Development and validation of an analytical method for the determination of select 4-position ring-substituted...

4-Phosporyloxy-N, N-dimethyltryptamine (psilocybin) is a psychedelic tryptamine found in certain mushroom species that has shown efficacy in the treatment of various psychiatric disorders. In conjunction with the renewed interest in therapeutic effects of psychedelics, there has been an increase in psilocybin-like designer tryptamines appearing in non-medical drug markets. The present study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detecting and quantifying 4-position ring-substituted tryptamines and their 4-hydroxy metabolites in plasma. Specifically, we investigated 4-phosphoryloxy-N, N-dimethyltryptamine (psilocybin), 4-acetoxy-N, N-dimethyltryptamine (psilacetin), 4-propionoxy-N, N-dimethyltryptamine (4-Pro-DMT) and their shared metabolite 4-hydroxy-N, N-dimethyltryptamine (psilocin), along with 4-methyl carbonato-N, N-di-n-propyltryptamine (4-MeCO3-DPT) and its metabolite 4-hydroxy-N, N-di-n-propyltryptamine (4-HO-DPT). Mass spectrometry analysis employed electrospray ionization (ESI) in positive mode, with two multiple reaction monitoring (MRM) transitions per analyte. Plasma samples were acidified with ascorbic acid, followed by protein precipitation with acetonitrile. Linearity was achieved across a concentration range of 0.5-100 ng/mL for all analytes, except psilocybin, which displayed linearity from 5 to 100 ng/mL. Validation results demonstrated acceptable bias (±20%) and imprecision (<20%) for all analytes. Matrix effects, evaluated in 10 samples (CV <18.3%), indicated minimal interference, although ion enhancement was observed for psilocin (31.9%) and psilocybin (45.7%). Extraction efficiency across all tryptamines was approximately 50%. The assay method was used to quantitate plasma samples from male rats treated with 1.0 mg/kg s.c. of the prodrug psilacetin, and collected before and 5, 30, 60, 120 and 240 min after injection. No psilacetin was detected, and psilocin concentrations ranged from non-detected up to 32.7 ng/mL. Overall, we successfully developed a sensitive and specific method for the detection and quantification of six tryptamines in plasma, providing a robust tool for future research and clinical applications.