Forensic Analysis of Hallucinogenic Mushrooms and Khat (Catha Edulis FORSK) Using Cation-Exchange Liquid Chromatography
Total Page：16
File Type：pdf, Size：1020Kb
Forensic Science International 195 (2010) 160–164 Contents lists available at ScienceDirect Forensic Science International journal homepage: www.elsevier.com/locate/forsciint Forensic analysis of hallucinogenic mushrooms and khat (Catha edulis FORSK) using cation-exchange liquid chromatography Tim Laussmann *, Sigrid Meier-Giebing Centre for Education and Science of the Federal Finance Administration, Customs Laboratory Cologne, Merianstrasse 110, 50765 Cologne, Germany ARTICLE INFO ABSTRACT Article history: Hallucinogenic mushrooms (e.g. Psilocybe and Panaeolus species) as well as leaves and young shoots of Received 3 August 2009 the khat tree (Catha edulis FORSK) are illicit drugs in many countries. The exact concentration of the Received in revised form 2 December 2009 hallucinogenic alkaloids psilocin and psilocybin in mushrooms and the sympathomimetic alkaloids Accepted 6 December 2009 cathinone and cathine in khat is usually essential for jurisdiction. Facing an increasing number of Available online 4 January 2010 mushroom and khat seizures by German customs authorities, a convenient comprehensive quantitative HPLC method based on cation-exchange liquid chromatography for these rather ‘‘exotic’’ drugs has been Keywords: developed which avoids time-consuming multi-step sample preparation or chemical derivatization Psilocin procedures. Using this method a number of different hallucinogenic fungi species and products that are Psilocybin Hallucinogenic mushroom mainly distributed via the internet have been analysed (dried and fresh Psilocybe cubensis SINGER as well Cathinone as P. cubensis collected from ‘‘grow boxes’’, Panaeolus cyanescens BERKELEY AND BROOME and so-called Cathine ‘‘philosopher stones’’ (sclerotia of Psilocybe species)). Highest total amounts of psilocin have been Khat detected in dried P. cyanescens reaching up to 3.00 Æ 0.24 mg per 100 mg. The distribution of khat alkaloids in different parts of the khat shoots has been studied. High concentrations of cathinone have not only been detected in leaves but also in green parts and barks of stalks. Additionally, the sample treatment for fresh mushroom and khat samples has been optimised. Highest amounts of alkaloids were found when fresh material was freeze-dried. ß 2009 Elsevier Ireland Ltd. All rights reserved. 1. Introduction so far are based on RP HPLC [1–8]. Due to the phosphate group the polarity of psilocybin is much higher compared to psilocin. During the last decade, the recreational use of hallucinogenic Therefore, it is difficult to establish appropriate separation mushrooms has become an increasing problem in Europe. The conditions especially if conventional C18 columns are used. In mushrooms are sold via the internet or in so-called ‘‘smart shops’’. order to solve this problem, an ion pairing reagent has been They are supplied in a fresh or air-dried state or as powder in employed [3]. However, long column equilibration times are capsules for later use [1,2]. Additionally, ‘‘grow-kits’’ can be necessary which are usually not acceptable in routine analysis. A purchased which consist of inoculated substrate in plastic boxes. comprehensive summary of analytical methods for the determi- Most of the hallucinogenic fungi on the market belong to the nation of alkaloids in hallucinogenic mushrooms has been Psilocybe genus. They mainly contain two hallucinogenic alkaloids, published recently together with a RP HPLC method [8]. psilocin and its phosphorylated derivative psilocybin [1–8] which Khat (Catha edulis FORSK., Celastraceae) is a shrub or small tree that are controlled substances in many countries. These alkaloids are is mainly cultivatedin East Africa and the Arabian Peninsula [10–12]. present at total concentrations of approximately 1–2% in dried Fresh leaves and shoots are chewed as a sympathomimetic mushrooms and the amount of the phosphorylated compound stimulant. Two different kinds of khat are on the market: the psilocybin usually exceeds the amount of psilocin [1–8]. ‘‘shrub-drug’’ or ‘‘leaf-drug’’ and the ‘‘tree-drug’’ [10]. The ‘‘shrub- Liquid chromatography is the method of choice for the drug’’ mainly consists of khat leaves, while the ‘‘tree-drug’’ contains quantitative analysis of these compounds. Gas chromatography only shoots with small leafs. Usually the ‘‘tree-drug’’ which is known can not be recommended since the poorly volatile and heat-labile to be more potent and has a longer shelf life is supplied in Germany. psilocybin tends to decompose by loss of the phosphate group Most of the European consumers belong to ethnological minorities during injection [6]. All liquid chromatography methods described which have the traditional habit of using khat. The major psychoactive component of khat is the alkaloid S-(-)-a-aminopro- piophenone also known as cathinone. Additionally, the less active * Corresponding author. Tel.: +49 221 97950188; fax: +49 221 97950 227. alkaloids S,S-(+)-norpseudoephedrine (cathine) and R,S-(-)-nore- E-mail address: [email protected]finv.de (T. Laussmann). phedrine can be found in the plant material [9–12]. Cathinone as 0379-0738/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2009.12.013 T. Laussmann, S. Meier-Giebing / Forensic Science International 195 (2010) 160–164 161 well as cathine are controlled substances in many countries. These 100 mg psilocybin, 0.05 mg/100 mg psilocin, P. cyanescens 2.10 mg/100 mg alkaloids are usually present at total concentrations of around 1 % in psilocybin, 1.44 mg/100 mg) before preparation. Recovery was calculated as measured amount of alkaloid divided by expected amount of alkaloid. leaves of the ‘‘shrub-drug’’ and between 2 % and 5 % in the ‘‘tree- Preparation of khat plant material: Usually, khat is delivered as bundles which drug’’ (calculated per dry weight) [10]. Additionally, the relative are wrapped in fresh and wet leaves of false banana (Ensete ventricosum WELW., amount of cathinone is much higher in the young, almost leafless Musaceae) [12] and, in most cases, wet tissue papers. Khat leaves and shoots were shoots (51% of total phenylpropylamine content) [11]. separated from packing material upon arrival and deep-frozen at below À80 8C. Frozen khat material was either used fresh or air-dried at either 20–25 8Cor608Cor HPLC [11–14] and GC [15–17] methods have been described in freeze-dried in order to determine optimal methods of sample treatment before order to perform a quantification of the khat alkaloids. However, extraction. Samples were homogenised with the laboratory mill for 30 s at sample preparation for RP HPLC is time-consuming as several pre- 10,000 rpm (interval mode). Approximately 2.50 g of the homogenised material purification steps are required. Prior to GC analysis samples have to were immediately transferred into 50.0 mL 100 mM HCl containing 20 mg/ml be treated with a derivatization reagent. Since cathinone is sensitive internal standard (caffeine) and extracted for 60 min at 20–25 8C using a magnetic stirrer. Usually the extract showed a reddish colour. In order to decrease the acidity to alkaline and oxidative conditions [9,11] extensive sample of the extraction solvent, an aliquot of the extract was diluted 1:10 with water preparation should be avoided. One previously developed method containing 20 mg/mL internal standard (caffeine) and filtered (0.2 mm syringe successfully employed cation-exchange chromatography for analy- filter) prior to HPLC analysis. For the determination of recovery of the measured sis of herbal phenalkylamines in human blood plasma [14]. alkaloids a defined amount between 0.41 mg and 1.55 mg of cathinone, cathine and ( )-norephedrine was added to 1.25 g to 2.50 g frozen khat reference material During the last five years the number of seizures of khat and Æ (alkaloid content: 0.94 mg/g cathinone, 0.29 mg/g cathine and 0.04 mg/g (Æ)- hallucinogenic mushrooms increased drastically. Therefore, it was norephedrine) before sample preparation. Recovery was calculated as measured necessary to develop a fast and convenient routine method for the amount of alkaloid divided by expected amount of alkaloid. Additionally, fresh khat quantitative analysis of these rather ‘‘exotic’’ drugs without time- shoots were separated into leaves, soft, green parts of the stalks, woody, reddish parts consuming sample preparation and/or derivatization steps. The of the stalks and bark of the reddish parts in order to determine the distribution of the alkaloids in khat plant material. method was originally designed for analysis of khat alkaloids but has also been found to be useful for analysis of psilocybin and psilocin from mushrooms. Another advantage is that the method 3. Results avoids hazardous HPLC eluents which becomes more and more important with regards to health, safety and environmental 3.1. Method performance regulations in ecologically managed laboratories. Accuracy: Accuracy of the HPLC methods was determined by 2. Experimental the analysis of samples supplied for an interlaboratory comparison 2.1. Chemicals and standards between several German and European forensic institutes. Different GC and HPLC methods have been employed which were Potassium dihydrogen phosphate, phosphoric acid (85%), sodium chloride, caffeine, methanol and ethanol (non-denatured) were obtained from Merck not specified in detail. Corresponding Z-scores of the values (Darmstadt, Germany) and were of p.a. quality. Psilocybin and psilocin standards obtained with the described methods were + 0.23 for the were purchased