Food Control 25 (2012) 270e273

Contents lists available at SciVerse ScienceDirect

Food Control
journal homepage: www.elsevier.com/locate/foodcont

Short communication

Detection of pesticides in crops: A modied QuEChERS approach

Daniela Marchis*, Gian Luca Ferro, Paola Brizio, Stefania Squadrone, Maria Cesarina Abete
C.Re.A.A. e National Reference Centre for Surveillance and Monitoring of Animal Feed, c/o Istituto Zooprolattico Sperimentale del Piemonte, Liguria, Valle dAosta, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The general term pesticide includes a large number of substances that belong to many different
Received 27 June 2011 chemical classes. Pesticides are applied to crops at various stages of cultivation to provide protection
Received in revised form against weeds and pests, and during post-harvest storage to preserve quality. The list of which pesticides
19 October 2011
are authorized for use in Europe is available on EU pesticides Database. The QuEChERS approach is
Accepted 26 October 2011
a method designed for the analysis of pesticides in fruits and vegetables. This method is based on an
extraction and clean-up step; it has been designed to be Quick Easy Cheap Effective Rugged and Safe. The
Keywords:
aim of this study was to modify the QuEChERS method to be applied in the analysis of 9 organophos-
QuEChERS
Raw material
phate and 1 pyrethroid pesticides in raw materials for animal feeding introducing an additional liquid
Feed eliquid partition step. This additional step allowed us to concentrate the samples, avoiding any solvent
GCeMS evaporation, prior to the instrumental analysis. Once the method was optimized, it was carried out
a pesticides quantization study using a GCeMS SIM multi-residue analysis. 45 samples of maize and soy
coming from Northern Italy (Piedmont Region) were analysed during ten months. In 30 samples
organophosphate pesticides were found up to 12.4 mg kg
1 of Chlorpyrifos, while no Deltamethrin was
detected.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
The analysis of undesirable contaminants in various food and
feed samples is nowadays a problem of primary concern for quality
control laboratories, considering human and animal health risks
related to the accumulation of these substances. Contaminants of
animal feed can cause harmful health effects in animals, and may
be dangerous for people through secondary exposure of consumers
to animal products (Kan & Meijer, 2007). Organic Persistent
Pollutants (POPs), which include some pesticides, have been known
to be contaminants that persist for a long time in the environment.
In fact they accumulate in fatty tissues of living organisms including
humans, and they are commonly found at increasing concentra-
tions upwelling the food chain (Stockholm Convention, 2010). The
European Union enacted laws regarding pesticides composition,
manufacture, storage, transport and use, in order to ensure public
health and animal safety (Commission Regulation EC No 178/2006;
Commission Regulation EC No 149/2008; Commission Regulation
EC No 260/2008; Commission Regulation EC No 839/2008;
Regulation EC No 396/2005; Regulation EC No 299/2008). The
* Corresponding author. Istituto Zooprolattico Sperimentale, via Bologna 148,
10154 Torino, Italy. Tel./fax: 39 011 2686 228.
E-mail address: daniela.marchis@izsto.it (D. Marchis).
EURL pesticides database contains the list of which pesticides are
authorized and for which matrices (EURL Pesticides, 2010).
Regulation 767/2009 EC set up denitions for the following
matrices: feeding stuffs, compound feeding stuffs, complete
feeding stuffs, complementary feeding stuffs, mineral feeding stuffs
and molassed feeding stuffs. It is clear that the general term feed
includes a great variety of complex matrices. The analysis of
pesticides in animal feed is an important issue because of the large
variety of interfering compounds that can be co-extracted during
the sample pre-treatment and considering the wide range of
agrochemicals that have to be determined. In pesticides testing, the
development of multi-residue methods is the main strategy
applied. However, considering that pesticides belong to different
classes with different physical and chemical properties, it is hard to
develop methods that cover all the analytes under study (Gilbert-
Lpez, Garca-Reyes, & Molina-Daz, 2009).
In 2003, Anastassiades developed a method for the multi-class,
multi-residue extraction of pesticides in fruits and vegetables. This
method was called QuEChERS, which stands for Quick, Easy, Cheap,
Rugged and Safe, and it is based on dispersive solid phase extrac-
tion (dSPE). In dSPE analytes are extracted with an aqueous
miscible solvent with a high amount of salt and/or buffering agents,
in order to induce liquid phase separation and stabilize acid and
base pesticides. QuEChERS approach is very exible and it serves as
a template for modication depending on the analyte properties,

0956-7135/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2011.10.055
 D. Marchis et al. / Food Control 25 (2012) 270e273
matrix composition, equipment and analytical technique available
in the laboratory. Ruggedness of the QuEChERS approach has been
extensively evaluated by the originators (Koesukwiwat, Lehotay,
Miao, & Leepipatpiboon, 2010; Lehotay, de Kok, Hiemstra, & van
Bodegraven, 2005, Lehotay et al., 2010).
Rashid, Nawaz, Barker, Ahmad, and Ashraf (2010) have
successfully applied the QuEChERS method for the determination
of organochlorine pesticides in soil, obtaining low limit of deten-
tion and, consequently, low limit of quantication.
The aim of our study was to modify the QuEChERS extraction
conditions of 9 organophosphate pesticides and 1 pyrethroid in raw
materials for feed, in order to reach the possible lower levels for
some of the pesticides studied, using a GCeMS single quadrupole.
Indeed, organophosphates and pyrethroids appear to be the most
commonly used pesticides in the Italian cereal production.
Acetonitrile is the typical extraction solvent in the QuEChERS
method, although it tends to provide a large solvent expansion
volume during GC vaporization, it can interfere with nitrogen
specic GC detectors, and it is less volatile than the other common
organic solvents. This is one of the reasons why we intended to
obtain the extract using a different kind of solvent. A nal step was
added, in order to avoid the acetonitrile evaporation and, at the
same time, to concentrate the analytes.
Finally, the modied QuEChERS method was applied to analyse
45 real samples of maize and soy coming from Northern Italy
(Piedmont Region), to investigate the presence of pesticides in
these matrices.
2. Materials and methods
2.1. Chemicals and reagents
The pesticides reference material (Diazinon, Chlorpyr-
iphoseCH3, ParathioneCH3, PirimiphoseCH3, Malathion, Chlor-
pyrifos, Parathion, PirimiphoseC2H5, Ethion, Deltamethrin) all at
10 mg kg
1 in cyclohexane were purchased from Dr. Ehrenstorfer.
Anhydrous magnesium sulphate and sodium hydrogen citrate ses-
quihydrate were purchased from Fluka. Acetonitrile pesticide
grade  99.9% (GC), sodium chloride  99.8% and sodium citrate
tribasic dihydrate were purchased from Riedel-de Han. The
dispersive SPE (dSPE) Clean-Up tubes (900 mg of anhydrous
magnesium sulphate, 150 mg Superclean PSA and 50 mg Discovery
DSC-18) were purchased from Supelco. The Iso-octane (GC grade)
was purchased from Merck. Working standard solutions of organ-
ophosphate pesticides and deltamethrin were prepared in cyclo-
hexane at 2 mg kg
1, and then they were used for spiking blank
maize samples and for preparation of matched calibration solutions.
2.2. Raw material samples and samples fortication
The maize raw material was homogenized in a grinder and
stored at room temperature in the dark. Pesticide free maize was
spiked as follows: a portion of homogenized maize (5 g) was
weighed into a falcon test tube and spiked at the required forti-
cation level (0.02 mg kg
1) adding an appropriate volume of mixed
standard solution containing all the 10 pesticides. This mixture was
then shaken, in order to reach a proper homogeneity of pesticides
in maize. The tubes containing fortied maize samples were left
open into the fume hood for a while, just to allow evaporation of
the solvent excess.
2.3. Sample pre-treatment
The extraction procedure based on QuEChERS method was
assessed as follows: an aliquot of 10 mL of CH3CN 10 mL of H2O
271
and the internal standard (IS), Ethion, were added to the centrifuge
test tube containing the homogenized maize sample (5.0 g).
Anhydrous MgSO4 (4.0 g), NaCl (1 g), Na3Citr2H2O (1 g), and
Na2HCitr1.5H2O (0.5 g) were added. The content was shaken
vigorously and then centrifuged at 3000 rpm for 5 min. The
centrifuge step helped the separation of acetonitrile from the
aqueous layer. The supernatant was stored overnight at
20  C, to
allow sugar and some of the fats content in the sample to precip-
itate. An aliquot of 6 mL of the upper acetonitrile layer was trans-
ferred in a dSPE clean-up tube containing PSA, MgSO4 and C18. The
tube was shaken vigorously and centrifuged at 3000 rpm for 5 min.
QuEChERS extraction procedure was modied adding a liquid-
eliquid partition, to get simultaneously a better extraction and
clean-up the samples: 5 mL of acetonitrile extract was treated with
different amounts of water, from 3 mL to 15 mL, and extracted with
different amounts of iso-octane, from 500 mL to 1500 mL. Another
treatment was also tested, using citrate buffer pH 5e5.5, from 3 mL
to 15 mL, instead of water and subsequent extraction with iso-
octane, from 500 mL to 1500 mL.
A portion of the upper layer iso-octane was transferred in a vial
for the injection into the instrument.
2.4. GCeMS conditions
A bench top Thermo DSQ spectrometer equipped with a Focus
GC gas chromatograph was employed to carry out the analyses.
The upper limit of the quadrupole mass analyser was of m z
1
1050. An electron ionization source was used to acquire the mass
spectra. The ion source was held at 250  C, the lament voltage was
set at 70 eV, the voltage intensity was 100 mA. The analytes were
acquired in Selected Ion Monitoring (SIM) mode. Separations of
organophosphate compounds and of the pyrethroid pesticide were
performed through an Optima 5MS (5% diphenyl 95% dime-
thylpolysiloxane) capillary column (30 m, 0.25 mm i.d., 0.25 mm
lm thickness, MachereyeNagel, DE). Separation was carried out
injecting samples (1 mL) in splitless mode at 250  C. Carrier gas
(helium) ow was kept regular at 1 mL min
1. The oven tempera-
ture was programmed as follows: isothermal at 120  C for 1 min,
from 120  C to 170  C at 25  C min
1, hold for 2 min, from 170  C to
200  C at 4  C min
1, hold for 1 min, from 200  C to 320  C at
25  C min
1 and isothermal at 320  C for 5 min. The transfer line
was set at 270  C.
2.5. Analytical performance
2.5.1. Selectivity
20 blank maize samples were extracted and analysed to deter-
mine the specicity of the method and to search for interfering
peaks under the same conditions. The occurrence of possible
interferences from co-extracted compounds were tested by moni-
toring the selected ion chromatograms characteristic for each
investigated compound at the retention time interval expected for
their elution.
2.5.2. Linearity
Linearity was tested for all the 10 pesticides studied. Seven
concentration levels for each analyte in three replicates were ana-
lysed to establish calibration curves. The linearity of each analyte
was tested using the least square regression method and the
coefcient of determination (R2) was calculated.
2.5.3. Limits
Limits of detection (LOD) were determined analysing 20 blank
maize our samples. For each analyte was detected the higher
signal (noise) observed in a range of time corresponding to the
272 D. Marchis et al. / Food Control 25 (2012) 270e273

Table 1 analytes are expected to elute. This fact points out that the
European law limits (food); LOD and LOQ values for the organophosphate extraction method seems to be selective and free from positive
compounds.
interference of co-extracted compounds, at least at the retention
Organophosphate EC law limits LOD LOQ time windows of interest.
compound (mg/kg) (mg/kg) (mg/kg) Calibration curves of the 10 analytes were built by plotting peak
Diazinon 0.02 0.0024 0.0079 areas against seven concentration levels (1 mg kg
1, 0.500 mg kg
1,
0.250 mg kg
1, 0.125 mg kg
1, 0.625 mg kg
1, 0.312 mg kg
1, and
ChlorpyrifoseCH3 3 0.0002 0.0006
ParathioneCH3 0.02 0.0060 0.0180
PirimiphoseCH3 5 0.0004 0.0011 0.156 mg kg
1). All the analytes showed to have R2 values greater
Malathion 8 0.0006 0.0021 than 0.99, ranging from 0.9936 (Ethion) to 0.9992 (Deltamethrin).
Chlorpyrifos 0.05 (barley 0.2) 0.0030 0.0100 In Table 1 LOD and LOQ values obtained for the organophos-
Parathion 0.05 0.0060 0.0200 phate pesticides and the pyrethroid are showed. All the LOQ values
PirimiphoseC2H5 No limit 0.0020 0.0060
Ethion 0.01 0.0018 0.0061
observed were below the law limits indicated for some of the
Deltamethrin 2 0.0008 0.0027 pesticides.

retention time 0.5%. The mean value of the noise was calculated Table 3
Results obtained from the analysis of 45 in eld samples.
for all the analytes. The LOD was calculated as the mean of the noise
value measured for each analyte, plus three times the standard sample Matrix Pesticides detected
deviation (SD) calculated. number
Limits of quantication (LOQs) were obtained from the LODs 1 Grain maize Pirimiphos Methyl 0.0011 mg/kg
values, applying the following formula: LOQ 3.3*LOD. 2 Minced soy <LOQ
3 Grain maize <LOQ
4 Soy protein <LOQ
2.5.4. Recovery 5 Soybean seeds Pirimiphos Methyl 0.0011 mg/kg
Recoveries were estimated by spiking raw material at 6 Soybean seeds Pirimiphos Methyl 0.0011 mg/kg
0.020 mg kg
1 for all the analytes; 20 replicates at two levels (0.020 7 Grain maize Not detected
and 0.060 mg kg
1) were analysed under repeatability conditions 8 Crashed maize Pirimphos Methyl 0.0148 mg/kg
9 Grain maize <LOQ
(same day, same technician) for all the pesticides tested. 10 Soybean meal <LOQ
11 Soy Pirimphos Methyl 0.163 mg/kg
12 Grain maize Pirimiphos Methyl 0.0011 mg/kg
3. Results and discussion 13 Crashed maize <LOQ
14 Laminate maize no GMO Pirimiphos Methyl 0.0011 mg/kg
3.1. QuEChERS extraction and simultaneous clean-up 15 Whole soy akes Pirimiphos Methyl 0.0011 mg/kg
16 Grain maize <LOQ
17 Crashed maize Pirimphos Methyl 0.0165 mg/kg
Data collected about the clean-up step performed with water
Chlorpyrifos 0.009 mg/kg
showed a certain increment of the extraction recoveries for all the 18 Grain maize <LOQ
analytes by increasing the amount of water used. Treatment with 19 Grain maize <LOQ
citrate buffer of the extracts was performed, too. Both water and 20 Soybean meal GMO Pirimiphos Methyl 0.0011 mg/kg
citrate buffer provided a rather similar increase of recoveries, and 21 Soy Pirimiphos Methyl 0.0011 mg/kg
22 Soy Pirimphos Methyl 0.0011 mg/kg
no signicantly differences were observed. Therefore, it was Chlorpyrifos 0.206 mg/kg
determined to perform the extraction step in water. Three different 23 Organic soy Pirimiphos Methyl 0.0011 mg/kg
amounts of iso-octane (500 mL, 1000 mL and 1500 mL) were tested; 24 Soy Pirimiphos Methyl 0.0011 mg/kg
an increase of the extraction recoveries increasing the volume of 25 Grain maize Pirimiphos Methyl 0.0011 mg/kg
26 Soybean meal GMO Pirimiphos Methyl 0.0011 mg/kg
iso-octane was observed.
27 Soybean meal Pirimiphos Methyl 0.0011 mg/kg
The clean-up step was performed as follows: 5 mL of acetoni- 28 Soybean meal Pirimiphos Methyl 0.0011 mg/kg
trile, 15 mL of water and 1500 mL of iso-octane. 29 Grain maize Pirimphos Methyl 0.0011 mg/kg
Chlorpyrifos 0.0074 mg/kg
30 Soybean meal Pirimiphos Methyl 0.0011 mg/kg
3.2. Analytical performances 31 Soybean meal Pirimiphos Methyl 0.0011 mg/kg
32 Grain maize Pirimiphos Methyl 0.0011 mg/kg
Ion chromatograms obtained from the extracts of maize our 33 National green maize <LOQ
34 Maize Pirimphos Methyl 0.0011 mg/kg
exhibited no interfering peaks at the retention times, where the
Chlorpyrifos 12.4 mg/kg
35 Soy Pirimiphos Methyl 0.0011 mg/kg
Table 2 Chlorpyrifos 0.0100 mg/kg
Recovery values for all the analytes and RSD% calculated for organophosphate 36 Soy akes <LOQ
compounds. 37 Grain maize Pirimphos Methyl 0.0011 mg/kg
Chlorpyrifos 0.0314 mg/kg
Organophosphate compounds % Recovery % RSD 38 Soy Pirimphos Methyl 0.0011 mg/kg
Diazinon 72 6.0 Chlorpyrifos 0.102 mg/kg
Chlorpyrifos-CH3 80 4.6 39 Soy in grain <LOQ
Parathion-CH3 52 5.8 40 Soy in grain Pirimphos Methyl 0.0011 mg/kg
Pirimiphos-CH3 86 5.2 Chlorpyrifos 0.0261 mg/kg
Malathion 53 6.5 41 Soybeans <LOQ
Chlorpyrifos 105 8.2 42 Grain maize Pirimiphos Methyl 0.0011 mg/kg
Parathion 74 7.2 43 Grain maize Pirimphos Methyl 0.0104 mg/kg
Pirimiphos C2H5 72 4.8 Chlorpyrifos 0.0057 mg/kg
Ethion 73 5.9 44 Soy Pirimiphos Methyl 0.0011 mg/kg
Deltamethrin 66 4.7 45 Grain maize <LOQ
 D. Marchis et al. / Food Control 25 (2012) 270e273
Recoveries were estimated by spiking at 0.020 mg kg
1 20
maize samples. Samples were extracted using the modied
QuEChERS method with simultaneous clean-up. The results
showed that the recoveries ranged between 60% and 105%, except
for malathion which showed a mean recovery of 53%. Relative
standard deviation percentage (RSD%) values were below of 9% for
all the analytes. Data about recoveries and RSD% are reported in
Table 2.
Finally, the modied QuEChERS method studied was applied
to analyse 45 in eld samples. The matrices tested were maize
(21 samples) and soy (24 samples). Results are reported in
Table 3 Organophosphate compounds were detected in 30
samples, while no deltamethrin was found. Pirimiphos Methyl
was quantied at LOQ level in 26 samples and in four samples it
was found at higher level than the LOQ. Chlorpyrifos was found
at LOQ level in one sample and at higher level then LOQ in eight
samples.
In samples 17, 22, 29, 34, 35, 37, 38, 40, 43 (9 in total) both
Pirimiphos Methyl and Chlorpyrifos were identied.
4. Conclusion
A rapid and efcient procedure for extraction and simultaneous
liquideliquid partition clean-up has been developed for determi-
nation of 10 pesticides in cereals.
It was introduced a new liquideliquid partition step, adding
both water and iso-octane to the acetonitrile extracts. Water was
added prior to the partition step to facilitate the separation of the
two layers. This process produced cleaner and more concentrate
extracts. The nal extract was suspended in iso-octane rather than
acetonitrile, in order to avoid possible troubles for the large
expansion volume of acetonitrile in the GC injector. As a result, the
addition of this nal step in the QuEChERS extraction method,
would allow the simultaneous extraction of both organophosphate
and organochlorurate pesticides (data not shown).
Indeed, the law limits for those pesticides xed in European
Regulations, were efciently reached by applying this method. The
range of recoveries, precision observed and the RSD% values
showed to be satisfying; values were not adjusted for recover,
considering that the QuEChERS method was designed for screening
purposes.
Chlorpyriphos and Pirimiphos Methyl were detected at LOQ
values and at higher levels in 30 samples. For these two
compounds, there are no law limits on feed, whilst limits on food
were xed. The EU limit set for Pirimiphos Methyl in cereals is
5 mg kg
1 and samples 8, 11, 17, 43 showed to be all far below
0.2 mg kg
1, therefore they could all be considered as compliant.
The European Union law xed a really low limit for Chlorpyrifos in
cereals (0.05 mg kg
1), and only samples 17, 29, 37, 40 and 44 could
be considered as compliant.
273
Acknowledgements
This study has been carried out with the nancial support of the
Italian Ministry of Health.
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