 5-HIAA has been shown to be similar to that of PAH in humans. The present study compared 5-HIAA and PAH clearances in rats. It showed that the clearance of 5-HIAA in rats was identical to that of inulin and creatinine. This suggests that in this species, 5-HIAA is only filtered and not secreted by the tubules in contrast to what is observed in humans.

Octanoic acid produces accumulation of monoamine acidic metabolites in the brain: interaction with organic anion transport at the choroid plexus.

Effects of octanoic acid on monoamines and their acidic metabolites in the rat brain were analyzed by HPLC. Octanoic acid (1,000 mg/kg i.p.) elevated homovanillic acid levels by 54% in the caudate and 338% in the hypothalamus but increased 5-hydroxyindoleacetic acid (5-HIAA) levels in both the caudate and the hypothalamus by approximately 50% compared with the control. A lower dose of octanoic acid (500 mg/kg) increased 5-HIAA levels by 29% in the caudate and 20% in the hypothalamus. However, it did not produce any changes in the concentration of homovanillic acid in either the caudate or the hypothalamus. Treatment with octanoic acid also failed to change the level of dopamine, serotonin, and 3,4-dihydroxyphenylacetic acid in the caudate and the hypothalamus. The role of carrier-mediated transport in the clearance of 5-HIAA from the rabbit CSF was also evaluated in vivo by ventriculocisternal perfusion. Steady-state clearance of 5-HIAA from CSF exceeded that of inulin and was reduced in the presence of octanoic acid. Because this transport system in the choroid plexus is normally responsible for the excretion of the serotonin metabolite from the brain to the plasma, accumulation of endogenously produced organic acids in the brain, secondary to reduced clearance by the choroid plexus, could be a contributing factor in the development of encephalopathy in children with medium-chain acyl-CoA dehydrogenase deficiency who have elevated levels of octanoic acid systematically.