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

doi:10.1016/j.jchromb.2003.08.030

Journal of Chromatography B

Volume 796, Issue 2, 5 November 2003, Pages 421-427

https://doi.org/10.1016/j.jchromb.2003.08.030 Get rights and content

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 β-glucuronidase (5000 units/ml urine); incubation, pH 6 at 37 °C for 2 h. The complete hydrolysis of PCG in urine was obtained under these conditions, while the enzymatic hydrolyses with three types of β-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 μg/ml of psilocin was detected in the “magic mushroom” user’s urine after the enzymatic hydrolysis, though psilocin was not detected without hydrolysis.

Introduction

Indole derivatives, psilocin (PC) and its phosphate psilocybin (PB), have structural similarity to a neurotransmitter, serotonin, and thereby exhibit a highly hallucinogenic potency. They are both principally contained in some species of the genus Psilocybe, which go under the name of “magic mushrooms (MMs)”, including Psilocybe cubensis, Psilocybe mexicana, Psilocybe subcubensis, Psilocybe semilanceata, Psilocybe argentipus (Japanese name: Hikageshibiretake), etc. and naturally occur throughout the world.

The mushrooms were originally used as godly traditional medicine for centuries in the religious ceremony by the shamans in Central and South America. Nowadays, they have extensively been used for recreational purposes as hallucinogenic substances in various countries including the European and American countries, and even in Japan. It has resulted in increasing abuse of MMs and sometimes in hallucinogenic intoxication by overdosing. Therefore, not only the contained hallucinogenic ingredients PB and PC, but also the fruit body of the MMs are controlled in Japan.

For confirmation of MM intake, the detection of PC in human urine has been generally performed by gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS), because PB is expected to be rapidly and readily dephosphorylated into PC by the intrinsic phosphates in animals [1], [2]. PC is also expected to be metabolized by conjugation with glucuronic acid, demethylation, deamination, oxidation, etc. [3], though the metabolic pathways for PC and PB are not currently known. The expected metabolic pathway of psilocybin is shown in Fig. 1. Several publications have reported analyses of urinary PC by GC-MS [4], [5] or HPLC-ECD [6] and described that the enzymatic hydrolysis with β-glucuronidase led to the evident increase in the amount of detected urinary PC [4], [5], [6], suggesting the fairly high elimination of PC as psilocin glucuronide (PCG) in urine. Thus, the direct analysis of PCG may be promising. Actually, several LC-MS techniques have been developed for the direct determination of glucuronide conjugates [7], [8], [9], [10], [11], [12], [13]. Our research group has also reported the application of LC-MS to the direct analyses of glucuronide conjugates of forensic interests including morphine glucuronides [7], oxazepam glucuronide [8] and ethyl glucuronide [11] in body fluids. However, the direct LC-MS analysis of PCG is not practical for the reliable confirmation of the MM intake because the standard PCG is not currently available. The cleavage of PCG into PC by hydrolysis followed by the determination of PC would be currently preferable, and the complete hydrolysis of PCG is, therefore, indispensable for the confirmation of the MM intake by urine analysis.

For the hydrolysis of PCG in urine, some studies have reported the investigation of the conditions [5], [6]. However, it was not clarified whether PCG present in urine was completely cleaved or not, because only the released PC was determined before and after the hydrolysis. The optimization of the hydrolysis for the complete cleavage of PCG would be required for a more accurate and sensitive analysis.

In the present study, PCG was initially identified in urine collected from a “magic mushroom user” by employing liquid chromatography tandem mass spectrometry (LC-MS-MS), which allows a more accurate identification, in addition to LC-MS. The optimization of the complete hydrolysis of PCG has been explored by directly monitoring PCG as well as PC throughout the hydrolysis with β-glucuronidase.

Section snippets

Materials

PC and PB were obtained from the Ministry of Health, Labour and Welfare of Japan, and those standard stock solutions were prepared in methanol (1 mg/ml). The solutions were stored at −20 °C until used, and adjusted to the appropriate concentration with distilled water or drug-free human urine immediately prior to use.

β-Glucuronidases (EC 3.2.1.31) originating from bovine liver (Type B-1), Helix pomatia (Type H-1) and Escherichia coli (Type IX-A) were purchased from Sigma (St. Louis, MO, USA), and

Identification of psilocin glucuronide (PCG) in urine

A urine specimen from the user (referred to as specimen A) as well as those from drug-free volunteers as blank urine samples were prepared according to the procedure described in Section 2 followed by LC-MS, and the resultant chromatograms from specimen A and the blank urine specimens were compared. A relatively large peak specific to specimen A (referred to as peak A) appeared in Fig. 2 at the retention time of 2.9 min in the extracted mass chromatogram at m/z 381, which corresponds to the

Conclusion

For optimization of the hydrolysis conditions for PCG in human urine, urinary PCG was, for the first time, identified in an preliminary experiment using LC-MS and LC-MS-MS. Also, the optimal hydrolysis conditions were explored by monitoring not only PC but also PCG by LC-MS-MS. The proposed optimal hydrolysis conditions are as follows: hydrolysis, enzymatic hydrolysis; enzyme, E. coli β-glucuronidase (5000 units/ml urine); incubation, pH 6 at 37 °C for 2 h. These established conditions allow the

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

Abstract

Introduction

Section snippets

Materials

Identification of psilocin glucuronide (PCG) in urine

Conclusion

Biochem. Pharmacol.

Toxicol. Appl. Pharmacol.

Biochem. Pharmacol.

Forensic Sci. Int.

J. Pharm. Biomed. Anal.

J. Chromatogr. B

J. Chromatogr. B