Forensic Science International, 45 (1990) 47-51 47

Elsevier Scientific Publishers Ireland Ltd.

RAPID TLC IDENTIFICATION TEST FOR KHAT (CATHA EDULIS’J

T. LEHMANN, S. GEISSHOSLER and R. BRENNEISEN*

Institute of Phamacy, University of Berne Kwitzerlund)

(Received April 12th, 1989)

(Revision received August 3rd. 19891
(Accepted August 7th, 19891

Summary

A rapid and sensitive method for identification of Catha edulis (khat) basing on a simple
extraction and TLC separation is described. The test is specific for the main khatamines cathi-
none and norpseudoephedrine.

Key words: Khat (Catha edulis); Cathinone; Norpseudoephedrine; Thin layer chromatography;
Identification

Introduction

Khat, the leaves or short tops of the evergreen plant Cutha edulis Forsk.
(Celastraceael, is well known in East Africa (Kenya, Ethiopia, Madagascar)
and the Arabian peninsula (North Yemen) for being chewed habitually by
many people. At present, khat has begun to be introduced to certain devel-
oped countries, e.g. Great Britain, were use of khat among East African and
Yemeni immigrants is substantial [l]. Shipments of khat - mostly by air
transportation - have been observed too by customs authorities in France
and the United States [1,2]. The widespread use and popularity is due to its
CNS-stimulating phenylpropylamines, especially to cathinone (a-aminopro-
piophenone, ‘natural amphetamine’) and to a much lesser extent to norpseudo-
ephedrine (‘cathine’l and norephedrine [l-6]. After the World Health
Organisation recommended that cathionine be placed under international
control, the amphetamine-like substance has been included in Schedule I of
the UN Convention on Psychotropic Substances. The recently found phenyl-
pentenylamines merucathinone, merucathine and pseudomerucathine [3,7 - 91
play only a minor role concerning the psychoactive effects of khat [lo].
In the guidelines for future investigations which were formulated by the
UN Expert Group on the Botany and Chemistry of Khat in 1979 [ll], an
‘urgent need for specific qualitative means of identification of khat material
and quantitative analysis of the important plant constituents’ was empha-
sized. Whereas a HPLC method for the quantitation of all known khatamines

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has already been proposed [3,12,13], to our knowledge no simple and reliable
test for identification of khat samples has been published until now. The
method of Rijhm and Schmid [14] is based on a very time consuming
extraction (6-15 hl and is therefore not suited for a rapid identification.
Furthermore the proposed thin layer chromatography (TLC) system results
in inefficient separation of complex khat extracts (e.g. MeOH extracts). A
macroscopic, microscopic and histochemical examination of khat leaves does
not show enough characteristics and is inappropriate for forensic purposes.
Nordal and Laane [15] suggested a combination of morphological, histochemi-
cal and chromatographic (TLC and GC) methods. Therefore, our aim was to
establish a specific and sensitive test for khat based on a rapid, simple and
inexpensive TLC method. With minimum laboratory equipment it can be
used for forensic and law enforcement applications. The proposed TLC
method was developed and tested by repeated analysis of numerous Catha
samples of different type, origin and age which have been screened before
for khatamines by HPLC and GC profiling [8,12,13,19].

Material and Methods

Most of the khat samples (Catha edulis) were bought between 1982 and
1986 at the khat markets of Addis Ababa and Awedai/Harrar (Ethiopia), Nai-
robi and Mombasa (Kenya), Sanaa (North Yemen) and Anivorano
(Madagascar). The specimens were deep-frozen within 24 h, transported by
air in a cooler to the laboratory and stored at - 20°C until used. Dried khat
samples originating from South Africa and Catha transvaalensis were
obtained through the Lowveld Botanic Garden, Transvaal and the Johannes-
burg Botanic Garden, Johannesburg (South Africa). Catha spinosa and
Ephedra dystachia were cultivated at the Botanical Garden of the University
of Berne. Duplicates of all analyzed specimens are being kept in the herbar-
ium of the Institute of Pharmacy, University of Berne.
One leaf (medium size, corresponding to about 500 mg fresh or 150 mg
dried plant material1 of a khat sample and 2.5 ml MeOH were ground thor-
oughly in a mortar. The suspension was then transferred to a 2.5-ml vial
with cap and sonicated for 10 min. After passing through a 0.2~pm cellulose
filter (or through the tip of a Pasteur pipette filled with cotton wool) the
clear and greenish solution was concentrated to about 100 ~1 under a stream
of nitrogen and again filtered. If no ultrasonic bath is available the following
alternative extraction procedure is recommended: one leaf of a khat sample
and 5 ml MeOH were ground properly in a mortar. The suspension was then
transferred to a test tube with stopper and shaken vigorously for 10 min.
After filtration through a 5-cm paper filter the solution was concentrated to
about 250 ~1 on a water bath (70°C) and again filtered through the tip of a
Pasteur pipette filled with cotton wool.
Five microlitres of the filtrate were applied bandwise (5-mm line) to a
TLC plate (Macherey-Nagel Alugram Sil G/UV,,, 0.25 mm, 40 x 80 mm or an
 49

equivalent product) and developed in EtOAc-MeOH-NH, 25% 85 : 10 : 5

[15,16]. A methanolic solution of about 1 mglml of cathinone hydrochloride
(synthesized according to [6]) and norpseudoephedrine or norephedrine
hydrochloride (Sigma Chemical Comp., St. Louis, U.S.A.) was used as stand-
ard. Prior to visualization the plate must be dried at room temperature or,
more quickly, by short use of a hot air blower. The plate was observed
under visible and UV light (254 nm), sprayed with fresh ninhydrin reagent
(0.3 g in 100 ml n-butanol + 3 ml cone. acetic acid) and heated at llO°C for 2
min.

Results and Discussion

An extraction without partition and with MeOH only as solvent was cho-
sen to get complex TLC patterns showing the khat sample zones of the typi-
cal khatamines and other constituents (Figs. la-c). Under visible light (Fig.

m 53D(tiD~
mm0 -
0.0 _-
3 2 3 4 5 6 7 8 9 10

Rr

1 2 3 4 5 6 7 8 9 10

Fig. 1. Thin-layer chromatograms of Cutha edulis (khatl and the main khatamines in comparison
with other Cutha species and Ephedra (al VIS detection; (bl UV,,, detection; (cl Ninhydrin detec-
tion. (11 Catha edulis “Kenya”; (21 Catha edulis “Ethiopia”: (31 Catha edulis “North Yemen”; (4)
Catha edulis “South Africa”; (51 Catha edulis “Madagascar”; (61 Catha transvaalensis; (71 Catha
spinosa; 631 Ephedra dystachia; (91 Cathinone ( = Cl; (101 Norephedrine/norpseudoephedrine (=
Nl. ? ?, green; ? ?, yellow; I , light-yellow; H , reddish; H , grey-yellow; I , grey-green;
violet; I , brown-green; ? ?, white: ? ?, grey; ? ?, dark-blue.
50

la1 khat samples show a brown-green zone of polar products (flavonoids) on

the starting line and at R, 0.06 a grey zone, at Rf 0.62 a light-yellow and at
0.72 a yellow zone, probably artifacts (decomposition products of the un-
stable cathinonel can be observed. At Rf 0.82 a green zone locates the colour-
ing principle of the plant (chlorophyll).
Under UV,,, cathinone is visualized as a dark-blue zone at Rf 0.43 (Fig. lb)
and in higher concentrations shows a violet zone after spraying with ninhy-
drin reagent (Fig. 1~1. Norpseudoephedrine and norephedrine are not or only
weakly visible under UVZs4, due to a hundred times lower E-value than
cathinone, but can be visualized with ninhydrin as pink zone at R, 0.25-0.3
(not separated). The minimum detectable amount of cathinone and norpseu-
doephedrine is about 250 ng (VU,,,) and 3 pg (ninhydrinl, respectively. If 5 ~1
of the filtered extracts are used for TLC, this corresponds to a relative
detection limit of 0.5 pg cathinone and 6 pg norpseudoephedrinelnorephed-
rine/lOO mg dried khat leaves. The sensitivity of the test is therefore suffi-
cient to identify even one leaf of a khat sample with low phenylalkylamine
content, e.g. khat from Yemen and Madagascar [13]. The phenylpentenyl-
amines can not be separated from the corresponding phenylpropylamines
and are always below the detection limit.
As cathinone has only been found in Catha edulis a positive identification
of this compound is a specific indication for khat. Nevertheless, positive
results should be confirmed by an alternative technique, such as high-perfor-
mance liquid chromatography (HPLC), gas chromatography (GC) or gas chro-
matography/mass spectrometry (GC/MS) [3,8,12,13,16].
Catha spinosa (endemic in North Yemen [17]1 and Catha transvaalensis
(endemic in South Africa [18]) have a morphology similar to Catha edulis but
produce different TLC patterns (Figs. la-cl. A phytochemical study of these
relatively unknown Catha species is in progress [19].
The total content of khatamines and especially the concentration ratios of
cathinone, norpseudoephedrine and norephedrine are dependent on the khat
type, origin, time of harvesting, drying method, age and storage conditions
[3,6,8,12,13]. Dried and old khat samples usually contain only very small but
still TLC detectable amounts of cathinone due to enzymatic reduction of
cathinone to norpseudoephedrine and norephedrine [3,6,12]. In such plant
material norpseudoephedrine is the dominating khatamine [12,13]. But in any
case only a quantitative analysis of major and minor khatamines, for exam-
ple by HPLC profiling [12,13], provides more information about the “history”
and the psychotropic potency of a given khat sample. Other plants with
norpseudoephedrine as constituent, such as Ephedra species, are
morphologically different from khat and give not the same pattern (Figs.
la- lcl.
Rapid but unspecific colour tests (Marquis’, modified Marquis’, Froehde’s,
Simon’s, Chen-Kao’s or Mandelin’s reagent [20-22311 for the presumptive
identification of phenylalkylamines do not give positive results with
cathinone and norpseudoephedrine. Therefore the commercially available
 51

Merck colour test for amphetamine (part of the Drug Identification Kit no.
11850) or the UN colour test for amphetamines (part of the UN Drug
Identification Kit), both use the modified Marquis’ reagent, are not suitable
for testing khat samples.

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