1-s2.0-S0379073809004927-main

Prévia do material em texto

Forensic Science International 195 (2010) 160–164
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
A R T I C L E I N F O
Article history:
Received 3 August 2009
Received in revised form 2 December 2009
Accepted 6 December 2009
Available online 4 January 2010
Keywords:
Psilocin
Psilocybin
Hallucinogenic mushroom
Cathinone
Cathine
Khat
A B S T R A C T
Hallucinogenic mushrooms (e.g. Psilocybe and Panaeolus species) as well as leaves and young shoots of
the khat tree (Catha edulis FORSK) are illicit drugs in many countries. The exact concentration of the
hallucinogenic alkaloids psilocin and psilocybin in mushrooms and the sympathomimetic alkaloids
cathinone and cathine in khat is usually essential for jurisdiction. Facing an increasing number of
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
developed which avoids time-consuming multi-step sample preparation or chemical derivatization
procedures. Using this method a number of different hallucinogenic fungi species and products that are
mainly distributed via the internet have been analysed (dried and fresh Psilocybe cubensis SINGER as well
as P. cubensis collected from ‘‘grow boxes’’, Panaeolus cyanescens BERKELEY AND BROOME and so-called
‘‘philosopher stones’’ (sclerotia of Psilocybe species)). Highest total amounts of psilocin have been
detected in dried P. cyanescens reaching up to 3.00 � 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.
� 2009 Elsevier Ireland Ltd. All rights reserved.
Contents lists available at ScienceDirect
Forensic Science International
journal homepage: www.e lsev ier .com/ locate / forsc i in t
1. Introduction
During the last decade, the recreational use of hallucinogenic
mushrooms has become an increasing problem in Europe. The
mushrooms are sold via the internet or in so-called ‘‘smart shops’’.
They are supplied in a fresh or air-dried state or as powder in
capsules for later use [1,2]. Additionally, ‘‘grow-kits’’ can be
purchased which consist of inoculated substrate in plastic boxes.
Most of the hallucinogenic fungi on the market belong to the
Psilocybe genus. They mainly contain two hallucinogenic alkaloids,
psilocin and its phosphorylated derivative psilocybin [1–8] which
are controlled substances in many countries. These alkaloids are
present at total concentrations of approximately 1–2% in dried
mushrooms and the amount of the phosphorylated compound
psilocybin usually exceeds the amount of psilocin [1–8].
Liquid chromatography is the method of choice for the
quantitative analysis of these compounds. Gas chromatography
can not be recommended since the poorly volatile and heat-labile
psilocybin tends to decompose by loss of the phosphate group
during injection [6]. All liquid chromatography methods described
* Corresponding author. Tel.: +49 221 97950188; fax: +49 221 97950 227.
E-mail address: tim.laussmann@bwz-k.bfinv.de (T. Laussmann).
0379-0738/$ – see front matter � 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.forsciint.2009.12.013
so far are based on RP HPLC [1–8]. Due to the phosphate group the
polarity of psilocybin is much higher compared to psilocin.
Therefore, it is difficult to establish appropriate separation
conditions especially if conventional C18 columns are used. In
order to solve this problem, an ion pairing reagent has been
employed [3]. However, long column equilibration times are
necessary which are usually not acceptable in routine analysis. A
comprehensive summary of analytical methods for the determi-
nation of alkaloids in hallucinogenic mushrooms has been
published recently together with a RP HPLC method [8].
Khat (Catha edulis FORSK., Celastraceae) is a shrub or small tree that
is mainly cultivated in East Africa and the Arabian Peninsula [10–12].
Fresh leaves and shoots are chewed as a sympathomimetic
stimulant. Two different kinds of khat are on the market: the
‘‘shrub-drug’’ or ‘‘leaf-drug’’ and the ‘‘tree-drug’’ [10]. The ‘‘shrub-
drug’’ mainly consists of khat leaves, while the ‘‘tree-drug’’ contains
only shoots with small leafs. Usually the ‘‘tree-drug’’ which is known
to be more potent and has a longer shelf life is supplied in Germany.
Most of the European consumers belong to ethnological minorities
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
alkaloids S,S-(+)-norpseudoephedrine (cathine) and R,S-(-)-nore-
phedrine can be found in the plant material [9–12]. Cathinone as
mailto:tim.laussmann@bwz-k.bfinv.de
http://www.sciencedirect.com/science/journal/03790738
http://dx.doi.org/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
alkaloids are usually present at total concentrations of around 1 % in
leaves of the ‘‘shrub-drug’’ and between 2 % and 5 % in the ‘‘tree-
drug’’ (calculated per dry weight) [10]. Additionally, the relative
amount of cathinone is much higher in the young, almost leafless
shoots (51% of total phenylpropylamine content) [11].
HPLC [11–14] and GC [15–17] methods have been described in
order to perform a quantification of the khat alkaloids. However,
sample preparation for RP HPLC is time-consuming as several pre-
purification steps are required. Prior to GC analysis samples have to
be treated with a derivatization reagent. Since cathinone is sensitive
to alkaline and oxidative conditions [9,11] extensive sample
preparation should be avoided. One previously developed method
successfully employed cation-exchange chromatography for analy-
sis of herbal phenalkylamines in human blood plasma [14].
During the last five years the number of seizures of khat and
hallucinogenic mushrooms increased drastically. Therefore, it was
necessary to develop a fast and convenient routine method for the
quantitative analysis of these rather ‘‘exotic’’ drugs without time-
consuming sample preparation and/or derivatization steps. The
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
avoids hazardous HPLC eluents which becomes more and more
important with regards to health, safety and environmental
regulations in ecologically managed laboratories.
2. Experimental
2.1. Chemicals and standards
Potassium dihydrogen phosphate, phosphoric acid (85%), sodium chloride,
caffeine, methanol and ethanol (non-denatured) were obtained from Merck
(Darmstadt, Germany) and were of p.a. quality. Psilocybin and psilocin standards
were purchased from Lipomed (Bad Säckingen, Germany). Cathinone hydrochloride,
cathine hydrochloride, (�)-norephedrine hydrochloride, tryptamine and hydrochloric
acid (100 mM and 37%) were supplied by Sigma–Aldrich (Steinheim, Germany).
2.2. High-performance liquid chromatography
A Merck–Hitachi HPLC System (Merck–Hitachi, Darmstadt, Germany) compris-
ing a controller (D 7000), an autosampler (L 7200), a degasser (L 7612), a high-
pressure pump (L 7100), a heated column thermostat (L 7300) and an UV-diode
array detector (8.0 ml flow cell, 10 mm path length,L 7450) was used in all
experiments. A Luna 5 mm SCX 100A 150 mm � 4.60 mm cation exchange column
(Phenomenex, Aschaffenburg, Germany) was employed for separation. The mobile
phase consisted of a 95:5 mixture of buffer (50 mM KH2PO4, 100 mM NaCl) and
ethanol. The pH of the mobile phase was adjusted to 3.0 with 50 mM phosphoric
acid. Ten microliter (mushroom analysis) or 20 mL (khat analysis) of the sample
were applied on the column. Separation was performed at a flow rate of 1.5 mL/min
(35 8C). Psilocybin and psilocin were quantified at 220 nm. Cathine and (�)-
norephedrine were detected at 216 nm while cathinone was measured at 257 nm. The
method was calibrated with solutions of standard compounds containing between
1.0 mg/mL and 40.0 mg/mL psilocybin and psilocin and 10 mg/mL tryptamine (internal
standard) and between 0.6 mg/mL and 24.0 mg/mL cathinone, cathine and (�)-
norephedrine and 20 mg/mL caffeine (internal standard).
2.3. Sample preparation
Preparation of mushrooms: Fresh mushrooms were collected from ‘‘grow boxes’’
and cut into longitudinal quarters in order to ensure a maximum of homogeneity of
the samples for the subsequent comparison of four different methods of sample
treatment before extraction. The four sample groups were either analysed
immediately (fresh), air-dried at either 20 8C or 60 8C or freeze-dried. Dried
samples were homogenised with a laboratory mill Grindomix GM 200 (Retsch,
Haan (Rheinland), Germany) for 30 s at 10,000 rpm (interval mode). Approximately
100 mg of the homogenate were extracted with 100 mL methanol containing
10 mM HCl (0.833 mL 37% HCl per L methanol) and 10 mg/mL internal standard
(tryptamine) at 20–25 8C for 60 min in an ultrasonic bath. In case of fresh fungi, 5 g
of the material were extracted in the same solvent at 20–25 8C for 15 min using a
laboratory mixer (interval mode). The extracts were filtered prior to HPLC analysis
(0.2 mm syringe filter, Macherey–Nagel, Düren, Germany). In order to determine
the recovery of the measured alkaloids a defined amount between 0.25 mg and
0.92 mg of psilocybin and psilocin was added to 100 mg dried Psilocybe cubensis or
Panaeolus cyanescens reference material (alkaloid content: P. cubensis: 1.05 mg/
100 mg psilocybin, 0.05 mg/100 mg psilocin, P. cyanescens 2.10 mg/100 mg
psilocybin, 1.44 mg/100 mg) before preparation. Recovery was calculated as
measured amount of alkaloid divided by expected amount of alkaloid.
Preparation of khat plant material: Usually, khat is delivered as bundles which
are wrapped in fresh and wet leaves of false banana (Ensete ventricosum WELW.,
Musaceae) [12] and, in most cases, wet tissue papers. Khat leaves and shoots were
separated from packing material upon arrival and deep-frozen at below �80 8C.
Frozen khat material was either used fresh or air-dried at either 20–25 8C or 60 8C or
freeze-dried in order to determine optimal methods of sample treatment before
extraction. Samples were homogenised with the laboratory mill for 30 s at
10,000 rpm (interval mode). Approximately 2.50 g of the homogenised material
were immediately transferred into 50.0 mL 100 mM HCl containing 20 mg/ml
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
of the extraction solvent, an aliquot of the extract was diluted 1:10 with water
containing 20 mg/mL internal standard (caffeine) and filtered (0.2 mm syringe
filter) prior to HPLC analysis. For the determination of recovery of the measured
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
(alkaloid content: 0.94 mg/g cathinone, 0.29 mg/g cathine and 0.04 mg/g (�)-
norephedrine) before sample preparation. Recovery was calculated as measured
amount of alkaloid divided by expected amount of alkaloid. Additionally, fresh khat
shoots were separated into leaves, soft, green parts of the stalks, woody, reddish parts
of the stalks and bark of the reddish parts in order to determine the distribution of the
alkaloids in khat plant material.
3. Results
3.1. Method performance
Accuracy: Accuracy of the HPLC methods was determined by
the analysis of samples supplied for an interlaboratory comparison
between several German and European forensic institutes.
Different GC and HPLC methods have been employed which were
not specified in detail. Corresponding Z-scores of the values
obtained with the described methods were + 0.23 for the
determination of the sum of psilocybin and psilocin and + 0.15
for the determination of cathinone. Cathine and (�)-norephedrine
were not evaluated. Additionally, the results obtained by the new
method were compared with results obtained by described alterna-
tive methods (RP-HPLC) in our laboratory. For the analysis of dried
mushroom reference material, the method by Beug et al. [3] was
employed. Analysis of fresh frozen khat reference material was
performed with the method described by Geisshüsler and Brenneisen
[12]. Concentrations of psilocybin, psilocin, cathinone, cathine and
R,S-(-)-norephedrine were identical within SD to the concentrations
found with the described cation-exchange HPLC method.
Selectivity and specificity: Representative chromatograms are
shown in Fig. 1. A total number of 7 hallucinogenic mushroom
samples and 14 khat samples from different seizures were
analysed without internal standards. There were no peaks detected
that interfered with the internal standards tryptamine (mush-
rooms) and caffeine (khat). Peak purity of the measured alkaloids
and the internal standards was controlled by correlation of the UV
spectra with library spectra. No peak impurities could be detected.
Additionally, dried samples of six common fungi (Amanita
muscaria (L.) HOOK., Cantharellus cibarius FR., Lentinula edodes (BERK.)
PEGLER, Boletus-species, Pleurotus ostreatus (JACQ.) QUÉL., Agaricus
bisporus (J.E. LANGE) PILÁT) and ten herbs (Mentha x piperita L.,
Matricaria chamomilla L., Camellia sinensis L. (as ‘‘black tea’’),
Erythroxylum coca LAM., Salvia officinalis L., Salvia divinorum EPLING &
JATIVA, Melissa officinalis L., Plantago lanceolata L., Crataegus species,
Achillea millefolium L.) were analysed. There were no peaks
detectable that interfered with psilocybin and psilocin or with
kath alkaloids, respectively.
Linearity and limits of detection and quantification: Linearity,
limits of detection and limits of quantification were calculated
from the calibration curve by replicate measurements of calibra-
tion standards according DIN 32645 using a statistics software
(SQS 99, Dr. Kleiner Software, Moos, Germany). Hallucinogenic
Fig. 1. HPLC chromatograms of original Panaeolus cyanescens (A: 220 nm, internal
standard: tryptamine) and khat samples (B: 257 nm, C: 216 nm, internal standard:
caffeine). Cathinone was determined at 257 nm, cathine and R,S-(-)-norephedrine
at 216 nm. The amount of internal standard (caffeine) has been optimised for
determination of cathinone at 257 nm (B).
T. Laussmann, S. Meier-Giebing / Forensic Science International 195 (2010) 160–164162
alkaloids in mushrooms were calibrated as wt.% and khat alkaloids
as wt.%. The calibration curve was found to be linear for all
measured compounds. Limits of detection were 0.7 ng on column
(equivalent to 0.007 mg/100 mg dry weight) for psilocybin, 0.6 ng
Table 1
Alkaloid concentration (mg/100 mg� SD) in Psilocybe cubensis SINGER obtained from ‘‘grow
case).
Psilocybin
Fresh 0.038� 0.005
Fresh, calculated on dry weighta 0.342� 0.047
Dried at 20 8C–25 8C 0.339� 0.020
Dried at 60 8C 0.058� 0.020
Freeze-dried 1.048� 0.050
a fresh fungi contained 89%�2% water (n = 5 independent seizures)
b total psychotropic psilocin content calculated as the sum of psilocin and psilocin w
(0.006 mg/100 mg dry weight) for psilocin, 1.3 ng (0.013 mg/mg
fresh weight) for cathinone, 0.7 ng (0.007 mg/mg fresh weight) forcathine and 0.9 ng (0.009 mg/mg fresh weight) for (�)-norephe-
drine. The limits of quantification were 2.8 ng (0.028 mg/100 mg dry
weight) for psilocybin, 2.2 ng (0.022 mg/100 mg dry weight) for
psilocin, 5.1 ng (0.051 mg/mg fresh weight) for cathinone, 2.5 ng
(0.025 mg/mg fresh weight) for cathine and 3,5 ng (0.035 mg/mg fresh
weight) for (�)-norephedrine.
Precision: Repeatability data of the method were calculated
from 6 determinations of control material performed by the same
person on the same day. Relative standard deviations (RSDs)
of � 1.2% (psilocybin), � 1.2% (psilocin), � 1.7% (cathinone), � 3.1%
(cathine) and � 2.7% ((�)-norephedrine) were determined. The
intermediate precision (between days repeatability) was calculated
from replicate determinations of control material on different days.
RSDs of � 3.9% (psilocybin, n = 15), � 3.8% (psilocin, n = 6), � 3.4%
(cathinone, n = 6), � 8.2% (cathine, n = 6) and � 3.4% ((�)-norephe-
drine, n = 6) were determined.
Recovery: Recovery was determined after addition of defined
amounts of reference substance to a reference material (dried
mushrooms, frozen khat) before extraction (n = 3). Relative
recovery (� SD) was (97.3 � 1.1) % (psilocybin), (98.8 � 1.7) %
(psilocin), (98.6 � 1.6) % (cathinone), (97.4 � 1.9) % (cathine) and
(93.3 � 5.8) % ((�)-norephedrine). Additionally, samples were sub-
jected to multiple extractions (n = 4 for khat and mushrooms) in order
to show the efficiency of the described one-step extraction. The
samples were extracted, filtered and re-extracted three times. After
the first extraction only (0.2 � 0,1) % of total psilocybin and
(2.2 � 1.1) % of total cathinone remained in the samples. The
amounts of psilocin, cathine and (�)-norephedrine were below the
detection limit after the first extraction.
3.2. Preparation of mushroom samples
Fresh mushrooms were collected from ‘‘grow-boxes’’, cut into
longitudinal quarters and sorted into four groups. This procedure
was necessary to ensure a maximum homogeneity of mushroom
material between the four groups. In order to find optimal sample
treatment conditions the four following methods were tested:
direct extraction of fresh mushrooms, extraction of mushrooms
dried to constant weight at different temperatures (20 8C and
60 8C) and extraction of freeze-dried mushrooms. The results are
summarised in Table 1. Highest alkaloid concentrations were
obtained when samples were freeze-dried prior to extraction.
Drying at room temperature or extraction of fresh fungi obviously
led to partial decomposition of the alkaloids so that only about half
of the total calculated psilocin content was recovered. Partially,
psilocybin seems to be dephosphorylated to psilocin (see Table 1,
line 3) during the procedure. Drying at elevated temperature
(60 8C) is not recommendable since 90% of total calculated psilocin
content is decomposed. Extraction time was varied between
10 min and 4 h. A maximum of psilocybin and psilocin was found
after 60 min of extraction. The extracted alkaloids were stable for
several hours when kept at a dark place at room temperature.
-boxes’’ using different methods of sample treatment prior to extraction (n = 5 in each
Psilocin Total psilocin contentb
0.012�0.003 0.039�0.005
0.108�0.024 0.353�0.047
0.252�0.006 0.496�0.020
0.044�0.025 0.086�0.039
0.105�0.015 0.858�0.051
hich could be released from psilocybin by dephosphorylation
Table 2
Alkaloid concentration (mg/100 mg (dry weight)� SD, n = number of independent seizures) in different mushroom species and products sold by retail.
psilocybin psilocin calculated total psilocind
Psilocybe cubensisa, dried fungi (n = 74) 1.151�0.228 0.126� 0.066 0.953�0.137
Psilocybe cubensisa, fresh fungi (n = 7) 0.792�0.095 0.066� 0.035 0.635�0.073e
Psilocybe cubensisa, ‘‘grow-box’’ (n = 8)b 0.509�0.292 0.127� 0.081 0.436�0.192
Panaeolus cyanescensc, dried fungi (n = 24) 2.514�0.908 1.194� 0.573 3.001�0.239
Psilocybe tampanensisa, ‘‘philosopheŕs stones’’, fresh sclerotia (n = 4) 0.119�0.062 0.058� 0.043 0.143�0.076
a species as declared on the packing of the product
b one grow box (18 cm�12 cm�6 cm) produced 13.7 g�8.4 g (weight after drying at room temperature, n = 8) hallucinogenic fungi
c species identified by microscopy of spores and cheilocystidia
d total psychotropic psilocin content calculated as the sum of psilocin and psilocin which could be released from psilocybin by dephosphorylation
e concentrations determined after drying at 20–25 8C
Table 3
Measured alkaloid concentration (mg/g� SD) in khat plant material (‘‘tree-drug’’) using different methods of sample preparation prior to extraction (n = 3 in each case).
cathinone cathine R,S-(-)-norephedrine
Fresh 1.042�0.103 0.410� 0.014 0.049�0.009
Fresh, calculated on dry weighta 3.859�0.381 1.519� 0.052 0.181�0.033
Dried at 20 8C–25 8C 2.362�0.165 1.549� 0.076 0.181�0.066
Dried at 60 8C 1.048�0.063 1.388� 0.056 0.219�0.052
Freeze-dried 4.384�0.293 1.545� 0.082 0.155�0.024
a fresh khat plant material contained 73�2% water (n = 9 independent seizures)
T. Laussmann, S. Meier-Giebing / Forensic Science International 195 (2010) 160–164 163
3.3. Alkaloid content in different mushroom products
Several different hallucinogenic mushroom species are sold via
the internet or in ‘‘smart shops’’. The most popular is Psilocybe
cubensis SINGER, which can be obtained fresh, dried or as inoculated
substrates in order to grow the fungi at home (‘‘grow-box’’). The
results of quantitative alkaloid determination are summarised in
Table 2.
Thirty confiscated grow-boxes were activated as described in the
package leaflet by addition of distilled water. The boxes were put
into special plastic bags that were supplied with the grow-boxes.
These bags have integrated air filters to keep conditions sterile.
Between 12 g and 215 g fresh fungi were collected from eight grow-
boxes. In conclusion, one grow-box can produce up to about 200 g
fresh fungi, corresponding to about 20 g dried fungi with a maximal
total hallucinogenic alkaloid content of 0.86 mg/100 mg (deter-
mined after freeze-drying). Based upon a mean hallucinogenic dose
of 10 mg Psilocin [1] the amount of fungi harvested from one grow
box is equivalent to up to 17 single doses. However, 12 grow-boxes
did not produce any fungi and 10 grow-boxes produced only small
fungi with a total fresh weight of less than 2 g. These problems were
due to contamination and subsequent excessive growth of micro
organisms and moulds. In our view similar problems would occur if
the grow-boxes were activated by the consumer at home.
Additionally, so called ‘‘philosopher’s stones’’ are supplied which
are sclerotia formed by Psilocybe fungi. The alkaloid content of these
sclerotia or so-called ‘‘truffles’’ is about 7 times lower than that of
fruiting bodies of P. cubensis. Recently, the fungi species Panaeolus
cyanescens BERKELEY AND BROOME with an extraordinarily high total
alkaloid concentration of up to 3.00� 0.24 g/100 mg appeared on the
market. A clear negative correlation between contents of psilocin and
psilocybin can be observed in Panaeolus cyanescens samples: the higher
the psilocybin content in an individual sample, the lower the psilocin
Table 4
Distribution of khat-alkaloids in different parts of fresh khat material (‘‘tree-drug’’). Kh
% by weight of the
complete khat shoots
Cathinone
mg/g % of total
Leaves 10.2�2.9 1.326�0.353 19.4�7.5
Green part of stalks 31.2�10.8 1.032�0.208 44.6�7.9
Reddish part of stalks 58.7�21,4 0.456�0.128 36.0�4.1
content (data not shown). This fact is obviously due to the conversion of
psilocybin to psilocin by dephosphorylation.
3.4. Preparation of khat samples
Four different methods of sample treatment prior to extraction
were employed: deep-freezing, drying at room temperature,
drying at elevated temperature (60 8C) or freeze-drying. Based
upon the measured alkaloid concentrations after these types of
sample treatment it can be recommended to immediately freeze-
dry or deep-freeze khat plant material until itis analysed. When
freeze-dried, the plant material loses 73 � 2% (n = 9 bundles) of the
initial weight. Drying at room temperature or elevated temperatures
can not be recommended since this treatment obviously leads to
decomposition of cathinone (Table 3).
Extraction time was varied between 2 min and 4 h. Cathinone,
cathine and R,S-(-)-norephedrine were completely extracted by
100 mM HCl from homogenised samples after 30 min of extrac-
tion. The extracted alkaloids were stable in 100 mM HCl for several
hours when kept at a dark place at room temperature.
3.5. Distribution of khat alkaloids in plant material
During the last years it has been an issue which parts of khat
plant material (‘‘tree-drug’’) could be defined as ‘‘consumable
parts’’. According to the literature [10], all parts of the tree drug can
be consumed. Therefore, khat shoots were analysed in total for
forensic purposes. However, in order to provide more information
on the distribution of the sympathomimetic alkaloids in the khat
plant material, fresh shoots were divided into three different parts:
leaves, green, soft parts of the stalk and reddish, woody parts of the
stalk (Table 4). The highest concentration of cathinone was found
in the leaves followed by the green stalks. Cathine was found to be
at material of three independent seizures was analysed in duplicates.
Cathine R,S-(-)-norephedrine
mg/g % of total mg/g % of total
0.610� 0.245 15.7�5.3 0.273�0.146 100.0
0.400� 0.112 31.4�7.0 <detection limit <detection limit
0.365� 0.108 52.9�8.3 <detection limit <detection limit
T. Laussmann, S. Meier-Giebing / Forensic Science International 195 (2010) 160–164164
almost homogeneously distributed in khat shoots while R,S-(-)-
norephedrine was only detected in leaves. Additionally, the bark of
reddish stalks which can be easily removed from the wood was
analysed. The bark contained 65% of cathinone and cathine found
in the reddish stalks.
Besides the ‘‘tree-drug’’, one single sample of already dried
leaves (‘‘leaf-drug’’) reached our lab. The dried sample contained
2.503 � 0.270 mg/g cathinone, 2.110 � 0.228 mg/g cathine and
0.065 � 0.007 mg/g R,S-(-)-norephedrine.
4. Discussion
A fast and convenient cation-exchange HPLC method for
routine quantitative determination of psychotropic alkaloids from
hallucinogenic fungi and khat plant material was developed which
avoids laborious sample preparation or derivatization procedures.
Several different seizures of hallucinogenic fungi and khat material
were analysed. It could be shown that highest alkaloid concentra-
tions were measured when samples were freeze-dried prior to
analysis.
A mushroom species, Panaeolus cyanescens, with extraordinari-
ly high concentrations of psilocybin and psilocin appeared on the
German market. Its total content of hallucinogenic alkaloids was
found to be about three times higher than that of the well known
species Psilocybe cubensis. Fungi harvested from ‘‘grow-boxes’’
usually do not reach the tryptamine alkaloid levels of commer-
cially produced hallucinogenic mushrooms. However, it could be
demonstrated that one single grow-box can produce enough
material for up to 17 hallucinogenic doses.
The distribution of khat alkaloids in the ‘‘tree-drug’’ was
analysed. Highest concentrations of cathinone were found in
leaves and green parts of the stalks. However, even the woody, red
parts of the stalks, especially the bark, contained a relevant amount
of the drug. Therefore, it is recommended to analyse the khat plant
material as a whole for forensic purposes. Interestingly, there were
only small amounts of R,S-(-)-norephedrine detectable in the
single sample of dried ‘‘leaf-drug’’. According to Schorno [10] the
amount of R,S-(-)-norephedrine in khat is highly variable and
depends not only on the age and quality of the leaves but also on
the geographic origin of catha-varieties. Khat from Kenya showed
higher amounts of R,S-(-)-norephedrine than khat from Yemen or
Madagascar [10,12].
Due to the different separation principle, application of cation-
exchange chromatography should generally be taken into account
as an alternative to established RP methods when analysing
alkaloids. It might be a special advantage that the samples can be
extracted, for example, by strong acidic aqueous solutions
avoiding possible co-extraction of interfering compounds that
might occur when organic extraction solvents are employed.
Aqueous solutions can be directly applied on the cation-exchange
column so that a step of buffer change e.g. by solid-phase
extraction is not necessary. Additionally, cation-exchange HPLC
with aqueous solvents avoids ecologically harmful organic
eluents that are usually necessary in RP HPLC which is a step
forward to achieve a ‘‘green lab’’ with respect to health, safety and
environmental regulations.
Acknowledgements
The authors wish to thank Mr. Günter Kolender, German
Mycological Society, for the microscopic determination of Panaeo-
lus cyanescens BERKELEY AND BROOME, Prof. Dr. Ewald Langer,
University of Kassel, Germany, for supplying a sample of dried
Amanita muscaria and Dr. Alexander Paul and Dr. Burkhard Grebe,
Customs Laboratory Cologne, Germany, for reading the manuscript
and helpful discussions.
References
[1] F. Musshoff, B. Madea, J. Beike, Hallucinogenic mushrooms on the German
market–simple instructions for examination and identification, Forensic Sci.
Int. 113 (2000) 389–395.
[2] K. Tsujikawa, T. Kanamori, Y. Iwata, Y. Ohmae, R. Sugita, H. Inoue, T. Kishi,
Morphological and chemical analysis of magic mushrooms in Japan, Forensic
Sci. Int. 138 (2003) 85–90.
[3] M.W. Beug, J. Bigwood, Quantitative analysis of psilocybin and psilocin in Psilo-
cybe cubensis (SINGER and SMITH) by high-performance liquid chromatography and
by thin-layer chromatography, J. Chromatogr. 207 (1981) 379–385.
[4] M. Wurst, M. Semerdžieva, J. Vokoun, Analysis of psychotropic compounds in
fungi of the genus psilocybe by reversed-phase high-performance liquid chro-
matography, J. Chromatogr. 286 (1984) 229–235.
[5] S. Borner, R. Brenneisen, Determination of tryptamine derivatives in hallucino-
genic mushrooms using high-performance liquid chromatography with photodi-
ode array detection, J. Chromatogr. 408 (1987) 402–408.
[6] M. Wurst, R. Kysilka, T. Koza, Analysis and isolation of indole alkaloids of fungi by
high-performance liquid chromatography, J. Chromatogr. 593 (1992) 201–208.
[7] J. Gartz, Extraction and analysis of indole derivatives from fungal biomass, J. Basic
Microbiol. 34 (1994) 17–22.
[8] N. Anastos, S.W. Lewis, N.W. Barnett, D.N. Sims, The determination of psilocin and
psilocybin in hallucinogenic mushrooms by HPLC utilizing a dual reagent acidic
potassium permanganate and tris(2,20-bipyridyl)ruthenium(II) chemilumines-
cence detection system, J. Forensic Sci. 51 (2006) 45–51.
[9] K. Szendrei, The chemistry of khat, Bull. Narc. 32 (1980) 5–35.
[10] H.X. Schorno, Khat, Suchtdroge des Islams, Pharm. Unserer Zeit 11 (1982) 65–73.
[11] R. Brenneisen, S. Geisshüsler, Psychotropic drugs, Pharm. Acta Helv. 60 (1985)
290–301.
[12] S. Geisshüsler, R. Brenneisen, The content of psychoactive phenylpropyl and
phenylpentenyl khatamines in Catha edulis FORSK. of different origin, J. Ethno-
pharmacol. 19 (1987) 269–277.
[13] K. Mathys, R. Brenneisen, HPLC and TLC profiles of phenylalkylamines of khat
(Catha edulis FORSK.) confiscated in Switzerland, Pharm. Acta Helv. 68 (1993) 121–
128.
[14] J. Beyer, F.T. Peters, T. Kraemer, H.H. Maurer, Detection and validated quantifica-
tion of nine herbal phenalkylamines and methcatinone in human blood plasma by
LC-MS/MS with electrospray ionization, J. Mass Spectrom. 42 (2007) 150–160.
[15] S.W. Toennes, G.F. Kauert, Excretion and detection of cathinone, cathine, and
phenylpropanolamine in urine after khat chewing, Clin. Chem. 48 (2002) 1715–
1719.
[16] F. Sporkert, F. Pragst, R. Bachus, F. Masuhr, L. Harms, Determination of cathinone,
cathine and norephedrine in hair of Yeminite khat chewers, ForensicSci. Int. 133
(2003) 39–46.
[17] B.M. El-Haj, A.M. Al-Amri, M.H. Hassan, H.S. Ali, R.K. Bin Khadem, The use of
cyclohexanone as a ‘‘derivatizing’’ reagent for the GC-MS detection of amphe-
tamines and ephedrines in seizures and the urine, Forensic Sci. Int. 135 (2003)
16–26.
	Forensic analysis of hallucinogenic mushrooms and khat (Catha edulis Forsk) using cation-exchange liquid chromatography
	Introduction
	Experimental
	Chemicals and standards
	High-performance liquid chromatography
	Sample preparation
	Results
	Method performance
	Preparation of mushroom samples
	Alkaloid content in different mushroom products
	Preparation of khat samples
	Distribution of khat alkaloids in plant material
	Discussion
	Acknowledgements
	References