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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/$15.000 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 0379-0738(95)01855-D
40
L. Ripani et al. 1 Forensic Science International
78 (1996) 39-46
maximum amount of cathin’e allowed for individual use (this concept is called ‘medium daily dose’) was established in March 7 1990, and corresponds to 0.06 g. Above this amount its possession is considered illegal (Fig. 1). 2. The chemistry of Khat
Khat consumers’ preference for fresh drug material is due to the presence of cathinone that is considered the main CNS-active component of the Khat leaves [2,3]. This compound was isolated and identified in 1975 by a group of researchers of the United Nat’lons Narcotics Laboratory working on a project initiated in the early seventies. Cathinone, being a ketoamine base, is extremely unstable and, in particular, it can be transformed into ( + )-norpseudoephedrine and ( - )-norephedrine by an enzymatic reduction (Fig. 2). It can also be oxidized to give 1-phenyl-1,2-propandione, while the cathinone dimers, such as 3,6-dimethyl-2,5-diphenylpyrazine are purely artifacts of the isolation [4]. In addition to the known phenylpropylamines cathinone, norephedrine and norpseudoephedrine (cathine), the presence of other amines such as merucathine, pseudomerucathine and merucathinone have been identified [5,6]. Pharmacological studies are still being conducted on these-compounds, but it is presently believed that the stimulating effect of the plant is principally due to cathine and cathinone.
Fig. 1. Typical
street drug presentation
(Khat).
L. Ripani et al. / Forensic Science International 78 (1996) 39-46
0\
- @Q 0\ CH-CH-CH,
YH0
CH-CH-CH,
’
41
AH
i;H 2
b$
<-)-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.
42
L. Ripani et al. / Forensic Science’International
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.
L. Ripani et al. 1 Forensic Science International
‘44
43
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
44
L. Ripani et al, / Forensic Science International
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
L. Ripani et al. / Forensic Science International
78 (1996) 39-46
45
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
46
L. Ripani et al. 1 Forensic Science international
18 (1996) 39-46
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.
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/$15.000 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 0379-0738(95)01855-D
40
L. Ripani et al. 1 Forensic Science International
78 (1996) 39-46
maximum amount of cathin’e allowed for individual use (this concept is called ‘medium daily dose’) was established in March 7 1990, and corresponds to 0.06 g. Above this amount its possession is considered illegal (Fig. 1). 2. The chemistry of Khat
Khat consumers’ preference for fresh drug material is due to the presence of cathinone that is considered the main CNS-active component of the Khat leaves [2,3]. This compound was isolated and identified in 1975 by a group of researchers of the United Nat’lons Narcotics Laboratory working on a project initiated in the early seventies. Cathinone, being a ketoamine base, is extremely unstable and, in particular, it can be transformed into ( + )-norpseudoephedrine and ( - )-norephedrine by an enzymatic reduction (Fig. 2). It can also be oxidized to give 1-phenyl-1,2-propandione, while the cathinone dimers, such as 3,6-dimethyl-2,5-diphenylpyrazine are purely artifacts of the isolation [4]. In addition to the known phenylpropylamines cathinone, norephedrine and norpseudoephedrine (cathine), the presence of other amines such as merucathine, pseudomerucathine and merucathinone have been identified [5,6]. Pharmacological studies are still being conducted on these-compounds, but it is presently believed that the stimulating effect of the plant is principally due to cathine and cathinone.
Fig. 1. Typical
street drug presentation
(Khat).
L. Ripani et al. / Forensic Science International 78 (1996) 39-46
0\
- @Q 0\ CH-CH-CH,
YH0
CH-CH-CH,
’
41
AH
i;H 2
b$
<-)-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.
42
L. Ripani et al. / Forensic Science’International
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.
L. Ripani et al. 1 Forensic Science International
‘44
43
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
44
L. Ripani et al, / Forensic Science International
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
L. Ripani et al. / Forensic Science International
78 (1996) 39-46
45
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
46
L. Ripani et al. 1 Forensic Science international
18 (1996) 39-46
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.