Psilocin glucuronide in whole blood: a stable and useful biomarker of psilocybin intake

Detecting psilocybin use is challenging because it rapidly converts to its psychoactive metabolite psilocin, and both compounds are unstable in blood. Bufotenin, a structural isomer of psilocin, may exhibit comparable instability in blood. For reliable detection, we developed and validated an LC-MS/MS method to simultaneously quantify psilocin, bufotenin, and their metabolites, psilocin glucuronide (PSG) and 5-hydroxyindole-3-acetic acid (5-HIAA), in human whole blood. We prepared blood samples by protein precipitation and lipid removal filtration. Analytes were separated using a biphenyl column. The method was validated according to AAFS guidelines with LOQs of 2.4 nM for psilocin, PSG, and bufotenin, and 30 nM for 5-HIAA. We assessed analyte stability in whole blood under conditions relevant to forensic sample handling. Psilocin degraded by 46%-66% at room temperature and 66%-76% at 4°C after one day, increasing to 88%-99% and 94%-100% after three days. At -20°C, degradation slowed, with up to 51% loss after one month and ≥91% after three months. In contrast, PSG remained stable for 14 days at both room temperature and 4°C, and for at least one year at -20°C, making it a reliable biomarker of psilocybin intake. Bufotenin showed moderate stability, while 5-HIAA was unsuitable as a biomarker due to its endogenous presence. Our method enables direct quantification of PSG, offering a straightforward and accurate alternative to indirect approaches. We demonstrated the method's applicability by analyzing 23 forensic blood samples that screened positive for psilocin or PSG, with PSG quantified in nearly all cases, even when psilocin was below LOQ. These findings confirm PSG as a specific and stable biomarker of psilocybin use, and its integration into routine forensic workflows could significantly improve detection reliability.