356 M. Adam et al. J. Sep. Sci.

2008, 31, 356 – 363

Martin Adam Original Paper

Petr Dobi
Ð
AleÐ Eisner
Karel Ventura
Department of Analytical
Chemistry, Faculty of Chemical
Technology, University of
Pardubice, Pardubice, Czech
Republic
Headspace single-drop microextraction of herbal
essential oils
A method employing the headspace single-drop microextraction (HS-SDME) is pre-
sented for the determination of essential oils in dried herbal leaves. By optimising
the key experimental parameters, a linear response for the individual target com-
pounds was obtained in the concentration range from LOQ to 4 mg/mL (r2 = 0.9912 –
0.9998), with LODs from 3.3 up to 20.5 lg per 100 g of dried leaves, and the repeat-
ability within the RSD of 2.1 – 8.9%. The HS-SDME-based procedure, enabling a rapid
and simple analysis of essential oils in herbs, was applied to selected real samples
(nine essential oils in four different samples) in combination with GC-FID identifica-
tion and quantification of the target volatiles.
Keywords: Essential oils / Herbs / Single-drop microextraction /
Received: August 8, 2007; revised: November 5, 2007; accepted: November 5, 2007
DOI 10.1002/jssc.200700377

1 Introduction
The evaluation of aroma-active compounds is important
in agricultural science and food chemistry. In agricul-
ture, the determination of essential oil compounds and
their composition is necessary for the quality control of
breeding parameters, cultivation and production of aro-
matic plants. A quality control of raw material, inter-
mediate and end products is furthermore required in the
food industry for the production of spices, essential oils
or flavoured foods [1]. Analysis of the essential oil from
the plant leaves is therefore desirable.
Essential oils are complex mixtures of volatile substan-
ces usually present at low concentrations [2] that are
used with great benefit in aromatherapy, both in con-
junction with conventional medicine and as an alterna-
tive therapy [3]. Until recently, essential oils have been
studied mostly from their flavour and fragrance view-
points only for flavouring foods, drinks and other goods.
At present, however, essential oils and their components
are gaining increasing interest because of their relatively
safe status, their wide acceptance by consumers and
their exploitation for potential multipurpose functional
use [4, 5]. Many authors have reported on antimicrobial,
antifungal, antioxidant and radical-scavenging proper-
Correspondence: Dr. Martin Adam, Department of Analytical
Chemistry, Faculty of Chemical Technology, University of Par-
dubice, Nam. Cs. legii 565, Pardubice, 532 10, Czech Republic
E-mail: martin.adam@upce.cz
Fax: +420-466-037-068
Abbreviations: HS-SDME, headspace single-drop microextrac-
tion; SDME, single-drop microextraction
ties [6, 7] of the spices and essential oils and, in some
cases, a direct food-related application has been tested
[8]. Moreover, most of the essential oils derived from
plants are known to possess insecticidal, antifungal, anti-
bacterial and cytotoxic activities [9].
A wide variety of analytical methods are used to
extract the volatile compounds from plant material.
Common techniques include hydro-distillation and
steam-distillation, dynamic or static headspace, super-
critical fluid extraction (SFE) and solid-phase microex-
traction (SPME) [10 – 15]. Headspace sampling (HS) is
another alternative acceptable to liquid injection when
essential oils must be selectively introduced to a gas chro-
matograph to avoid transfer of nonvolatile constituents
which may increase run times or complicate the proper
separation [16].
In 1996, liquid-phase microextraction (LPME), also
known as single-drop microextraction (SDME), was intro-
duced by Jeannot and Cantwell [17, 18]. This technique
was developed as a solvent minimised preconcentration
technique. SDME is based on the distribution effect of
the analytes between a microdrop of organic solvent and
sample matrix. The organic solvent drop is suspended at
the tip of a microsyringe needle and exposed to the sam-
ple. The target compounds are then transferred from the
sample matrix into the drop. After extraction, the micro-
drop is retracted back into the microsyringe and injected
into either gas- or high-performance liquid chromato-
graph for further analysis [19]. SDME has the advantages
of high extraction speed and a fair simplicity. It uses
inexpensive apparatus and virtually eliminates solvent
consumption [20]. Moreover, this technique does not
require any pretreatment step.

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J. Sep. Sci. 2008, 31, 356 – 363
In the headspace single-drop microextraction (HS-
SDME) mode, the organic solvent is exposed to the head-
space above the liquid or solid sample [21, 22]. In this
case, the extraction solvent with high-boiling point and
low vapour pressure is required [23, 24]. HS-SDME is
potentially applicable in many areas of analytical chem-
istry including environmental, pharmaceutical, forensic
and food analysis in which volatile compounds are fre-
quently determined [25]. It has been demonstrated that
LPME shows comparable extraction efficiency and repro-
ducibility as the widely used SPME technique [26].
The aim of the present work was to optimise the exper-
imental conditions of SDME in the headspace mode
when examining this method for the determination of
herbal essential oils. The extraction efficiencies were
optimised by adjusting the key parameters such as
extraction time and temperature, amount of sample for
extraction or extraction solvent chosen. Finally, by
employing the HS-SDME in combination with the GC-
FID, the new procedure proposed was applied to deter-
mine both absolute and relative content of selected
essential oils in selected herbal samples.
2 Experimental
2.1 Plant material and reagents
The plant materials used in this study (dry leaves of Men-
tha piperita, Lavandula augustifolia, Melissa officinalis and Tri-
folium pratense) were obtained from Botanicus (Ostr
,
Czech Republic); all plants being collected during July
2006. The leaves were separated from the rest of the
plants, dried at room temperature and then kept in dark
glass wide-neck bottles at 48C prior to use. Before the
proper analysis, the samples had to be pretreated with
respect to maximal specific surface, which was accom-
plished by their grinding in a mortar.
Standards of essential oils, such as 1,4-cineole (98%),
eucalyptol (98%), limonene (98%), menthol (99%), men-
thone (99%), myrcene (90%), nerol (98%), a-thujone (96%)
and thymol (99%), used for the identification of target
compounds, were purchased from Sigma – Aldrich
(Prague, Czech Republic). p-Xylene (analytical grade) used
as an extraction solvent was then obtained from Lach-
Ner (Brno, Czech Republic).
2.2 Instrumentation
All extracts obtained by HS-SDME were analysed by a GC-
,
FID system (model HP-5890’; Hewlett-Packard, Avondale,
,
PA, USA) equipped with a capillary column ( Ultra No. 2’;
25 m60.32 mm id, 0.52 lm film thickness of phenylme-
thylsilicone). Chromatograms were evaluated by using
the CSW integration software (Data Apex; Prague, Czech
Republic). The extraction temperature was adjusted by a
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Sample Preparations 357
,
Julabo thermostat (model EC-5’; Julabo Labortechnik,
Seelbach, Germany).
2.3 Headspace SDME extraction procedure
All extractions were performed in 10 mL glass vials
sealed with PTFE-faced septum caps (Supelco; Bellefonte,
PA, USA). Prior to extraction, the samples analysed were
pulverised in the mortar, transferred into the sampling
vial and preheated at the extraction temperature for
10 min. HS-SDME was performed with a commercially
available 5 lL GC microsyringe with the skew needle
Hamilton Syringe (75N; Hamilton Bonaduz AG, Bonaduz,
Switzerland). Before each extraction, the microsyringe
was washed several times with the solvent in order to
eliminate the bubbles in the barrel and the needle. After
the needle passed through the septum, the needle tip
had to be kept about 0.5 cm above the surface of the ana-
lysed sample (0.5 g). The syringe was clamped and the
plunger depressed to cause the solvent (2.0 lL) to form a
drop suspended at the tip. The sample was continuously
heated at the same temperature as that of the water bath
during extraction. The temperatures selected were 40,
50, 60, 70, 80 and 908C. When the extraction was finished
after a special period of time, the acceptor drop was
retracted into the microsyringe.
2.4 Chromatographic analysis
After extraction, the microsyringe was removed from the
sample vial and immediately inserted into the gas chro-
matograph. The separation conditions were as follows:
initial column temperature 608C, increased to 1508C at
58C/min and, finally, increased to 2808C at 308C/min
(hold for 2 min). The injector and detector temperatures
were maintained at 220 and 2908C, respectively. The
nitrogen (purity 5.0, Linde, Prague, Czech Republic) was
used as the carrier gas with 50 kPa column head pressure
at a flow rate of 3.15 mL/min (split ratio 1:10). Hydrogen
and air (both Linde) passed at 30 and 300 mL/min were
used in the FID mode. The extracted analytes were quan-
tified using the standard addition method.
3 Results and discussion
There are different factors that may affect the extraction
process; especially, the selection of suitable extraction
solvent and its volume, extraction temperature and
time. Thus, it is crucial to perform the respective optimi-
sation in order to obtain the good recovery strategy
forms. In this work, nine compounds have been chosen
as representatives of essential oils, examining their
behaviour under extraction conditions studied. Figure 1
depicts the GC-FID chromatogram of essential oils stand-
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358 M. Adam et al.
ards dissolved in p-xylene (A) together with an example
of the real HS-SDME extract of M. piperita (B). The chroma-
tographic conditions, including the column selection,
were used according to our previous experience [27]. Also
herein, these conditions were found as adequate for the
evaluation of the HS-SDME chromatograms of analysed
samples.
3.1 Optimisation of extraction conditions
To develop an optimal HS-SDME procedure for the deter-
mination of herbal essential oils, several variables
related to the extraction steps were of interest in order to
achieve a maximal efficiency of extraction of the com-
pounds studied as well as sufficient selectivity with
respect to other components present in the matrix.
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci. 2008, 31, 356 – 363
Figure 1. GC chromatograms of
(A) essential oils standards dis-
solved in p-xylene and (B) SDME
extract of the M. piperita. 1, Myr-
cene; 2, 1,4-cineole; 3, eucalyptol;
4, limonene; 5, a-thujone; 6, men-
thone; 7, menthol; 8, nerol; 9, thy-
mol.
3.1.1 Selection of extraction solvent, drop volume
and sample amount
Special attention had to be paid to the selection of the
extraction solvent of high purity and a low toxicity
which should not evaporate under the extraction condi-
tions. Besides this, due to chromatographic identifica-
tion, the retention time of the solvent of choice should
be different from those of target compounds. According
to classical steam distillation method, p-xylene was
selected as the solvent for collection of essential oils. As
confirmed experimentally, its peak was well separated
from those of the essential oil components.
Generally, the use of a large organic drop results in an
increase in the analytical response of the instrument;
however, larger drops are usually difficult to manipulate
and the respective SDME procedures are less reliable. In
addition, as the analytes get into the drop through the
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J. Sep. Sci. 2008, 31, 356 – 363
diffusion process one can observe that the larger the
drop volume, the longer the time to reach the equili-
brium and hence, the organic drop volume has to be
carefully optimised in order to achieve the desired sensi-
tivity. For the solvent volume optimisation, the effect of
the drop volume ranging from 1 to 3 lL of target com-
pounds was investigated. Our results have indicated that
the increase in the solvent drop volume improves the
extraction efficiency of the SDME technique and the pre-
cision obtained with a larger solvent volume is still satis-
factory. However, when the drop volume was above 2 lL,
such microdrops were difficult to manipulate and less
stable to be kept in the needle tip. By considering these
factors, the following investigations were carried out
with the drop volume of 2.0 lL p-xylene.
Other parameters in our focus were the sample vol-
ume and headspace volume and therefore, the influence
of sample weight on the composition of the extracted
compounds was also studied. Because, for constant vol-
ume vials, the sample volume is in direct relationship
with headspace volume, it was necessary to find the most
suitable ratio between headspace volume and sample vol-
ume. As ascertained, the increased amount of sample in
the vial led to a higher concentration of target volatile
compounds in the headspace. On the other hand, after
saturation of the microdrop with volatile analytes, the
increase in the sample amount had no further effect on
the mass transfer into the extracting solvent. Thus, based
on these observations, the optimal sample weight was
chosen to be 0.5 g of dried herbal leaves per 10 mL dosage
of sample vial. At this level, the headspace volume is
equal to one half of the sampling vial.
3.1.2 Effect of temperature and sorption time
profiles
First of all, it was necessary to evaluate the evaporation
losses of the solvent during the extraction process. For
this experiment, a 2 mL microdrop of p-xylene was
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Sample Preparations 359
Figure 2. Effect of temperature on the
evaporation of 2 lL microdrop of p-xylene.
formed at the tip of microsyringe inside the vial contain-
ing an inert matrix (sea sand) where the headspace vol-
ume was the same as that for sample extraction. During
these studies carried out at various temperatures (see
below), the microdrop formed in different time periods
was retracted into the microsyringe and the remaining
amount of the p-xylene was determined by visual control.
A more precise evaluation was then performed by the GC
analysis when the peak area of the residual p-xylene had
been related to the peak area of the 2 lL of p-xylene. The
results of these experiments are summarised in Fig. 2. As
can be seen, the solvent evaporation is more pronounced
due to the increase in temperature. For the further
experiments, however, a compromise between the sol-
vent evaporation and the increased concentration of tar-
get analytes at higher temperature has to be found yet.
According to the previous paragraph, the effect of tem-
perature upon the extraction efficiency and appropriate
sorption time profile is to be studied together since the
temperature may significantly affect the mass transfer of
analytes from the sample to headspace and from head-
space to the extraction phase. In order to accelerate the
extraction process, the sample was preheated at the
extraction temperature when various extraction temper-
atures ranging from 40 to 908C were investigated. Figure
3 makes a comparison of the results obtained at various
temperatures and appropriate extraction time in
dependence of the individual sorption time profile for
the extraction of the mixed herbal sample (M. piperita
and L. augustifolia, ratio 1:1). This extraction sequence
included the time period corresponding to the maximal
yield of target compounds at the individual tempera-
tures (i. e. 5 min for 408C; 4 min for 508C; 2.5 min for
608C; 90 s for 708C; 60 s for 808C and 408s for 908C). The
results have shown that the extraction efficiencies of
most target compounds increase with the extraction
temperature up to 708C, which can be explained by the
fact that higher temperatures increase the volatility of
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360 M. Adam et al.
essential oils and their desorption from the herbal
matrix. Here, it can be stated that the higher the temper-
ature the more profound is the effect of solvent evapora-
tion together with the volatility of individual essential
oil compounds, both resulting in lower extraction effi-
ciency and lesser amounts of extracted compounds.
According to this, the temperature of 708C was chosen in
further experiments.
Finally, the SDME procedure proposed was examined
with respect to the extraction time and its role in the
microextraction arrangement used in these investiga-
tions. In most methods based on SDME, the extraction
efficiency increases with longer extraction time. For the
HS-SDME method, the amount of extracted analytes
increases with the extended exposure of microdrop in
the headspace and, subsequently, the equilibrium state
is achieved rapidly and the resultant extraction time is
very short. This is the most important advantage of HS-
SDME technique.
In this work, an extraction time profile for selected
essential oils in a p-xylene microdrop varying from 0.5 to
i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci. 2008, 31, 356 – 363
Figure 3. Effect of extraction temper-
ature upon the HS-SDME efficiency
of target essential oils for mixed
herbal sample M. piperita and L.
augustifolia (1:1). Extraction condi-
tions were as follows: 5 min for 408C;
4 min for 508C; 2.5 min for 608C; 90 s
for 708C; 60 s for 808C and 40 s for
908C, respectively.
Figure 4. Effect of extraction time at
708C upon the HS-SDME efficiency of
target essential oils in mixed herbal
sample M. piperita and L. augustifolia
(1:1).
3 min was studied in order to assess the optimum extrac-
tion time (at 708C). Figure 4 shows the influence of
extraction time on efficiency for four selected essential
oils and as can be seen, the best results were obtained
with extraction for 90 s being chosen as the optimum
selected for all subsequent experiments. A decrease
observed for this time period might be attributed to sol-
vent evaporation and a back extraction from the micro-
drop into the headspace.
3.1.3 Validation of the method
In our study, analytical performance of the method was
evaluated via the linearity range, LOQ and LOD, and the
precision when using the optimised conditions
described above. The validation was performed for nine
essential oil compounds (listed in the Section 2). Figure
1A shows typical chromatograms obtained for the mixed
standard solution after HS-SDME. The results concerning
LODs and LOQs, linear dynamic range together with the
correlation coefficients (r2) for all target compounds are
then given in Table 1. The linear response range was
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J. Sep. Sci. 2008, 31, 356 – 363 Sample Preparations 361

Table 1. Analysed essential oils and its basic characteristics together with the LOD and LOQ levels

Compound b.p. (8C)a) q (g/cm3) MR Formula TR (min) LOD (lg)b) LOQ (lg)b) R2

1,4-Cineole 173 0.900 154.25 C10H18O 9.3 3.32 11.07 0.9916

Eucalyptol 176 – 177 0.921 154.25 C10H18O 9.8 15.80 52.68 0.9992
Limonene 176 – 177 0.843 136.24 C10H16 9.9 11.28 37.59 0.9998
Menthol 212 0.890 156.27 C10H20O 16.4 18.33 61.10 0.9912
Menthone 207 – 210 0.893 154.25 C10H18O 14.9 14.30 47.68 0.9976
Myrcene 167 0.858 136.23 C10H16 8.7 10.84 36.12 0.9973
Nerol 225 – 230 0.877 154.25 C10H18O 18.8 20.52 68.41 0.9931
a-Thujon 201 0.914 152.23 C10H16O 12.1 16.59 55.29 0.9919
Thymol 232 0.965 150.22 C10H14O 20.9 18.21 60.70 0.9950
a)
b.p. is the boiling point at the atmospheric pressure (101.325 kPa).
b)
Calculated as content per 100 g of dried leaves.

Table 2. Absolute and relative essential oil contents of M. officinalis and M. piperita

Essential oil M. officinalis M. piperita

Abs.cont. (mg)a) RSD (%) Rel.cont. (%)b) Abs.cont. (mg) RSD (%) Rel.cont. (%)

1,4-Cineole 4.84 2.24 0.86 aLOQ – –

Eucalyptol 96.34 5.36 17.20 aLOQ – –
Limonene aLOQ – – 245.46 3.00 25.05
Menthol aLOQ – – 82.80 2.64 8.45
Menthone 129.48 4.70 23.12 219.43 8.51 22.39
Myrcene aLOQ – – 17.72 7.78 1.81
Nerol 62.42 2.11 11.15 aLOQ – –
a-Thujone 15.63 2.27 2.79 22.82 8.81 2.33
Thymol aLOQ – – 81.29 3.21 8.29

For calculation of RSD values all analyses were performed in triplicate.

a)
Abs.cont. – absolute content related to 100 g of dried leaves.
b)
Rel.cont. – relative content, 100% is the total content of the essential oils determined by the steam distillation method
(560 mg per 100 g of dried leaves of M. officinalis and 980 mg per 100 g of dried leaves of M. piperita, respectively).

examined over the concentration range from LOQs to
2 mg/mL for 1,4-cineole, menthol, myrcene, nerol, a-thu-
jone and thymol, whereas an interval from LOQs up to
4 mg/mL was seen for the remaining compounds, i. e.
eucalyptol, limonene and menthol, respectively. The
,
LODs, estimated with the aid of the 3:1 S/N ratio’ crite-
rion, were found to be within 3.32 – 20.52 lg per 100 g of
dried sample leaves for all studied essential oils; the
respective LOQs, calculated as the S/N = 10, were then
11.07 – 68.41 lg per 100 g of dried sample leaves. Such val-
ues indicate that the proposed method is sensitive
enough to determine the essential oils in the herbal sam-
ples. Finally, the precision of the method under opti-
mised conditions (i. e. sample weight 0.5 g; 10 mL sam-
pling vial; extraction time 90 s; temperature 708C; drop
volume 2 lL p-xylene) was determined by analysing the
samples in triplicate and, when expressed as the RSD (in
% rel.), typical results were between 2.1 and 8.9% (see
Tables 2 and 3). Also these results have demonstrated
that HS-SDME is a reliable technique for the determina-
tion of herbal essential oils.
3.1.4 Total content of essential oils
Because it is difficult to determine the total content of
essential oils in herbs, the proper analyses were perform-
ed by common steam distillation method. The total con-
tents found in essential oils were as follows: M. officinalis
0.56% (m/m), M. piperita 0.98% (m/m), L. augustifolia 0.54%
(m/m) and T. pratense 0.04% (m/m), when the average RSD
value (from three replications) of steam distillation was
2.5% for each sample. These experiments were carried
out using approx. 40 g of dried sample leaves and 600 mL
of water with duration for 4 h. The results obtained were
then used for the evaluation of the relative contents of
individual essential oils determined by the HS-SDME
method.
3.2 Application to real samples
The optimised HS-SDME method was applied to the anal-
ysis of various herbal samples such as M. piperita, L. augus-
tifolia, M. officinalis and T. pratense in an effort to determine

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362 M. Adam et al. J. Sep. Sci. 2008, 31, 356 – 363

Table 3. Absolute and relative essential oil contents of L. augustifolia and T. pratense dried leaves

Essential oil L. augustifolia T. pratense

Abs.cont. (mg)a) RSD (%) Rel.cont. (%)b) Abs.cont. mg) RSD (%) Rel.cont. (%)

1,4-Cineole 10.11 6.65 1.87 11.45 5.44 28.63

Eucalyptol 208.00 8.13 38.52 aLOQ – –
Limonene aLOQ – – aLOQ – –
Menthol 81.44 2.64 15.08 3.25 7.43 8.13
Menthone 16.18 5.94 3.00 6.77 7.18 16.93
Myrcene 9.63 7.78 1.78 aLOQ – –
Nerol 59.90 4.24 11.09 aLOQ – –
a-Thujone 7.31 8.54 1.35 5.11 8.96 12.78
Thymol aLOQ – – aLOQ – –

For calculation of RSD values all analyses were performed in triplicate.

a)
Abs.cont. – absolute content related to 100 g of dried leaves.
b)
Rel.cont. – relative content, 100% is the total content of the essential oils determined by the steam distillation method
(540 mg per 100 g of dried leaves of L. augustifolia and 40 mg per 100 g of dried leaves of T. pratense, respectively).

the absolute and relative contents of target essential oils.
Again, three replicate measurements were carried out
for all the samples. The individual compounds were iden-
tified with the aid of GC-FID and quantified using the
standard addition method. For this method, the appro-
priate amount of selected standards of essential oils
(based on the retention time evaluation) was added as a
fresh sample into the extraction vial before the proper
HS-SDME procedure. After extraction was finished, a
chromatogram was recorded and compared to that
obtained without standard addition. All results were cal-
culated as the amount of the respective compound
related to the total content of essential oils determined
by steam distillation. Results from these experiments are
shown in Tables 2 and 3.
4 Concluding remarks
The study presented in the previous sections demon-
strates the development and application of an HS-SDME
method for the extraction of herbal essential oils. When
combined with GC-FID, SDME was found to be suitable
for the determination of target compounds in dried
leaves. Although the method described was demon-
strated on selected herbs, it seems to be applicable to a
wider range of herbal samples. This study confirms that
the SDME approach is simple, rapid, economical and
requires no preliminary sample preparation step, and –
at the same time – implies that SDME would be an alter-
native sample preparation to the traditional LLE and SPE.
The procedure itself is also environmentally friendly,
because only a small volume of a solvent is used.
Within the study elaborated, some important experi-
mental parameters affecting the extraction efficiency of
essential oils were studied in detail and the optimal con-
ditions are as follows: sample weight 0.5 g; volume of the
sampling vial, 10 mL; extraction time, 90 s; extraction
temperature, 708C, and the solvent of choice, p-xylene.
These conditions were then used in a model analysis of
nine selected essential oils in four different samples of
herbs and the respective results have shown a good effec-
tiveness and reliability of the method tested.
The authors would like to acknowledge financial supports from
the Ministry of Education, Youth and Sports of the Czech Republic
(project MSM 0021627502) and from the Czech Science Founda-
tion (No. 203/05/2106).
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