Analysis of Psilocin, Bufotenine and LSD in Hair

Journal of Analytical Toxicology 2015;39:126 –129 doi:10.1093/jat/bku141 Advance Access publication December 23, 2014 Article Analysis of Psilocin, Bufotenine and LSD in Hair Rafaela Martin*, Jennifer Schürenkamp, Angela Gasse, Heidi Pfeiffer and Helga Köhler Institute of Legal Medicine, University Hospital Münster, Röntgenstr. 23, D-48149 Münster, Germany *Author to whom correspondence should be addressed. Email: A method for the simultaneous extraction of the hallucinogens psilocin, bufotenine, lysergic acid diethylamide (LSD) as well as iso-LSD, nor-LSD and O-H-LSD from hair with hydrochloride acid and methanol is presented. Clean-up of the hair extracts is performed with solid phase extraction using a mixed-mode cation exchanger. Extracts are measured with liquid chromatography coupled with electrospray tandem mass spectrometry. The method was successfully validated according to the guidelines of the ‘Society of Toxicological and Forensic Chemistry’ (GTFCh). To obtain reference material hair was soaked in a solution of the analytes in dimethyl sulfoxide/methanol to allow incorporation into the hair. These fortified hair samples were used for method development and can be employed as quality controls. Introduction Hair analysis is a useful tool for retrospective drug screening, for example in the context of workplace drug testing or tests of driving ability. In Germany and Italy, it is part of the procedure for granting or re-granting the driver’s license because drug abuse is incompatible with the ability to drive (1, 2). Hair analysis can also be used if an incident happened some time ago and no other sample material other than hair is available. Furthermore, the time and length of drug intake can be estimated by correlating the position of the analytes in the hair samples with the growth rate of hair (1). Lysergic acid diethylamide (LSD) and psilocin are hallucinogenic substances that are consumed mostly for recreational purposes. The latter is contained in some mushroom species (‘magic mushrooms’) while bufotenine is a position isomer of psilocin, that is for example excreted by bufo toads but is also formed endogenously (3, 4). In some countries, it is consumed as a drug (5). LSD, psilocin and bufotenine are controlled substances in many countries. Up until now no methods have been published for analyzing psilocin or bufotenine in hair and it is therefore not known if these substances are incorporated in hair at all. There are only three cases published with positive LSD findings in human head hair with concentrations of 1 – 17 pg/mg (6, 7). Furthermore, LSD was found in pubic hair two times with concentrations of 0.66 and 26 pg/mg (8, 9). In one of these cases, a trace of iso-LSD (impurity of LSD) was also detected (8). The metabolites nor-LSD and O-H-LSD have not been found in human hair to date, although a trace of nor-LSD was detected in rat hair after the intraperitoneal administration of the very high dose of 2 mg/kg for ten consecutive days (6). This article describes a method to extract psilocin, bufotenine and LSD as well as some of its metabolites from hair. Furthermore, hair was soaked with the analytes to incorporate them into the hair and thus prepare fortified hair that was used for method development and quality control. Experimental Chemicals and reagents Bufotenine (1 mg/mL methanol) and LSD (1 mg/mL acetonitrile) were purchased from Lipomed (Weil am Rhein, Germany). Psilocin and psilocin-d10 as solids and stock solutions of iso-LSD (100 mg/mL acetonitrile), O-H-LSD (100 mg/mL acetonitrile) and LSD-d3 (25 mg/mL acetonitrile) were obtained from Cerilliant (Round Rock, TX, USA). Nor-LSD/nor-iso-LSD (combined; pure nor-LSD was not available) as a solid was supplied by Sigma-Aldrich (Steinheim, Germany). Oasisw MCX solidphase extraction (SPE) cartridges (60 mg, 3 mL) were purchased from Waters (Milford, MA). Standard solutions Stock solutions at a concentration of 1 mg/mL of psilocin-d10 and psilocin in methanol and nor-LSD/nor-iso-LSD in acetonitrile were prepared. The internal standard (IS) working solution contained 1 mg/mL psilocin-d10 and 25 ng/mL LSD-d3 in acetonitrile. For calibration, a working solution with 1 mg/mL psilocin and bufotenine, 25 ng/mL LSD and iso-LSD as well as 50 ng/ mL O-H-LSD and nor-LSD/nor-iso-LSD in acetonitrile as well as a 10-fold dilution of this solution were prepared. Preparation of fortified hair A solution containing 1 mg/mL LSD, iso-LSD, nor-LSD/ nor-iso-LSD and O-H-LSD, 10 mg/mL bufotenine and 20 mg/mL psilocin in methanol/dimethyl sulfoxide (DMSO) (1:1, v/v) was prepared. Then, 1 g dark blond, blank hair was stored in 20 mL of this solution under a nitrogen atmosphere protected from light for 7 days. Then, the solution was decanted and the hair was rinsed nine times with 20 mL methanol. Finally, the hair was dried at room temperature. HPLC–MS-MS conditions Samples were analyzed with high performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS-MS). A Waters Alliance 2695 Separation Module with a Pursuit C18 (3 mm, 150  2.0 mm, Varian) column was used for chromatographic separation. The mobile phase consisted of acetonitrile (A) and 2 mM ammonium acetate buffer (B), both with 0.1% formic acid. The micromass Quattro Micro was operated with positive electrospray ionization in the multiple reaction monitoring mode using HPLC– MS-MS conditions described by Martin et al. (10). Extraction and clean-up For decontamination hair samples were washed with 20 mL methanol. It was decanted and the hair was washed in 20 mL acetone, which was decanted as well. After drying at room temperature, the hair was cut in pieces of 1 –3 mm length. Extraction and clean-up were performed and protected from light. After the # The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: addition of 50 mL of 0.1 M ascorbic acid, 50 mg hair were extracted with 2 mL of a 1:1-mixture of hydrochloric acid (0.01 M) and methanol in an ultrasonic bath for 6 h. The temperature of the water bath was kept under 508C due to the instability of psilocin. The samples were centrifuged at 4,000 rpm for 2 min, and the supernatant was decanted into a new test tube. Then, 20 mL IS working solution were added to the supernatant and the methanol was evaporated at 408C in a stream of nitrogen. Furthermore, 1 mL of 0.01 M hydrochloric acid was added to the extracted hair and vortexed to desorb analytes still sticking to the hair. The test tube was centrifuged for 2 min, the supernatants were combined and 4 mL phosphate buffer (0.1 M, pH 6) were added. Clean-up of the hair extracts was performed with SPE. Oasisw MCX columns were conditioned with 2 mL methanol and 0.1 M phosphate buffer, respectively. After sample application, the columns were washed with 2 mL water and 2 mL acetonitrile, followed by a drying step of 5 min in a stream of nitrogen. Then, the columns were washed wit (...truncated)