Anal Bioanal Chem (2009) 395:23872395
DOI 10.1007/s00216-009-3133-x
ORIGINAL PAPER
Pesticide extraction from table grapes and plums using ionic
liquid based dispersive liquidliquid microextraction
Lidia M. Ravelo-Prez & Javier Hernndez-Borges &
Antonio V. Herrera-Herrera &
Miguel ngel Rodrguez-Delgado
Received: 22 July 2009 / Revised: 2 September 2009 / Accepted: 3 September 2009 / Published online: 25 September 2009
# Springer-Verlag 2009
Abstract Room temperature ionic liquids (RTILs) have
been used as extraction solvents in dispersive liquidliquid
microextraction (DLLME) for the determination of eight
multi-class pesticides (i.e. thiophanate-methyl, carbofuran,
carbaryl, tebuconazole, iprodione, oxyfluorfen, hexythiazox,
and fenazaquin) in table grapes and plums. The developed
method involves the combination of DLLME and high-
performance liquid chromatography with diode array
detection. Samples were first homogenized and extracted
with acetonitrile. After evaporation and reconstitution of
the extract in water containing sodium chloride, a quick
DLLME procedure that used the ionic liquid 1-hexyl-3-
methylimidazolium hexafluorophosphate ([C6MIM][PF6])
and methanol was developed. The RTIL dissolved in a
very small volume of acetonitrile was directed injected in
the chromatographic system. The comparison between the
calibration curves obtained from standards and from
spiked sample extracts (matrix-matched calibration)
showed the existence of a strong matrix effect for most
of the analyzed pesticides. A recovery study was also
developed with five consecutive extractions of the two
types of fruits spiked at three concentration levels. Mean
recovery values were in the range of 72100% for table
grapes and 66105% for plum samples (except for
thiophanate-methyl and carbofuran, which were 6475%
and 5866%, respectively). Limits of detection (LODs)
were in the range 0.6515.44 g/kg for table grapes and
0.9026.33 g/kg for plums, representing LODs below the
L. M. Ravelo-Prez : J. Hernndez-Borges :
A. V. Herrera-Herrera : M. . Rodrguez-Delgado (*)
Departamento de Qumica Analtica, Nutricin y Bromatologa,
Facultad de Qumica, Universidad de La Laguna (ULL),
Avenida Astrofsico Francisco Snchez s/n,
38206 La Laguna (Tenerife), Spain
e-mail: mrguez@ull.es
maximum residue limits (MRLs) established by the
European Union in these fruits. The potential of the
method was demonstrated by analyzing 12 commercial
fruit samples (six of each type).
Keywords Room temperature ionic liquids . Dispersive
liquidliquid microextraction . Pesticides . Table grapes .
Plums . High-performance liquid chromatography
Introduction
A wide number of pesticides of different chemical nature are
currently being used for agricultural purposes all over the
world. Their use provides clear benefits in terms of an increase
in the agricultural production, but also serious drawbacks, like
the presence and bioaccumulation of some of them through
the food chain, becoming eventually a risk/threat to both
animal and human life. Therefore, the evaluation of pesticide
residues in food is nowadays a priority objective to ensure
food safety and quality. For this reason, there is currently a
growing demand for fast and reliable pesticide analytical
methodologies as well as monitoring programs in both
agriculture and food production.
Since the concentrations of pesticides in food commod-
ities like fruits are normally low, extraction/pretreatment
procedures as well as high-sensitivity instrumentation are
necessary for their final determination. The most popular
pretreatment methods are liquidliquid extraction and solid
phase extraction (SPE) [1, 2]. However, recent research
activities are oriented towards the development of efficient,
economical, and miniaturized sample preparation methods,
with a minimization of the amount of organic solvent used.
In view of this aspect, several novel microextraction
techniques are being developed in order to reduce the
2388
analysis step, increase the sample throughput, and to
improve the quality and the sensitivity of the analytical
method [3, 4]. One of the emerging techniques in this area
is dispersive liquidliquid microextraction (DLLME),
which was recently introduced by Rezaee et al.[4]. This
technique, which is based on a ternary component solvent
extraction system (i.e. extraction solvent, disperser solvent,
and aqueous samples containing analyte of interest) is
attracting more attention due to its main advantages such as
rapidity, high enrichment factor, low cost, and simplicity of
operation, among others [4, 5].
Despite the fact that DLLME has been successfully
applied in several occasions for the extraction of pesticides
from different samples, mainly water [68], the extraction
of compounds from solid matrices by DLLME has received
very little attention. In fact, to the best of our knowledge,
there only exist six works in the literature concerning the
use of DLLME for the extraction of pesticides from solid
matrices, such as tea leaves [9, 10], soils [10, 11], apples
[12], watermelons and cucumbers [13], and bananas [14].
The search for new extraction solvents is a key trend
in DLLME evolution. In this sense, room temperature
ionic liquids (RTILs), which are ionic media resulting
from the combination of organic cations and various
anions [15], have attracted much attention taking into
account their special features such as high viscosity, low
vapor pressure, good thermal stability, a wide range of
miscibility with water and other organic solvents, non-
flammability, and dual natural polarity [1517]. The use of
ionic liquids (ILs) in DLLME provides a fast and easy to
operate procedure that avoids using highly toxic solvents
[18]. However, up to now, IL-DLLME has almost not
been explored for the extraction of pesticides. In fact,
there only exist four works in the literature concerning this
application [14, 1820] and all these studies have been
developed for water samples except one of them in which
pesticides were extracted from banana samples [14]. In
this work [14], parameters influencing the IL-DLLME
procedure (i.e., sample pH, sodium chloride percentage,
IL amount, and volume of disperser solvent) were
optimized by means of an experimental design, for the
selective extraction of a group of eight pesticides (i.e.,
thiophanate-methyl, carbofuran, carbaryl, tebuconazole,
iprodione, oxyfluorfen, hexythiazox, and fenazaquin)
prior to their high-performance liquid chromatography
with diode array detection (HPLC-DAD) separation. The
IL 1-hexyl-3-methylimidazolium hexafluorophosphate
([C6MIM][PF6]) was employed as extraction solvent.
The combined use of IL-DLLME with HPLC-DAD
allowed the determination of all these pesticides below
the maximum residue limits (MRLs) established by the
European Union (EU) in bananas (European Union MRLs
Sorted by Pesticide, 2009. Available at http://ec.europa.eu/
L.M. Ravelo-Prez et al.
sanco_pesticides/public/index.cfm?event=substance.selec
tion&ch=, April 2009). This work constituted the first
application of RTILs as extraction solvents for DLLME
for the extraction of pesticides from non-aqueous samples.
In order to demonstrate and to extend the applicability
of this previously developed IL-DLLME procedure for
the extraction of pesticides from other fruit samples
below their MRLs, the IL-DLLME-HPLC-DAD method,
previously developed, was applied to the analysis of
pesticides in table grapes and plums. Suitable recovery as
well as accuracy and precision studies were developed.
Because of the complexity of these samples and their
influence on the response of the chromatographic system,
matrix-matched calibration was also carried out. Finally,
the method was applied to the analysis of twelve samples
(six of each type: tables grapes and plums) in order to
evaluate the presence of these compounds in these
samples. This work, which shows the good performance
of the previously developed method and extends its
applicability, represents the second publication in the
literature regarding the extraction of pesticides from non-
aqueous samples using IL-DLLME.
Materials and methods
Chemicals and reagents
Pesticide analytical standards of thiophanate-methyl, carbo-
furan, carbaryl, iprodione, hexythiazox, and fenazaquin
were from Riedel-de-Han (Sigma-Aldrich, Madrid, Spain);
purity was higher than 98.0%. Tebuconazole and oxy-
fluorfen were from Fluka (Sigma-Aldrich, Madrid, Spain);
purity was higher than 99.6%. Individual stock solutions of
approximately 500 mg/L were prepared by dissolving each
compound in ACN and stored in glass-stopper bottles at
4 C. Mixtures of appropriate concentration were prepared
by appropriate combination and dilution with ACN. The
working solutions were prepared daily by dilution of these
mixtures with ACN.
Methanol and ACN HPLC-grade were from Scharlau
(Barcelona, Spain). Anhydrous magnesium sulfate, sodium
chloride, sodium hydrogencitrate sesquihydrate, and sodium
citrate tribasic dihydrate were from Sigma-Aldrich while
hydrochloric acid was from Merck (Darmstadt, Germany).
The IL 1-hexyl-3-methylimidazolium hexafluorophosphate
([C6MIM][PF6]) was provided by Fluka (Switzerland);
purity was higher than 97.0%.
HPLC-DAD conditions
HPLC analyses were performed on a Waters Alliance
HPLC system (Milford, MA, USA), equipped with two
Pesticide extraction from table grapes and plums 2389

pumps (model 1525), an autosampler (model 717 plus) and Software

a DAD (model 2996). For data storage and evaluation
Empower 2 software from Waters and a personal computer
were used. Separations were carried out using a Nova-Pak
C18 column (150 3.9 mm, 4 m) and a Guard-Pak C18
pre-column (4 m), both from Waters. Gradient HPLC
elution was performed with 100% Milli-Q water as mobile
phase A and 100% ACN as mobile phase B. The initial
mobile phase gradient condition was 75:25 (v/v) of solvents
A and B, respectively. The elution was isocratic for the first
3 min and was altered gradually to 45:55 (v/v) over 4 min
(curve 3). Then, the eluent composition was changed to
40:60 (v/v) over 5 min (curve 7) and later to 100% B for
10 min (curve 5). Then, the elution was isocratic for 5 min
and finally, the initial eluent composition was restored in
5 min (curve 6) and maintained for 5 min more. The flow
rate was set at 1.0 mL/min and the injection volume was
20 L. The working wavelengths were 205, 215, and
220 nm. Milli-Q was obtained from a Milli-Q gradient
system A10 from Millipore (Bedford, MA, USA).
The StatGraphics Plus Software Version 5.1 (Statistical
Graphics, Rockville, USA) was used for data processing.
Table grape and plum samples
Table grapes and plums from Chile were bought in local
markets and supermarkets of Tenerife (Canary Islands,
Spain) during April, May, and June of 2009. One gram of
homogenized fruit (1 or 2 kg for plums and table grapes,
respectively) was weighed into a 50-mL centrifuge tube and
spiked with a small volume of an appropriate standard
mixture solution. Then, 5 mL of ACN were added and the
tube was closed and shaken vigorously by hand for 1 min.
To induce phase separation and pesticide partitioning, a
buffer-salt mixture (consisting of 2 g of anhydrous
magnesium sulfate, 0.5 g of sodium chloride, 0.5 g of
sodium citrate tribasic dehydrate, and 0.25 g of sodium
hydrogencitrate sesquihydrate) was added. The tube was

Table 1 Chemical structure, family, and MRLs for table grapes and plums of the selected pesticides
Peak number Pesticide Family Use Structure EUs MRLs (mg/kg)*
Table grape Plum
1 Thiophanate-methyl Benzimidazole Fungicide NHCSNHCO2CH3 0.1 0.3
NHCSNHCO2CH3

2 Carbofuran Carbamate Insecticide/nematicide OCONHCH3 0.02 0.02

O CH3

CH3

3 Carbaryl Carbamate Insecticide OCONHCH3 0.05 0.05

4 Tebuconazole Triazole Fungicide OH 2 0.5

Cl CH2 CH2 C C(CH3)3
CH2
N
N
N

5 Iprodione Dicarboximide Fungicide Cl O

10 3
N
N
CONHCH(CH3)2
Cl O

6 Oxyfluorfen Diphenyl ether Herbicide O CH 2 CH 3 0.1 0.05

F 3C O NO2

Cl

7 Hexythiazox Carboxamide Acaricide Cl 1 0.5

S O

N
CH3 CONH

8 Fenazaquin Quinazoline Acaricide N 0.2 0.3

N

O CH2CH2 C(CH3)3

a
Taken from EU MRLs sorted by pesticide. Available at <http://www.ec.europa.eu/food/plant/protection/pesticides/index_en.htm>.
2390 L.M. Ravelo-Prez et al.

closed and immediately shaken vigorously on a Vortex
mixer for 1 min. Then, the mixture was sonicated for 5 min
and centrifuged at 4,000 rpm for 10 min. The supernatant
was filtered through a Chromafil Xtra PET-45/25 filter
(pore size 0.45 m, Macherey-Nagel, Dren, Germany) and
evaporated at 40 C and 205 mbar using a Rotavapor R-200
(Bchi Labortechnik, Flawil, Switzerland). The residue was
dissolved with 10 mL of Milli-Q water at pH 2.7 (adjusted
with 1.0 M HCl) containing 28.9% NaCl (w/v) and
subjected to the DLLME procedure, as described below.
DLLME procedure
The solution previously obtained was placed in a 15-mL
glass centrifuge tube. A mixture of 88 mg of [C6MIM][PF6]
(extraction solvent) and 714 L of methanol (disperser
solvent) was injected at high rate into the sample solution
with a micropipette in order to induce the formation of a
cloudy solution, which consisted in fine droplets of IL
dispersed in the aqueous sample. The mixture was
subsequently centrifuged for 20 min at 4,000 rpm and the
upper aqueous phase was removed with a syringe (disper-
sive particles of IL were sedimented at the bottom of the
centrifuge tube). After this process, the IL phase (20 L)
was dissolved in ACN (the final volume was 125 L) and
20 L were injected into the HPLC system for analysis.
Results and discussion
The HPLC-DAD separation of the group of pesticides
selected in this work (i.e., thiophanate-methyl, carbofuran,
carbaryl, tebuconazole, iprodione, oxyfluorfen, hexythia-
zox, and fenazaquin) as well as their preconcentration using
IL-DLLME was previously optimized and studied in
banana samples [14]. These compounds are also currently
applied in the treatment of table grapes and plums. Table 1
shows some characteristics of these pesticides (family, use,
and chemical structure) as well as their MRLs (which are
given for the whole fruit) according to the EU legislation

Fig. 1 HPLC-DAD chromato-

grams of a spiked and b non- 0.18
spiked table grape sample after 2
0.16
optimum IL-DLLME conditions A
0.14
(pH 2.7, 28.9% NaCl (w/v), 1 3
0.12
88 mg [C6MIm][PF6] and
714 L methanol). Mobile 0.10
Absorbance (UA)

phase A, 100% Milli-Q water. = 205 nm

0.08
Mobile phase B: 100% ACN. = 215 nm
0.06
Flow rate: 1.0 mL/min. Gradient = 220 nm
4 5 6 7
program is shown in HPLC- 0.04
8
DAD conditions section. 0.02
Injection volume: 20 L. 0.00
Sample dissolved in ACN: 1
-0.02
thiophanate-methyl (100 g/kg),
2 carbofuran (20 g/kg), 3 car- -0.04
baryl (50 g/kg), 4 tebuconazol -0.06
(375 g/kg), 5 iprodione
(150 g/kg), 6 oxyfluorfen 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
(100 g/kg), 7 hexythiazox Time (min)
(375 g/kg), and 8 fenazaquin
0.18
(200 g/kg)
0.16
B
0.14

0.12

0.10
Absorbance (UA)

= 205 nm
0.08 = 215 nm
0.06 = 220 nm
0.04

0.02

0.00

-0.02

-0.04

-0.06

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
Time (min)
Pesticide extraction from table grapes and plums 2391

(European Union MRLs Sorted by Pesticide, 2009. Avail-
able at http://ec.europa.eu/sanco_pesticides/public/index.
cfm?event=substance.selection&ch=, July 2009). Among
these compounds, carbofuran and carbaryl cannot be used
in Europe for agricultural purposes after the end of the year
2008 since they have been excluded from Annex I of
Commission Directive 91/414/CEE (European Union Pes-
ticides Database, 2009. Available at http://ec.europa.eu/
sanco_pesticides/public/index.cfm?event=activesubstance.
selection&a=1, July 2009). Both of them were selected, in
order to investigate if in spite of its recent prohibition, they
are still being used.
The previously studied HPLC-DAD method was
found to be repeatable with acceptable sensitivity (limits
of detection, LODs, in the low microgram per liter
range). On the other hand, several IL-DLLME parame-
ters (sample pH, sodium chloride percentage, [C6MIM]
[PF6] amount, extraction solvent, and volume of methanol,
disperser solvent) were optimized by means of an
experimental design providing a selective and sensitive
extraction of the target analytes from banana samples after
a suitable ultrasound-assisted extraction with ACN and
salt addition (see Materials and methods section for
details).
When the previously developed IL-DLLME-HPLC-
DAD method was applied to table grape and plum samples,
it was found that it could also be satisfactorily applied to
these complex samples. Figure 1 shows the chromatograms
of the IL-DLLME extract of a spiked (Fig. 1a) and non-
spiked (Fig. 1b) table grape sample (similar chromatograms
were obtained for plum samples) taking into account
maximum UV absorbance wavelengths of the selected
pesticides (205 nm for all compounds except for carbaryl
and tebuconazole, 220 nm; and fenazaquin, 215 nm). As it
can clearly be seen, no residues of the selected pesticides
were found in the samples. Moreover, it was possible to
correctly identify and quantify the selected pesticides
without any problem at their corresponding wavelengths
despite the presence of different interferences associated to
the IL (retention time between 3 and 6 min) and IL
impurities (retention times were 12.23, 14.63, 15.00, and
16.78 min).

Table 2 Calibration data from standards prepared in table grapes and plums after DLLME-HPLC-DAD and assessment of the matrix effect

Peak Pesticide Retention Linear range Matrix Calibration curve (n=7) R2 Matrix
time (min) (mg/L) effecta

1 Thiophanate-methyl 7.02 0.0613.42 Standard y=(8.813104 3,476)x(1,7966,421) 0.996

Table grape y=(8.658104 1,572)x+(2,7932,903) 0.999 No
Plum y=(7.655104 4,167)x+(1,8357,698) 0.997 Yes
2 Carbofuran 7.47 0.0975.44 Standard y=(1.024105 2,293)x(1,1116,735) 0.998
Table grape y=(1.020105 5,462)x(3,87616,040) 0.997 No
Plum y=(9.627104 4,211)x+(1,52812,367) 0.998 Yes
3 Carbaryl 7.92 0.0101.02 Standard y=(4.576105 9,380)x(2,8824,574) 0.998
Table grape y=(4.171105 14,228)x(7706,937) 0.998 Yes
Plum y=(4.182105 16,771)x(5008,177) 0.998 Yes
4 Tebuconazole 10.96 0.1226.83 Standard y=(2.396104 1,107)x(2,2354,083) 0.996
Table grape y=(2.044104 1,488)x(7875,488) 0.995 Yes
Plum y=(2.083104 1,258)x(1554,640) 0.996 Yes
5 Iprodione 11.56 0.0412.31 Standard y=(1.290105 4,018)x(4,6005,020) 0.998
Table grape y=(1.277105 5,731)x(54187,160) 0.998 No
Plum y=(1.152105 5,341)x(2,8186,673) 0.998 Yes
6 Oxyfluorfen 17.30 0.0402.24 Standard y=(1.121105 3,909)x(1,8044,725) 0.998
Table grape y=(1.014105 5,300)x+(1,3626,406) 0.997 Yes
Plum y=(1.199105 6,470)x+(1,1797,819) 0.997 Yes
7 Hexythiazox 17.75 0.0895.01 Standard y=(5.713104 2,020)x(1485,469) 0.998
Table grape y=(5.804104 2,066)x+(5,1555,592) 0.999 Yes
Plum y=(5.607104 3,783)x+(1,84610,242) 0.995 No
8 Fenazaquin 19.09 0.0432.42 Standard y=(1.298105 4,848)x(3,3076,328) 0.996
Table grape y=(1.462105 7,426)x(6,4839,693) 0.997 Yes
Plum y=(1.165105 4,146)x+(1,4095,412) 0.999 Yes
a
Statistical difference is considered when p values for the comparison of the slopes or intercepts are 0.1 (see text for further explanation); R2 :
Quadratic correlation coefficient
2392 L.M. Ravelo-Prez et al.

Matrix effect evaluation
Previous experience with the extraction of pesticides from
complex matrices like fruits [14, 21, 22] has shown the
existence of a strong matrix effect that should carefully be
evaluated. In this sense, matrix effect can be clearly
observed by the comparison of the calibration curves
obtained from standards and spiked sample extracts
(matrix-matched calibration), because sample components
can influence the chromatographic signal, making it
decrease or even increase [23]. For that reason, matrix-
matched calibration was carried out in the present study by
dissolving a mixture of the standards in the final IL-
DLLME extract. The linearity of the method was tested by
analyzing the extracts of blank fruit samples of each type
(certified ecological samples which were free of pesticides)
fortified in the range from 0.010 to 6.83 mg/L (seven
calibration points) and also standards of the selected
pesticides dissolved in ACN (same calibration range). The
previous analysis of these blank ecological samples
revealed the absence of the selected group of pesticides or
any other interference from the matrix. In both cases, the
response of the detector was linear in the range of
concentration studied, with quadratic correlation coeffi-
cients (R2) higher than 0.995 for all the compounds. The
results of this study are shown in Table 2, which includes
the full calibration curve, including standard deviations of
the slopes and intercept, as well as the linear range for each
pesticide.
Statistical comparison between the calibration in ACN
and in each type of fruit extract was carried out using a
statistical program that calculates F and p values to
compare the slopes and the intercepts. When the value of
p for the comparison of the slopes or intercepts is 0.1,
statistical differences take place and thus, calibration with
the sample matrix is necessary. Table 2 also shows for
which pesticides a significant difference between the two
curves was found. As expected, for most of the pesticides, a
statistical difference was observed between both equations
and therefore, both table grapes and plums showed an

Table 3 Mean recoveries, RSD and LODs values of the selected pesticides in table grape and plum samples after the DLLME-HPLC-DAD
method

Pesticide Table grape Plum

Levela Mean recovery RSD LODb Levela Mean recovery RSD LODb
(g/kg) (%) (%)n=5 (g/kg) (g/kg) (%) (%)n=5 (g/kg)

Thiophanate-methyl 7.63 64 6.4 4.21 7.63 60 7.9 5.01

100 70 3.8 250 65 5.3
150 68 9.1 375 64 6.2
Carbofuran 12.1 69 1.9 3.87 12.1 58 8.5 4.36
20.0 73 4.4 20.0 63 3.0
30.0 75 6.8 30.0 66 2.8
Carbaryl 1.27 72 7.7 0.651 1.27 66 5.3 0.902
50.0 76 6.5 50.0 73 7.1
75.0 74 5.8 75.0 70 3.5
Tebuconazole 15.2 90 3.6 5.44 15.2 87 5.5 6.33
375 95 2.0 500 90 7.1
563 100 4.7 750 95 2.4
Iprodione 5.16 82 8.4 4.92 5.16 73 3.0 2.98
150 90 6.1 150 76 2.8
225 87 5.2 225 80 1.9
Oxyfluorfen 5.00 86 8.8 1.67 5.00 80 7.5 1.46
100 89 2.9 50.0 86 8.6
150 92 5.1 75.0 90 3.4
Hexythiazox 11.2 94 6.2 3.59 11.2 100 6.3 4.07
375 90 1.4 500 98 7.7
563 93 3.5 750 105 2.9
Fenazaquin 5.39 87 5.8 4.21 5.39 84 4.8 3.78
200 98 1.7 300 90 7.0
300 92 2.0 450 95 4.1
a
Spiked level
b
LOD of the whole method
Pesticide extraction from table grapes and plums 2393

important matrix effect. Only in the case of thiophanate-
methyl, carbofuran, and iprodione in table grapes as well as
hexythiazox in plums, both calibration curves were com-
parable, indicating that only for these compounds in these
samples no matrix effect was observable. Therefore,
quantification of the samples was carried out using the
specific calibration with each type of matrix to take into
account the mentioned matrix effect.
Recovery, precision, and accuracy studies
With the aim of evaluating the repeatability of the whole
method, a recovery study was carried out at three
concentration levels for the two types of fruits (the lowest
of these levels was very close to the LOQ of the method).
All experiments were carried out in quintuplicate (n=5) at
each level. Table 3 shows the results of this study as well as
the spiking levels and LODs of the whole method. As it can
be seen in the table, mean recovery values were in the range
of 72100% for table grapes and 66105% for plum
samples, except for thiophanate-methyl and carbofuran,
which were slightly lower, in the range 6475% and 58
66%, respectively, with relative standard deviation values
(RSD) lower than 9.1 in all cases. LODs (which were also
experimentally checked) ranged between 0.651 and
5.44 g/kg for table grapes and between 0.902 and
6.33 g/kg for plums. These LODs are well below the
harmonized EU MRLs established for these fruits. More-
over, these values are in the same order of magnitude as the
ones obtained for these pesticides in grapes or plums in
previous works using GC-MS [2426] or HPLC-MS [27
32] after conventional pretreatment procedures, generally
an extraction with an appropriate organic solvent or SPE. It
should be mentioned that in none of these studies the
simultaneous determination of this group of pesticides has
been developed. Besides, these LODs and recovery values
were also very similar to those obtained for banana samples
in our previous work [14], which clearly demonstrates the
utility of the method IL-DLLME method for the extraction
of these pesticides from different fruit matrices.

Table 4 Results of assays to check the accuracy of the proposed method for the selected pesticides in table grape and plum samples

Pesticide Table grape Plumb

Spiked level (g/kg) Found (g/kg)a Accuracy (%) t Spiked level (g/kg) Found (g/kg)a Accuracy (%) t

Thiophanate-methyl 45.0 41.12.3 91 1.72 129 13117 102 0.99

100 98.84.2 99 0.65 250 24821 99 1.12
125 12613 101 0.08 313 31115 99 0.21
Carbofuran 16.1 15.01.1 93 1.91 16.1 16.00.5 99 0.07
20.0 18.41.6 92 0.78 20.0 20.71.2 104 1.93
25.0 22.01.2 88 2.23 25.0 26.21.7 105 1.16
Carbaryl 25.6 27.12.9 106 2.36 25.6 22.93.3 89 2.23
50.0 52.05.3 104 1.89 50.0 48.55.4 97 0.77
62.5 61.39.4 98 1.43 62.5 61.06.6 98 0.54
Tebuconazole 195 19418 99 0.67 258 26023 101 2.01
375 37529 100 0.00 500 49716 99 1.12
469 47232 101 0.24 625 61930 99 1.69
Iprodione 77.6 75.80.9 98 2.05 77.6 78.21.8 101 0.54
150 14714 98 1.06 150 14915 99 0.22
188 19026 101 0.28 188 18918 101 1.05
Oxyfluorfen 52.5 51.43.5 98 1.11 27.5 25.91.4 94 1.89
100 98.57.0 99 0.23 50.0 48.24.3 96 1.34
125 12818 102 2.01 62.5 62.15.0 99 0.66
Hexythiazox 193 19514 101 0.78 256 25411 99 0.75
375 36929 98 1.26 500 4929.9 98 0.27
469 46612 99 0.17 625 62033 99 0.12
Fenazaquin 103 99.62.8 97 2.12 153 14918 97 1.59
200 20220 101 0.19 300 29721 99 0.44
250 24613 98 0.09 375 37816 101 0.85

t experimental t value
a
Average valuestandard deviation of five determinations (95% confidence level)
2394 L.M. Ravelo-Prez et al.

Validation for spiked fruit samples was also carried
out by developing a precision and accuracy study. For
this purpose, each fruit sample was fortified with the
pesticides at three different levels of concentration (see
Table 4) and analyzed with the proposed IL-DLLME-
HPLC-DAD method in quintuplicate (n=5). These levels
have been selected in order to demonstrate the applicabil-
ity of the method to concentrations equivalent or very near
to those of the MRLs established for table grape and plum
samples. A one-sample test (Students t-test) [33] was
used to compare the concentration found for each
pesticide with the spiked concentration. Results obtained
are also shown in Table 4. As it can clearly be seen in the
table, t values were lower than the tabulated one (2.78 for
n = 5) and therefore, no significant differences were
observed between the real sample and the experimental
values (the null hypothesis might be accepted). Accuracy
percentages were between 88% and 106% in all cases,
which clearly demonstrates the suitability of the proposed
method for the feasible quantification of these compounds
in both types of samples.
Analysis of different table grapes and plum samples
To further demonstrate the applicability of the proposed
methodology for the monitoring of these pesticide
residues in table grapes and plums, 12 samples (six of
each type) bought in local markets and supermarkets of
Tenerife (Canary Islands, Spain) were analyzed. After the
homogenization of ten individuals plums (approx. 1 kg)
and five grape bunches (approx. 2 kg) as indicated by
the Spanish legislation [34], 1 g of the homogenate was
taken as analytical sample. Only carbaryl (peak 3) and
iprodione (peak 5) were found in all these samples.
Carbaryl was found in two of table grape samples, at
concentration of 15 and 22 g/kg, while iprodione was
present in one of the analyzed plum samples at a
concentration of 52 g/kg). Figure 2 shows the chromato-

Fig. 2 Chromatograms of non-

spiked table grape (a) and plum 0.18
(b) samples after optimum IL- 0.16
DLLME-HPLC-DAD procedure. A
0.14
Peak identification 3 carbaryl and
5 iprodione 0.12
Absorbance (UA)

0.10
0.08 l = 205 nm
3
l = 215 nm
0.06
= 220 nm
0.04
0.02
0.00
-0.02
-0.04
-0.06

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
Time (min)

0.18
0.16 B
0.14
0.12
Absorbance (UA)

0.10
l = 205 nm
0.08 l = 215 nm
0.06 = 220 nm
5
0.04
0.02
0.00
-0.02
-0.04
-0.06

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
Time (min)
Pesticide extraction from table grapes and plums
grams of two of the analyzed samples (one of each type)
that contained carbaryl (Fig. 2a) and iprodione (Fig. 2b).
Identification of the pesticides was carried out by spiking
the samples with the selected pesticides and also by
comparison of the retention time and DAD spectra of both
samples and standards. The presence of such pesticides at
these levels in some of the samples does not represent a
threat for the consumer, since they are below the MRLs
established by the EU. The presence of carbaryl, currently
forbidden in the EU, can be associated to the fact that it is
used in other countries (the countries of origin of the
analyzed fruit).
Conclusions
In this work, a new method is proposed for the analysis of a
group of eight multi-class pesticides residues in table grapes
and plums using IL-DLLME-HPLC-DAD. This method
involves an ultrasound-assisted extraction of the fruits followed
by an IL-DLLME procedure using the ionic liquid [C6MIM]
[PF6] as extraction solvent. The method is very simple, fast,
and reliable, with a very low consumption IL (88 mg), sample
(1.0 g) and solvents. After suitable validation of the method in
terms of linearity, precision, accuracy, and selectivity, twelve
fruit samples (six of each type) were analyzed. The results
revealed that in three of these samples carbaryl or iprodione
were found. However, the concentrations of the studied
pesticides were within the permissible limits.
Acknowledgments L.M.R.P. and A.V.H.H. wish to thank the
Spanish Ministry of Education for the FPU grant at the University
of La Laguna. J.H.B. also thanks the Spanish Ministry of Science and
Innovation for the Ramn y Cajal contract at the University of La
Laguna. This work has been supported by the Spanish Ministry of
Science and Innovation (Project AGL2008-00990/ALI).
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