Optimized glucuronide hydrolysis for the detection of psilocin in human urine samples

A sensitive analytical method for detecting psilocin in urine was developed by optimizing hydrolysis conditions. Enzymatic hydrolysis using Escherichia coli beta-glucuronidase (5000 units/ml urine) at pH 6 and 37°C for 2 hours completely converted psilocin glucuronide to psilocin, whereas enzymes from bovine liver, Helix pomatia, and Ampullaria gave incomplete conversion. Acid and alkaline hydrolysis were not applicable. In a magic mushroom user's urine, 3.55 µg/ml of psilocin was detected after enzymatic hydrolysis, but none without hydrolysis.

Study at a glance

Abstract

In order to develop a sensitive and reliable analytical method for psilocin (PC) in urine samples, the hydrolysis conditions including the acid, alkaline and enzymatic hydrolyses have been investigated by monitoring not only PC but also psilocin glucuronide (PCG) by liquid chromatography tandem mass spectrometry (LC-MS-MS); PCG was initially identified in a "magic mushroom (MM)" user's urine by liquid chromatography mass spectrometry (LC-MS) and LC-MS-MS. The proposed conditions optimized for the hydrolysis are as follows: hydrolysis, enzymatic hydrolysis; enzyme, Escherichia coli beta-glucuronidase (5000 units/ml urine); incubation, pH 6 at 37 degrees C for 2h. The complete hydrolysis of PCG in urine was obtained under these conditions, while the enzymatic hydrolyses with three types of beta-glucuronidases originated from bovine liver (Type B-1), Helix pomatia (Type H-1) and Ampullaria provided uncompleted hydrolysis of PCG. Also, neither the acid nor alkaline hydrolysis was found to be applicable. According to the present method, 3.55 microg/ml of psilocin was detected in the "magic mushroom" user's urine after the enzymatic hydrolysis, though psilocin was not detected without hydrolysis.