PHYTOCHEMICAL ANALYSIS

Phytochem. Anal. 13, 162169 (2002)

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/pca.637

Focused Microwave-assisted Extraction of

Cocaine and Benzoylecgonine from Coca Leaves
A. Brachet, P. Christen* and J.-L. Veuthey
Laboratory of Pharmaceutical Analytical Chemistry, University of Geneva, Bd. dYvoy 20, 1211 Geneva 4, Switzerland

Extraction of cocaine and benzoylecgonine from coca leaves was performed by focused microwave-assisted
extraction (FMAE). Cocaine extraction was optimised with respect to the nature of the extracting solvent,
the particle size distribution, the moisture of the sample, the applied microwave power and the radiation
time. A central composite design was used to optimise the two latter parameters and to assess the robustness
of the extraction method around the best conditions. FMAE generated extracts similar to those obtained by
conventional solidliquid extraction but in a more efficient manner, i.e. 30 s were sufficient to extract
cocaine quantitatively from leaves. Analyses of cocaine and benzoylecgonine in coca leaves was carried out
by capillary GC-FID and GC-MS for peak identification, as well as by capillary electrophoresis with UV
detection. Copyright ! 2002 John Wiley & Sons, Ltd.
Keywords: focused microwave-assisted extraction; cocaine; benzoylecgonine; experimental design; coca leaves;
Erythroxylum coca.

INTRODUCTION
Microwave-assisted extraction (MAE) has received
increasing attention as a potential alternative to solid
liquid extractions (SLE) for the extraction of secondary
metabolites from plants. MAE depends on the dielectric
susceptibility of both solvent and matrix. Microwave
energy causes molecular motion by migration of ions and
rotation of dipoles, therefore, microwave heating depends
on the presence of polar molecules or ionic species
(Zlotorzynski, 1995; Letellier and Budzinski, 1999a).
MAE offers a rapid delivery of energy to the total volume
of solvent and subsequent rapid heating. Moreover, when
microwave radiation can be focused directly onto the
sample, heating is more efficient and thus homogeneity
and reproducibility improve greatly. Microwave ovens
can be operated either under pressure or at atmospheric
pressure.
Many reviews have been published on the application
of MAE for the extraction of organic compounds
(pesticides and organometallic compounds) from environmental matrices (Pare et al., 1994; Majors, 1995, 1999;
Zlotorzynski, 1995; Vandenburg et al., 1997; Lamble and
Hill, 1998; Letellier and Budzinski, 1999a; Camel, 2000),
but very few applications have been published in the
phytochemical and pharmaceutical areas (for a review
see Kaufmann and Christen, 2002). Ganzler et al., (1986)
presented the first use of microwave energy for the
extraction of compounds from soil, biological and plant
samples, and since then various procedures have been
described for the extraction of sparteine from seeds and
rat faeces (Ganzler et al., 1990), pyrrolizidine alkaloids
* Correspondence to: P. Christen, Laboratory of Pharmaceutical Analytical
Chemistry, University of Geneva, Bd. dYvoy 20, 1211 Geneva 4,
Switzerland.
Email: philippe.christen@pharm.unige.ch

Copyright ! 2002 John Wiley & Sons, Ltd.

from Senecio species (Bicchi et al., 1992), essential oils

from rosemary, peppermint leaves, sea parsley and garlic
(Pare et al., 1991; Chen and Spiro, 1994), fungal
metabolites, ergosterol and fatty acids from natural
products (Young, 1995), and drugs from biological fluids
(Meier et al., 1983; Torloni et al., 1993; Franke et al.,
1996).
Most of the applications described so far have
employed closed extraction vessels under pressure, and
few studies have involved the use of focused microwave
ovens at atmospheric pressure. The emergence of this
new commercial microwave system, which is especially
designed for extractions, is rather recent, and the few
studies that have appeared have mainly concerned
environmental matrices (Budzinski et al., 1996; Letellier
et al., 1996; Letellier and Budzinski, 1999a, b).
Erythroxylum coca var. coca is one of the four coca
varieties which contains a significant amount of cocaine
(0.50.8% dry weight) in the leaves (Turner et al., 1979;
Plowman and Rivier, 1983). Among the other tropane
alkaloids present in coca leaves, benzoylecgonine is of
particular interest as it is not only an endogenous coca
alkaloid but it is also a by-product of the hydrolysis of
cocaine arising during storage or extraction. Very little
information is available about the presence of benzoylecgonine in coca leaves (De Jong, 1940; Espinel and
Guzman, 1971; Jenkins et al., 1996) compared to the
large documentation about its determination as the major
metabolite of cocaine in urine (Fernandez et al., 1996;
Phillips et al., 1996; Hall et al., 1999; Jeanville et al.,
2000).
Cocaine from coca leaf is usually extracted by SLE
such as Soxhlet or reflux processes (Turner et al., 1979;
Plowman and Rivier, 1983; Moore and Casale, 1994;
Moore et al., 1994). However, these traditional methods
require a large quantity of solvent and are time
consuming. Recent investigations with modern extraction
techniques such as supercritical fluid extraction (SFE;
Received 1 May 2001
Revised 30 July 2001
Accepted 30 July 2001

MICROWAVE-ASSISTED EXTRACTION OF COCA LEAVES

Brachet et al., 2000) and accelerated solvent extraction

(ASE; Brachet et al., 2001) have been undertaken in
order to develop a more rapid, simple and selective
extraction method with lower solvent consumption. In the
present paper we report on the feasibility of using focused
microwave-assisted extraction (FMAE) for the quantitative extraction of cocaine and benzoylecgonine from coca
leaves. Various parameters affecting the efficiency of
extraction of cocaine have been investigated, and a
central composite design has been used to optimise
microwave power and radiation time and to evaluate the
robustness of the method by drawing response surfaces
(Mulholland, 1988; Filbey and Altria, 1994; Fabre,
1996).

EXPERIMENTAL
Materials. Leaves of Erythroxylum coca var. coca were
harvested in Tingo Maria (Peru), authenticated by T.
Plowman and kindly provided by L. Rivier from the
Forensic Institute of Lausanne (Switzerland). Coca
leaves were ground to a fine and homogeneous powder
using a Retsch (Haan, Germany) model MM 200 ballmill; ground material was sieved to yield average particle
sizes in the ranges 90150, 170220 and 220470 !m.
Cocaine hydrochloride and methadone hydrochloride
were obtained from Siegfried Handel (Zofingen, Switzerland) and Hanseler (Herisau, Switzerland), respectively.
Benzoylecgonine tetrahydrate and amphetamine sulphate
were purchased from Fluka (Buchs, Switzerland) and
from Sigma (St. Louis, MO, USA), respectively.
Analytical grade solvents (methanol, ethanol, hexane
and toluene) were purchased from Romil (Kolliken,
Switzerland), whilst ultra pure water was provided by a
Milli-Q RG unit (Millipore; Bedford, MA, USA). All
other chemicals, such as sodium bicarbonate, triethylamine, 85% phosphoric acid and Tris were obtained from
Fluka.
Focused microwave-assisted extraction at atmospheric pressure. FMAE was performed at atmospheric
pressure at a frequency of 2450 MHz using a Soxwave
(Prolabo, France) model 3.6 apparatus with programmable heating power from 25 to 250 W. Powdered airdried coca leaves (100 mg) were placed into a 250 mL or
a home-made 20 mL quartz extraction vessel with 30 or
5 mL of solvent, respectively. For some experiments,
water or an aqueous solution saturated with sodium
bicarbonate was added prior to the addition of solvent.
Multiple extractions (three samples simultaneously) were
performed under conditions set by the factorial design.
Samples were automatically stirred during the extraction
process. The potential loss of volatile compounds was
controlled by condensation of the vapours in a condenser
situated above the sample flask. After extraction, the
vessel was allowed to cool to room temperature: extracts
were centrifuged and the supernatant removed and
evaporated to dryness under vacuum at 40C. Residues
were dissolved in 2 mL of a solution of methadone
(45 !g/mL; GC internal standard) in methanol, and the
mixture was centrifuged at 1700g for 5 min.
Analytical methods. Extracts were analysed without
further purification by GC-FID and the identification of
Copyright ! 2002 John Wiley & Sons, Ltd.

163

the cocaine peak was confirmed by GC-MS as described

elsewhere (Brachet et al., 2000). Capillary electrophoresis with UV detection (CE-UV) was used to determine
the benzoylecgonine as well as the cocaine content in
basified extracts according to the method of Brachet et al.
(2001). An aliquot (200 !L) of coca extract in methanolic
solution (containing the GC internal standard) was
evaporated to dryness under a gentle stream of nitrogen
and the residue was dissolved in 200 !L of Tris
phosphate buffer:water (10:90, v/v) containing 36.7 !g/
mL of amphetamine as the CE internal standard. A shortend injection procedure in the CE-UV allowed a rapid
quantitative determination of cocaine and benzoylecgonine. Coefficients for regression models were calculated
using the NEMROD (LPRAI, Marseille, France) program; the response surface was drawn using Microsoft
Excel (version 7.0).

RESULTS AND DISCUSSION

In a first set of experiments, the influence of the
extracting solvent, of sample granularity, of filtration of
the sample, and of sample moisture with aqueous solution
or with the addition of an alkaline substance was
investigated by a univariate method. These preliminary
experiments were performed with a microwave power of
75 W for 20 min using a particle size distribution
between 90 and 150 !m (except where stated otherwise).
In further assays, the applied microwave power and the
radiation time themselves were optimised using a central
composite design in which the measured response was
the yield of cocaine extracted. The analysis of crude
extracts was carried out either by GC-FID or CE-UV.
Effect of extracting solvent
Solvent choice is dictated by the solubility of the analytes
of interest, by the interaction between solvent and matrix,
and finally by the microwave absorbing properties of the
solvent determined by its dielectric constant (Jassie et al.,
1997). The solvents studied were those used for the
conventional extraction of cocainea methanol:water
(71:29, v/v) mixture, used as a polar modifier in SFE
(Brachet et al., 2000), and hexane were included in order
to investigate a larger range of dielectric constants. As
shown in Fig. 1, non-polar solvents with low dielectric
constants (hexane and toluene) did not allow the
quantitative extraction of cocaine. Solvents such as
ethanol, methanol and water were sufficiently polar to
heat in the presence of microwaves. Methanol gave the
best recovery of cocaine, while ethanol presented the
lowest. No extraction selectivity was observed with pure
methanol, ethanol, water or with the methanol:water
mixture (data not shown). Even though water has the
highest dielectric constant, extraction using pure water or
in admixture with methanol did not yield the highest
amounts of cocaine: either water did not sufficiently
solubilise cocaine (which may be mainly stored as a salt
in plant cells; Bruneton, 1993; Ferreira et al., 1998) or
degradation of cocaine occurred under these particular
conditions. Pure methanol was selected as the extraction
solvent for further investigations because of its good
heating capacity under microwaves and its ability to
solubilise cocaine.
Phytochem. Anal. 13: 162169 (2002)

164

A. BRACHET ET AL.

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Effect of sample granulometry

Investigations were carried out to study the effect of
sample grinding. Figure 2 shows that the particle size
distribution had a strong influence on the efficiency of
extraction of cocaine, with enhanced extraction being
obtained with samples of smaller particle size. Finely
ground coca powder was more easily extracted because
of the larger surface area available to provide contact
between the sample and the solvent. A particle size
distribution between 90 and 150 !m was therefore chosen
for all further investigations.
Effect of on-line filtration of sample
After MAE, a filtering step is required to separate the
filtrate from the solid material. The apparatus used in this
study offered the possibility of filtering the sample
directly by means of a cartridge (which could be removed
above the microwave field after the extraction step).
However, it was demonstrated that the cartridge strongly
adsorbed cocaine (Fig. 3), and methanol extraction of the
cartridge was necessary to recover the adsorbed cocaine.

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It is therefore recommended to avoid using a cartridge

and to carry out the extraction directly in the vessel:
centrifugation should replace filtration.
Effect of sample moisture
As MAE depends on the dielectric susceptibility of both
solvent and matrix, better recoveries can be obtained by
moistening samples with a substance that possesses a
relatively high dielectric constant (compared to the
extracting medium). Water is usually used since it is a
good absorber of microwave energy. Microwaves interact selectively with free water molecules and cause
localised heating; water molecules transfer some of their
thermal energy to the analyte molecules which gives rise
to an elevation of temperature and significantly contributes to an enhanced analyte extraction (Letellier and
Budzinski, 1999b).
The effect of moistening the sample with pure water
was investigated and, at the same time, the effect of
basification was tested. Two ways of basifying the
medium were studied: dry leaves were either directly
basified inside the vessel or extracted with an alkaline
solvent. The first alkaline solution tested was an aqueous
saturated solution of sodium bicarbonate which is
generally used in SLE of cocaine (Moore and Casale,
1994; Moore et al., 1994). The second investigated
alkaline substance was triethylamine, since it is frequently added as a polar modifier in SFE to extract
cocaine quantitatively from hair (Edder et al., 1992;
Morrison et al., 1998). Before performing microwave
extraction, dry coca leaves were moisturised with 100 !L
of water or 100 !L of an aqueous saturated solution of
sodium bicarbonate and then extracted in methanol.
Addition of 100 !L of water to dry coca leaves and use of
methanol:triethylamine (85:15, v/v) mixture as extracting
solvent were also carried out. Figure 4 shows that the
impregnation of leaf material with water, followed by
FMAE in methanol, slightly increased the recovery of
Phytochem. Anal. 13: 162169 (2002)

MICROWAVE-ASSISTED EXTRACTION OF COCA LEAVES

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cocaine. On the other hand, the presence of an alkaline

solution gave a lower cocaine recovery than with pure
methanol. From these experiments, it may be concluded
that cocaine extraction under basic conditions increases
degradation of cocaine which is hydrolysed to benzoylecgonine, which is itself hydrolysed to ecgonine (Bastos
and Hoffman, 1976; Jatlow et al., 1980). Since the latter
compound has no characteristic adsorption in the UV it
cannot be detected by CE-UV. Moreover, Fig. 4 shows
that presence of triethylamine considerably reduced the
degradation of benzoylecgonine to ecgonine whilst still
permitting the hydrolysis of cocaine to benzoylecgonine.
The stability of benzoylecgonine may be due to the
amphoteric nature of the benzoylecgonine molecule
(Morrison et al., 1998).
Although methanol was demonstrated to be the best
extracting solvent, the effect of moisture with water or
aqueous basic solution was also investigated in a solvent
such as hexane which is not affected by microwave
energy. In this case, the solvent is used as a coolant and
for its ability to solubilise cocaine. Temperature elevation
results only from passive thermal energy diffusion from
the matrix to the surrounding solvent (Pare and Belanger,
1997). In a leaf/hexane mixture the leaves are heated by
the microwaves, while in a leaf/methanol mixture the
methanol and the leaves absorb the bulk of the
microwave energy. Figure 5 shows the effect of sample
moisture on cocaine recovery in hexane. Pure hexane
gave a very low cocaine recovery since air-dried coca
leaf material did not contain enough water to absorb
microwaves. When a small quantity of water was added
directly in hexane, a slight increase in cocaine recovery
was observed; the added water superheated and caused
the cell membranes to rupture allowing cocaine to
dissolve in the non-absorbing cooler solvent. However,
cocaine may be stored in leaves mainly in a salt form
(Bruneton, 1993; Ferreira et al., 1998) and only base
cocaine could be dissolved in hexane. It was thus
necessary to add an aqueous solution saturated with
sodium bicarbonate to allow a maximum of cocaine to be
solubilised in hexane. Under these conditions cocaine
recovery increased four times but it still remained lower
than was obtained with pure methanol. However, the
Copyright ! 2002 John Wiley & Sons, Ltd.

165

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addition of an aqueous solution saturated with sodium

bicarbonate in hexane produced a more selective
extraction of cocaine and other coca alkaloids, as shown
in Fig. 6. Far fewer methyl esters of fatty acids were
present following basified hexane extraction compared
with basified methanol extraction. It can also be noted
that methylecgonine and methylecgonidine were present
in lower quantities with the former extraction. Norcocaine was only observed in the basified hexane extract
while benzoylecgonine was completely absent (as
determined by CE-UV analysis).
Effect of microwave power and radiation time
In order to investigate the effects of applied microwave
power and radiation time (or extraction time), a central
composite design was implemented to determine optimal
conditions and to evaluate the robustness of the method by
drawing response surfaces. Sample volume was fixed at
5 mL methanol and granulometry was between 90 and
150 !m. Preliminary experiments were carried out in order
to select an appropriate experimental domain (data not
shown). However, the range of microwave power
corresponds to the minimum and maximum power
delivered by the system. The selected factors and their
corresponding ranges are reported in Table 1, and the eight
experiments that were carried out are shown in Table 2.
The replication of the centre point (n = 6; experiments 9
14 in Table 2) allowed an estimation to be made of the
experimental error. All other experiments were randomly
performed without replication. The measured response
was defined as the cocaine content expressed as percentage dry weight of leaves and was determined by GC-FID.
Multiple regression gave a mathematical relationship
between independent variables and the response. The
coefficient of determination (r2 = 0.83) was acceptable,
and the repeatability gave a very satisfactory relative
standard deviation (RSD) of 0.6% (standard deviation = 0.004%; n = 6) considering that it represents the
total analysis (FMAE and GC-FID analysis).
A response surface could be drawn from the mathematical model and can be visualised as a three-dimensional
plot by representing the response as a function of the two
factors. Figure 7 shows that microwave operating parameters had little influence on cocaine recovery as no
Phytochem. Anal. 13: 162169 (2002)

166

A. BRACHET ET AL.

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curvature is observed. No interactive effects between

radiation time and the applied power were noticeable:
increasing either time or the applied power increased the
efficiency of recovery of cocaine very slightly. Thus, the
Table 1. The selected factors and their corresponding
factors
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extraction method around the central conditions (125 W

for 8 min) was perfectly robust. However, final conditions
were selected which would allow energy savings and
would permit the safest implementation of cocaine
extraction. Therefore, a 30 s extraction time was chosen
with a microwave power of 125 W. Under these conditions, the cocaine content of the leaf material was
determined as 0.680 ! 0.004% by GC-FID analysis
(0.63 ! 0.03% by CE-UV analysis) and the benzoylecgonine content was 0.10 ! 0.02% (by CE-UV analysis).
As the extraction time had been considerably reduced
(from an original 20 min to a final 30 s), and thus the
exposure time to alkali minimised, the effects of the
addition of different quantities of an aqueous saturated
Phytochem. Anal. 13: 162169 (2002)

MICROWAVE-ASSISTED EXTRACTION OF COCA LEAVES

167

Table 2. Two-factor central composite design employed to

optimise microwave power and extraction time in
the FMAE of cocaine from coca leaves; the
experiments carried out showing corresponding
responses
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solution of sodium bicarbonate were again investigated.

It was found that the higher the volume of the base added,
the lower were the recoveries of cocaine (Fig. 8).
However, cocaine degradation was still lower than that
observed following 20 min of radiation (Fig. 4) confirming that in an alkaline aqueous solution the exposure time
should be minimal. For benzoylecgonine, recoveries
increased up to a pH of 8.7, but decreased at pH 9.6 since
benzoylecgonine hydrolysed to ecgonine. Moreover,
when extraction was performed with 100 !L of sodium
bicarbonate, repeatability of the extraction was lower
than with pure methanol and RSDs were 7.8 and 9.3%
(n = 6, CE-UV analysis) for cocaine and benzoylecgonine, respectively. In view of these findings, the final

Copyright ! 2002 John Wiley & Sons, Ltd.

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conditions were established as: particle size of plant

material, 90150 !m; solvent/volume, 5 ml methanol;
radiation time, 30 s; microwave power, 125 W.
In conclusion, FMAE at atmospheric pressure appears
to be an interesting and promising alternative for the
extraction of cocaine from coca leaves in comparison
with conventional extraction techniques. Results showed
that the capacity of the solvents to absorb microwave
energy played a role in the efficiency of extraction.
Methanol was found to be the best extraction solvent
because of its good capacity to heat under microwaves
and its ability to solubilise cocaine. Moistening the leaf
material with water slightly improved cocaine recovery,
while addition of an alkaline agent significantly
decreased cocaine recovery, mainly owing to degradation
of cocaine in methanol during the extraction process
under alkaline conditions. If extraction was performed in
hexane after impregnating leaves with an alkaline
aqueous solution, extraction was demonstrated to be
very selective but not quantitative.
Applied microwave power and time of radiation were
optimised by means of a central composite design, in
pure methanol and with a particle size distribution in the
range 90150 !m. The response surface showed that
these two factors have no effect on cocaine recovery
around the central conditions, meaning that the extraction
method is perfectly robust. With a microwave power of
125 W, it was demonstrated that an extraction of only
30 s was sufficient to extract quantitatively cocaine from
leaves. Thus, cocaine extraction time was considerably
shortened compared to that required by classical extraction. Cocaine and benzoylecgonine content of leaf
material (as determined by CE-UV) were in good
agreement with the values reported in the literature.
Moreover, FMAE gave cocaine extraction recoveries
comparable to those obtained using more recent extraction techniques (such as SFE and ASE) in a reduced time
because of the process simplicity. However, FMAE
requires filtration to separate the raw extract from the
solid material.

Phytochem. Anal. 13: 162169 (2002)

168

A. BRACHET ET AL.

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Y-0+(0 7[ -#= U())<-# NY! KIB^! N&+&'+.(# -#= .=&#+.X'-@

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Phytochem. Anal. 13: 162169 (2002)

MICROWAVE-ASSISTED EXTRACTION OF COCA LEAVES

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169

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Phytochem. Anal. 13: 162169 (2002)