MS identification of Catha edulis stimulant-active principles

Forensic Science Intern3ii

Forensic Science International

ELSEVIER

78 (1996) 39-46

GC/MS identification of Catha edulis stimulant-active principles L. Ripani, S. Schiavone, L. Garofano Chemistry and Biology Sections, Centro Carabinieri

Investigazioni

ScientiJiche, Roma, Italia

Received 24 January 1995; revision received 4 September 1995; accepted 7 September 1995

Abstract

The chemical composition of Khat (Cutha edulis Forsk. Celastraceae) was discovered in recent years by a study undertaken at the United Nations Narcotics Laboratory in Geneva. Considering two phenylpropylamines to be the main compounds responsible for the stimulant activity of the plant. we propose a rapid and efficient method for the extraction and determination of cathinone. cathine and norephedrine. Kqwwrl.s:

Crrtlrtr ddis;

GC/MS

analysis; Cathinone;

Cathine; Norephedrine;

Extraction

1. Introduction Catha edulis is a plant belonging to the Celastraceae family: it is a slender, straight short tree reaching a height of 4 m, with persistant oval, usually opposite finely toothed leathery leaves. This tree has grown naturally in East Africa for century and its leaves and buds are either chewed or used to make an infusion in much the same way as with tea [l]. Alkaloids having effects similar to those produced by amphetamine are thus introduced into the body. The phenylpropylamines responsible for the CNS-stimulating effects are cathine, i.e ( + )-norpseudoephedrine and cathinone, i.e. ( - )-aamino-propiophenone. In Italy, as of January 10, 1988, such alkaloids have been considered illegal drugs, but it must be mentioned that only cathine is reported in Table 1 of the n. 162/1990 Law which principally governs the control of drugs of abuse. The 0379-0738/96/$15.000 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 0379-0738(95)01855-D

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maximum amount of cathin’e allowed for individual use (this concept is called ‘medium daily dose’) was established in March 7 1990, and corresponds to 0.06 g. Above this amount its possession is considered illegal (Fig. 1). 2. The chemistry of Khat

Khat consumers’ preference for fresh drug material is due to the presence of cathinone that is considered the main CNS-active component of the Khat leaves [2,3]. This compound was isolated and identified in 1975 by a group of researchers of the United Nat’lons Narcotics Laboratory working on a project initiated in the early seventies. Cathinone, being a ketoamine base, is extremely unstable and, in particular, it can be transformed into ( + )-norpseudoephedrine and ( - )-norephedrine by an enzymatic reduction (Fig. 2). It can also be oxidized to give 1-phenyl-1,2-propandione, while the cathinone dimers, such as 3,6-dimethyl-2,5-diphenylpyrazine are purely artifacts of the isolation [4]. In addition to the known phenylpropylamines cathinone, norephedrine and norpseudoephedrine (cathine), the presence of other amines such as merucathine, pseudomerucathine and merucathinone have been identified [5,6]. Pharmacological studies are still being conducted on these-compounds, but it is presently believed that the stimulating effect of the plant is principally due to cathine and cathinone.

Fig. 1. Typical

street drug presentation

(Khat).

L. Ripani et al. / Forensic Science International 78 (1996) 39-46

0\

- @Q 0\ CH-CH-CH,

YH0

CH-CH-CH,

’

41

AH

i;H 2

b$

<-)-NUREPHEDRINE

(+,-NClRPSEUDaEPHEDRINE

c0x1

I

(-FCATHINONE

l-PHENYL-1,2-PRBPANDIONE

k

U OH II I

DIMERIZATIIIN

C-CH-CH,

BENZDYL ETHANOL

3,6-DIMETHYL-2,5-DIPHENYLPYRAZINE

Fig. 2. Decomposition products of ( - )-cathinone.

Accordingly, our effort focused on obtaining a rapid and efficient method for the extraction and determination of the above mentioned two active principles. In particular, we propose a capillary GC/MS method to be carried out without derivatisation.

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3. Experimental

78 (1996) 39-46

procedures

3.1. Materials

All the chemicals were RPE grade from Carlo Erba Reagenti and Lychrosolv grade from Merck. 3.2. Extraction

of Khat samples

To 5 g of finely chopped fresh leaves and stalks were added 100 ml of 0.1 N HCl and crushed in a mortar. After filtering, the acid solution was transferred to a separation funnel, it was made alkaline with 1 N sodium hydroxide and then extracted with three 50 ml portions of trichloromethane. The organic extracts were collected, dehydrated, filtered and evaporated to dryness using a nitrogen vacuum rotary evaporator. The residue was dissolved with 1 ml of trichloromethane and injected into the GC/MS instrument. 3.3. Capillary gas chromatography/mass

spectrometry

The analysis was performed with a Hewlett Packard Model 5890 Series II Gas Chromatograph interfaced with a Hewlett Packard Model 5889A Mass Spectrometer. The separation column was a 30 m long Hewlett Packard HP-5 M.S. (crosslinked 5%-phenylmethylsilicone), having an inner diameter of 0.25 mm and a stationary phase film thickness of 0.25 pm, directly interfacing the mass spectome-

10000090000

-

rloooo70000-

GO000 -

I

50000 400003oooG -

20000-

10000( I 10

I

I,,

15

I

I Time (min.)

Fig. 3. Total ion chromatogram

of Khat sample. Peaks: 1 = cathinone; 2 = cdthine; 3 = norephedrine.

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78 (1996) 39-46

A

CATHINONE

77 ,

51 , 63 ,

I I

I GO

11

105

I,

’

I 80

f

91 I “I’

100

25000 -

CATHINE 20000-

15000-

lOlmO-

5000 -

77

51 0 ’

,-I 40

1

I II’S

/

,

, GO

”

63 I

;

,

I

, 80

I

‘I’

91 ,

105 I

, 100

I,,L2’ 1

134 132,, r _ r----rL-;-.1120

150 T--&--I14K

h4asslCl1arge

Fig. 4. (a) Mass spectrum norephedrine.

of cathinone.

(b) Mass spectrum

of cathine.

(c) Mass spectrum

of

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78 (1996) 39-46

Abundance

GOOO-

5000-

4000-

WOO-

zooo-

lOOO-

108 51 I

;

63

91

117

132135

147

Fig. 4(c).

ter. The gas chromatograph worked with a split/splitless injector. Helium was used as the carrier gas with linear velocity u at 70°C of 35 cm/s for a flow rate of 0.87 ml/min and a split ratio of 5O:l. The oven was operated with the following temperature plan: 40°C as initial temperature; 25”C/min up to 95°C for 18 min and then 20”C/min up to 270°C for 10 min. The temperature of the transfer line GC/MS was 280°C while the source and the quadrupole temperatures of mass spectrometer were, respectively, 200°C and 100°C. 4. Results and discussion

The GC/MS analysis allowed us to separate and identify the alkaloids, cathinone, cdthine, and norephedrine, without derivatization (Figs. 3 and 4). In fact, it must be stated that the separation of Khatamines had required derivatization with trifluoroacetic anhydride and the coupled cathine/norephedrine had never been sufficiently separated using a GC technique [4]. In particular, the mass spectra of cathine and norephedrine can be differentiated from that of cathinone by the presence of the peak corresponding to m/e 79 that is indicative of an alkyl benzilic alcohol and that come from a double transposition of hydrogen in such compounds [7]. This peak is not present in the mass spectrum of cathinone in which we can find

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CATHINONE

&

NH

2

m/e 149

m/e 105

m/e 79

I

H3C - CH=tiH,

m/e 44

I

m/e 77

m/e 91

base peak

m””

j

CATHINE

z

&

cH3

2

+NH

mle 151

m/e 152

o- y=IJ 2

m/e 117

i

+NH

3

J CEO

2

m/e 118

Fig. 5. Fragmentation

0

0

+

m/e 107

-

m/e 105

,--~ :~ +; 0._I tie 77

of cathinone and cathine.

only the m/e 77 peak. The same pattern can be observed by comparing the mass spectra of 1-phenyl-ethanol and acetophenone: in the first compound the peaks corresponding to m/e 79 and 77 are present with an abundance ratio of 1.8: 1, while in the second one we can find only the peak corresponding to m/e 77, Other important information that identifies cathine can be obtained by the abundance of

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the m/e 107 peak with respect to the m/e 105 peak and by the presence of the m/e 117 and 118 peaks that are not formed in the fragmentation of cathinone (Fig. 5).

References [I] X. Schorno, Khat: Epidemic drugs from Islam. Pharm. uns Zeit., 11 (1982) 65. 121 P. Kalix, The pharmacology of Khat. Gen. Pharmacol., 15 (1984) 179. [3] P. Kalix, Khat: an amphetamine-like stimulant. J. Psychoactive Drugs, 26 (1994) 69. [4] R. Brenneisen, S. Geisshusler, Psychotropic Drugs. Analytical and Chemical Aspects of Cutha edulis Forsk. Phurm. Acta Helv., 60 (1985) 11. [5] R. Brenneisen, S. Geisshusler, X. Schorno, Merucathine: A new phenyl alkylamine Catha Ed&s. Plunta Med., 50 (1984) 531.’ [6] K. Szendrei, The chemistry of Khat. Bull. Narc., 32 (1980) 5. [7] F. McLafferty, Inferpretation of Muss Spectra. (Italian Edition), 1972, pp. 145, 192.