Abstract
Psilocin is an active substance and dephosphorylated product of psilocybin formed after ingestion of mushrooms. The low stability caused by the quick oxidation of this analyte requires sensitive methods for its determination in biological matrices. In this work, we described a method development, optimization, and validation for the quantification of psilocin in authentic oral fluid samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Liquid-liquid extraction (LLE) was performed using 100 µL of oral fluid samples collected with Quantisal™ device and t-butyl methyl ether (MTBE) as the extraction solvent. The method showed acceptable performance, with limits of detection and quantification of 0.05 ng/mL, and the calibration model was achieved between 0.05 and 10 ng/mL. Bias and imprecision results were below -14.2% and 10.7%, respectively. Ionization suppression/enhancement were lower than -30.5%, and recovery was higher than 54.5%. Dilution integrity bias was below 14.4%. No endogenous and exogenous interferences were observed analyzing oral fluid from 10 different sources and 55 pharmaceuticals and drugs of abuse, respectively. No carryover was observed at 10 ng/mL. Psilocin was stable in oral fluid at -20°C, 4°C and 24°C up to 24, 72 and 24 h, respectively, with variations lower than 17.7%. The analyte was not stable after 3 freeze/thaw cycles, with variations between -73% and -60%. This suggests the instability of psilocin in oral fluid samples, which requires timely analysis, as soon as possible after the collection. The analyte remained stable in processed samples in autosampler (at 10°C) for up to 18 h. The method was successfully applied for the quantification of five authentic samples collected from volunteers attending parties and electronic music festivals. Psilocin concentrations ranged from 0.08 to 36.4 ng/mL. This is the first work to report psilocin concentrations in authentic oral fluid samples.
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