Studies on Enzymatic Hydrolysis of Psilocin- <i>O</i> -glucuronide for Screening of Psilocin in Human Urine by Liquid Chromatography-Triple Quadrupole Tandem Mass Spectrometry

Psilocin is a psychedelic indole alkaloid and one of the major human metabolites of its phosphorylated precursor, psilocybin. Psilocin binds to the serotonin receptors, 5-HT2A, and dose-dependently induces hallucinogenic effects stronger than psilocybin. As a result, psilocin has been a drug of interest in the analytical toxicology community for years. In vivo, a majority of psilocin is metabolized to and excreted in urine as psilocin-O-glucuronide. Therefore, efficient conversion of psilocin-O-glucuronide to free psilocin directly impacts urine psilocin testing. Due to the chemical lability and light sensitivity of psilocin, the hydrolysis of psilocin-O-glucuronide requires relatively mild and carefully controlled reaction conditions, thus posing a significant challenge to analysts. In recent years, the development of recombinant β-glucuronidases offered a new approach for highly efficient deconjugation. However, to the best of our knowledge, little information on enzymatic hydrolysis of psilocin-O-glucuronide has been published since 2014. We utilized a certified LC-MS/MS-based psilocin screening method and investigated hydrolysis of psilocin-O-glucuronide in human urine catalyzed by one conventional β-glucuronidase (originating from E. Coli) and three recombinant enzymes (IMCSZyme, Kura Biotech-BGTurbo, and Kura Biotech-B-One). The scope of our study included effects of enzyme concentration, pH, incubation time, and incubation temperature on the hydrolysis efficiency, as well as the comparison of modern enzymatic to traditional Brønsted-Lowry acidic and basic hydrolysis methods. The recombinant β-glucuronidases demonstrated remarkable superiority in hydrolysis efficiency compared to the E. Coli-originated conventional β-glucuronidase. Kura Biotech-BGTurbo achieved the fastest hydrolysis completion at the lower temperatures (25-50 °C) while IMCSZyme offered the most consistent performance across a wide range of incubation temperatures (25-80 °C). Our findings provide quantifiable scientific references as a general guideline for other forensic toxicology laboratories to establish or optimize their own psilocin-O-glucuronide hydrolysis methods.