Food Analytical Methods

https://doi.org/10.1007/s12161-018-1333-8

A Fast and Easy QuEChERS-DLLME Method Combined with GC-MS

for Ethion and Bifenthrin Residues Determination and Study of Their
Dissipation Dynamics in Palm Dates
Sherif B. Abdel Ghani 1,2 & Saleh S. Alhewairini 1 & Svetlana Hrouzková 3

Received: 7 May 2018 / Accepted: 22 July 2018

# Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
New fast and efficient analytical method based on QuEChERS followed by dispersive liquid-liquid microextraction (DLLME)
was developed for extraction and pre-concentration of residues of ethion and bifenthrin insecticide/acaricides in palm date matrix.
GC-MS was used for quantification of the pesticides. The developed method was validated according to the European regula-
tions’ requirements. Method accuracy was 102.2% and 96.1% with a precision of 8.4% and 4.3% (intra-day) and 7.9 and 5.1%
(inter-day) for ethion and bifenthrin, respectively. The validated QuEChERS and QuEChERS-DLLME methods were compared.
Applying DLLME for preconcentration of pesticides lowered the limit of quantification nearly 20 times. By adding the DLLME
step using chlorobenzene as extractant, the method achieved better sensitivity and enrichment factor. Matrix effects were
evaluated. Developed methods were utilized in analysis of real samples originated from field trial and markets. For field trial,
pre-harvest interval (PHI) and dissipation kinetics were investigated. QuEChERS-DLLME method detected ethion in all market
samples and bifenthrin in two of them.

Keywords Ethion . Bifenthrin . QuEChERS . Dispersive liquid-liquid microextraction . Palm dates . Pre-harvest interval

Introduction acaridae mite species, with 27 of them reported as fruit and

Date palm, Phoenix dactylifera, is a widely cultivated tree in
tropical and subtropical latitudes, for its sugar-rich fruit called
date. Palm date is consumed fresh such as Barhi cultivar, soft
(Rottab), semi-dried (Sukari), and dried (Abremi). Date palm
is cultivated in over 40 countries in five continents with a total
production of about 7.8 million metric tons (FAO Statistics
2014). Palm date is consumed worldwide in about 180 coun-
tries (FAO Statistics 2014). Middle Eastern countries have the
highest consumption rate reaching 41.8 g/person/day (GEMS/
Food 2003). Date palm is infested by about 132 insect and
* Svetlana Hrouzková
svetlana.hrouzkova@stuba.sk
1
Plant Production and Protection Department, College of Agriculture
and Veterinary Medicine, Qassim University, P.O. Box 6622,
Buraydah, Al-Qassim 51452, Saudi Arabia
2
Department of Plant Protection, Faculty of Agriculture, Ain Shams
University, Cairo, Egypt
3
Faculty of Chemical and Food Technology, Institute of Analytical
Chemistry, Slovak University of Technology in Bratislava,
Radlinského 9, 812 37 Bratislava, Slovak Republic
related stalks’ pests (El-Shafie et al. 2017). Lesser date moth
(Batrachedra amydraula) and old world date mite
(Oligonychus afrasiaticus) are the most damaging fruit’s pests
(Blumberg 2008 and El-Shafie 2012). Insecticides with acar-
icidal activity are the most applied ones in order to control
both insect and mite date palm pests.
Pesticides are intentionally applied to crops to protect them
from loss and to enhance the quality of the product. Residues
of the applied pesticides remain in the produce causing, even-
tually, risk to human health. Legislations are being made and
updated every day to cope with the increasing number of
pesticides and the accumulated information about their health
and environmental impacts. High costs of monitoring pro-
grams for pesticide residues as well as environmental con-
cerns encourage analytical chemists to improve old methods
and to develop new ones. Miniaturization approaches de-
creased sample size and hence cost and time, and are environ-
mentally benign.
Conventionally, QuEChERS sample preparation is used for
analysis of dates as a high sugar content matrix and, some-
times, with low water content (Picó et al. 2018). This tech-
nique is a sample preparation without preconcentration of
analytes (Hrouzková 2017).

At present, microextraction techniques are of interest be-
cause of their high potential for fast, safe, and easy procedure
with low solvent consumption and low waste production
(Andraščíková et al. 2015). One of them, dispersive liquid-
liquid microextraction (DLLME) was developed by Assadi
and co-workers (Rezaee et al. 2006) to extract polyaromatic
hydrocarbons (PAHs) from water samples. The technique uses
a mixture of two solvents—dispersive solvent and extractive
solvent. The disperser is miscible with both the liquid sample
and the extractive solvent. Extractive solvent is not miscible
with water sample. The extracting mixture is dispersed into
the water sample using a syringe. The mixture forms a tem-
porary suspension with a cloudy appearance. This suspension
allows analyte transfer to extractive solvent via the high sur-
face area interface. Extractive solvent is separated by proper
centrifugation force then taken using a glass syringe (Rezaee
et al. 2006) or solidified by cooling (Khalili-Zanjani et al.
2007, and Farahani et al. 2008). Extractive solvent can be of
higher or lower density than water. Since the first introduction,
several papers reported different dispersive solvents such as
acetone, methanol, and acetonitrile (Berijani et al. 2006;
Panagiotou et al. 2009; Andraščíková and Hrouzková 2016)
and extractive solvents (Zhao et al. 2007; Pang et al. 2017),
dispersing ways (Du et al. 2010; Yang et al. 2011; Peng et al.
2015), and automation (Melwanki and Fuh 2008). The effect
of salting and pH was also extensively investigated (Peng et
al. 2015; Xu et al. 2015; Andraščíková and Hrouzková 2016).
Marketing of organic farming products is under higher de-
mands of quality than the conventional ones. Residues of in-
organic fertilizers and pesticides are of the main concern.
Multiseries hyper-markets also stipulate lower pesticide resi-
due content than known MRLs in products of their contract
producers. Sometimes, a pre-concentration step is required to
reach satisfactory detection of some compounds. Evaporation
is the most adopted method in routine laboratories. Semi-
volatile pesticides might be lost by such means. DLLME
can be used as a pre-concentration step after first step
QuEChERS extraction in food stuff to satisfy validation pa-
rameters. Up to now, different sample-pretreatment proce-
dures have been proposed due to the wide variety of pesticides
used and the inherent complexity of the matrices
(Andraščíková et al. 2013). In the past, DLLME required so-
phisticated non-disposable tools that necessitated cleaning be-
tween samples. In addition, complicated calculations might be
involved. These conditions constrain the implementation of
DLLME in high throughput or routine labs. Simplification is
one of the trends that invited attention in DLLME develop-
ment avenues (Leong et al. 2014).
The present study implemented GC-MS for the determina-
tion of ethion and bifenthrin pesticides’ residues in dates. The
aim of the paper was fast and easy method development and
validation with the target on trueness of the methodology in
terms of recovery assays. The main objective was to compare
Food Anal. Methods
two sample preparation methodologies, QuEChERS and
QuEChERS-DLLME methods in terms of calibration data,
LODs, LOQs, and precision at low concentration levels. The
methods were applied to real-life samples and in field trial
with the aim to estimate pre-harvest interval (PHI) and to
study the dissipation kinetics.
Experimental
Chemicals
Acetonitrile (HPLC grade) was purchased from Sigma-
Aldrich, France. Chloroform, chlorobenzene BzCl, and gla-
cial acetic acid were obtained from BDH Ltd. (Pool, England).
Carbon tetrachloride was procured from Merck, Darmstadt,
Germany. Ethion (99.9%) and bifenthrin (99.9%) (Fluka®)
pure reference standards were purchased from Sigma-
Aldrich (St Louis, MO, USA). Stock standard solution at
100 mg/L was prepared in acetonitrile. Working standard so-
lutions were prepared by dilution with acetonitrile.
Commercial formulations of ethion, Ethio, 40% emulsifiable
concentrate (EC) and bifenthrin, Bifenamin, and 10% EC
were bought from the local market. Anhydrous MgSO4 was
purchased from across (USA). NaCl was obtained from
Merck (Germany). Bulk PSA (silica gel base modified by
ethylenediamine-N-propyl phase that contains both primary
and secondary amines) was obtained from Supelco® (USA).
Matrix-Matched Standards and Samples
Dates, Barhi cultivar, grown in the University Farm without
application of pesticides (content of studied pesticides in dates
was checked before utilization by QuEChERS and GC-MS)
were used as a blank matrix in recovery experiments and for
the preparation of matrix-matched multi-level calibration stan-
dard solutions. The matrix-matched standard solutions were
prepared by adding working pesticide solutions in acetonitrile
of respective concentration to the extract of date samples. The
matrix-matched calibration solutions were used for the evalu-
ation of linear range, LODs and LOQs and repeatability.
Real date samples (fruit) were obtained from local super-
markets. Samples were destoned and comminuted and kept
frozen till analysis.
Sample Preparation
QuEChERS
Frozen samples of dates were comminuted using laboratory
mixer Retsch GM-200 (Retsch Gmbh, Haan, Germany). For
QuEChERS method, 10 g of frozen comminuted sample was
weighed in 50-mL centrifuge tube and 10 mL of 1% acetic
Food Anal. Methods
acid in acetonitrile (MeCN) was added. Tubes were hand
shaken for 1 min. A pre-weighed mixture of anhydrous mag-
nesium sulfate (4 g) plus sodium chloride (1 g) were added,
then the tubes were hand shaken again for 1 min. Samples
were centrifuged (Centra MP4R centrifuge, IEC, MA USA)
under cooling (5 °C) at 4400 rpm for 5 min. An aliquot of the
extract (upper layer) of volume 1 mL was transferred to a 2-
mL centrifuge tube containing 500 mg anhydrous magnesium
sulfate and 50 mg PSA, tubes were hand shaken for 1 min, and
then centrifuged for 5 min at 4400 rpm. The cleaned extract
was transferred into 1.5 mL PTFE capped glass vial (Agilent
Technologies, USA) and directed to chromatographic analy-
sis. The cleaned extract of untreated (blank) date samples was
used for matrix-matched standard solution preparations and
for the investigation of the matrix effects. The extracts of real
samples were diluted, where required, using blank extracts.
QuEChERS-DLLME Method
Three grams of comminuted frozen date sample was weighed
in a 15-mL centrifuge tube and 3 mL of 1% acetic acid in
acetonitrile was added. Tubes were hand shaken for 1 min.
A pre-weighed mixture of anhydrous magnesium sulfate
(1.2 g) plus sodium chloride (0.3 g) was added then the tubes
were hand shaken again for 1 min. Samples were centrifuged
under cooling (5 °C) at 4400 rpm for 5 min. Two milliliters of
aliquot of the extract (upper layer) was transferred to another
15 mL centrifuge tube. One-hundred microliters of BzCl was
added and mixed before 10 mL of cold deionized water was
added and tubes were hand shaken for 1 min. Tubes were
centrifuged at 4400 rpm for 5 min. Ten milliliters of the upper
layer was drawn using an automatic pipette. Sedimented BzCl
(100 μL) was transferred to insert vial using a 100-μL micro-
pipette and subjected to GC-MS analysis (Fig. 1).
Instruments
Determination of ethion and bifenthrin was realized using a
Shimadzu GC/MS-Q2010 Ultra (Shimadzu Corporation,
Japan) connected to a Shimadzu AOC-20i autosampler and
equipped with split/splitless injector (working in the split
mode at the split ratio of 1:5 or 1:10). Injector temperature
was 230 °C. A Restek (USA), Rxi-5 Sil MS fused silica col-
umn, 30 m length × 0.25 mm i.d. × 0.25 μm film thickness,
was used. Oven temperature program was 200 °C for 3 min,
raised to 300 at 15 °C/min, then held for 4 min. Injected
volume was 1 μL. Transfer line temperature was 285 °C.
Electron ionization mode was utilized (70 eV) and ionization
source at 235 °C with a solvent delay time of 3 min in the case
of MeCN and 4 min for BzCl. Initially, MS detector operated
in the scan mode (from 50 to 500 m/z) to select target ions.
Three ions were selected for ethion, namely 231, 171, and
153 m/z and four ions, 181, 166, 153, 182 m/z, for bifenthrin.
The first ion for each pesticide was the most abundant ion
(231 m/z for ethion and 181 m/z for bifenthrin), hence it was
utilized for quantification, while the other ions were used for
confirmation. Subsequently, all samples were analyzed in SIM
mode in 2 segments, one for each compound. Retention time
for ethion was 7.48 min and for bifenthrin was 8.63 min.
Validation of the Method
Recovery and Precision Studies
Recovery experiments were performed at three levels for each
method. 10 (MRL level), 100, and 1000 μg/kg and 1, 5 and
10 μg/kg for QuEChERS and QuEChERS-DLLME, respec-
tively. Blank date puree samples were fortified with the re-
quired concentrations and left for 20 min before analytical
samples were taken for residue analysis. Five replicates were
performed for each concentration. Precision was calculated as
RSD % of recovery from spiked samples at the three spiked
levels in both methods. Inter-day precision was derived from
the analytical method validation data obtained on non-
consecutive days.
Linearity
To evaluate linearity, calibration curves were constructed for
both analytical methods utilizing matrix-matched standards.
For QuEChERS method, the curve was plotted using the con-
centrations of 5, 10, 50, 100, 250, 500, and 1000 μg/L (equiv-
alent to the same concentration in μg/kg in sample).
For QuEChERS-DLLME method, studied concentrations
were 5, 20, 100, 200, 400, and 1000 μg/L that correspond to
0.25, 1, 5, 10, 20, and 50 μg/kg in samples.
Each point was the average obtained by injecting three
replicates of matrix-matched standards, each was prepared
by spiking the newly prepared extract.
Field Trial
Field trials were carried out in the Experimental Farm of
College of Agriculture and Veterinary Medicine, Qassim
University, Qassim, Saudi Arabia. The recommended applica-
tion rate of Ethio® was 100 mL of formulation per 100 L
water, while it was 62.5 mL/100 L for Bifenamin®.
Spraying solution was prepared and was applied using a
Cifarelli motorized air-assisted mist blower sprayer. Two
heavily-laden date palm trees (Barhi cultivar) were selected
for this experiment. Weather conditions were recorded, tem-
perature was between 20.2 and 44.0 °C with average relative
humidity of 20.2% and average 13 h of daylight.
Representative samples were collected at 0 (2 h), 1, 3, 7, 11,
14, 21, 28, and 35 days after application. Date samples (about
2 kg) were collected and prepared following European

Fig. 1 DLLME pre-concentration step
Commission guidelines (European Commission Directive
(2002/63/EC and Commission Regulation (EC) No
178/2006). Untreated (blank) date fruits were collected before
pesticide application. Samples were transferred to the labora-
tory in paper bags. Stalks and stones were removed carefully
using fork and knife. Stones were weighed and the weight was
recorded. Stones’ weight was about 12.1% on average.
Destoned date fruits were comminuted using a laboratory
blender and then kept frozen (− 20 °C) till analysis.
Results and Discussion
Palm dates represent fruits with high sugar content and high
complexity of matrix. Although many pesticides are registered
for the application on palm dates, based on our preliminary
survey of 35 palm date samples, it was revealed that ethion
and bifenthrin are the most frequently used pesticides, as the
residues of these two pesticides occurred in real samples. The
screening by Picó et al. (2018) revealed the presence of ethion
in date samples. Ethion is an organophosphorus insecticide/
acaricide and bifenthrin is a pyrethroid with insecticidal/
acaricidal action. For both pesticides, a maximum residue lim-
it (MRL) of 0.01 mg/kg in palm dates is valid for European
Union, the same value is accepted in Saudi Arabia and Arab
Gulf countries. Bifenthrin belongs to a specific category of
pollutants that may affect the normal hormonal function or
possess endocrine-related functions, known as endocrine
disrupting compounds (Hrouzková and Matisová 2012). The
monitoring of endocrine disrupting pesticide residues in food
is of great interest as pesticides can represent a risk for con-
sumer and also safeguard the biodiversity in the environment
Food Anal. Methods
at lower concentrations as expected and set by MRLs.
Analyzing pesticide residues at low concentration levels re-
quires multistep sample preparation including cleaning and
preconcentration of the resulting extract. For this purpose,
the comparison of QuEChERS and QuEChERS combined
with DLLME with the focus on validation parameters was
realized.
Method Development and Validation
Two sample preparation methods were compared. Original
QuEChERS procedure (Anastassiades et al. 2003) was ap-
plied to 10 g of date samples and 10 mL of MeCN was utilized
with the cleaning procedure given in the BSample
Preparation^ section. The QuEChERS-DLLME method was
developed with the aim to reach high enrichment factor in
comparison to the original QuEChERS method, which pro-
vides no enrichment of analytes. Combination of QuEChERS
and DLLME (Andraščíková and Hrouzková 2016) was mod-
ified according to our needs and optimized for specific use for
high-sugar-content matrix, particularly dates, with the aim to
simplify the procedure and to reach high efficiency for this
particular application. To this end, the first step of the modi-
fied QuEChERS methodology (liquid-liquid extraction) was
followed by the second step—DLLME. For the first step,
reduction of extractive solvent volume was proposed. MeCN
was shown as an effective extractant for original QuEChERS,
therefore, for the first step of extraction, MeCN was used.
Three grams of comminuted sample was taken for the
QuEChERS-DLLME method as the minimum weight to pro-
duce 2 mL of extract to be used for the second step. Higher
volumes of the extract would stay unused, therefore, to keep
Food Anal. Methods
the method ecological, volumes were selected with the aim to
generate the lowest possible waste.
Three different solvents were investigated as the extracting
solvents for DLLME, namely chloroform CHCl3, carbon tet-
rachloride CCl4, and benzene chloride BzCl. The chemical
nature of extractive solvent critically determines the ability
to extract the target pesticides. For initial experiments, a
100 μL volume of solvent was employed. Different volumes
of water 6, 8, and 10 mL were tested. Initially, 6 mL of water
was added, but BzCl was not sedimented and both, chloro-
form and carbon tetrachloride, sedimented in substantially
higher volumes than added. Addition of 8 mL of water to
BzCl led to a higher volume sedimented than added (about
170 μL). The retrieved volumes are summarized in Table 1.
After addition of 10 mL of water, full sedimentation and phase
separation for the three solvents were obtained. A volume of
10 mL water was used in the subsequent experiments in order
to investigate and compare the efficiency and extraction be-
havior of the three solvents. Cold water (at the temperature cca
5–8 °C) was utilized to maintain low temperature of the cen-
trifuge tube and to enhance phase separation. Under such con-
ditions, the retrieved amount of chloroform and carbon tetra-
chloride was about 70 to 80 μL while in the case of BzCl it
was around 100 μL. Obtained enrichment factors were 30.63,
28.12, and 20.87 for ethion and 27.84, 25.6, and 19.06 for
bifenthrin in CHCl3, CCl4, and BzCl, respectively (Fig. 2).
Higher enrichment factors for CHCl3 and CCl4 are attributed
to the lower sedimented volume and volatility of both solvents
during transfer. This was not the case for BzCl where the
sedimented extract volume was the same as the added volume
of extractive solvent. The found enrichment factor was close
to the theoretical one. For that reason, BzCl was chosen as the
extractive solvent. BzCl has also a high boiling point (131 °C),
therefore, the volume of the extract will remain stable at am-
bient temperature longer than for more volatile solvents.
Comparison of Sensitivity-Direct Injection Vs.
Experiment Employing DLLME Step with BzCl
Sensitivity was tested over 6 levels of concentrations, namely
1, 2, 5, 10, 20, and 50 μg/L of both compounds in MeCN.
DLLME step was performed using BzCl. Standard solutions
with the expected concentrations (20 times higher than the
concentrations of solutions in MeCN prepared without extrac-
tion procedure), namely 20, 40, 100, 200, 400, and 1000 μg/L
of both compounds were prepared in BzCl and injected.
Measured areas employing DLLME step were plotted against
the areas of the 20 times more concentrated solutions without
DLLME step, representing 100% recovery.
R2 values of the obtained curves were more than 0.9988
proving good representation of the regression line and linear-
ity over this range. In order to evaluate correlation between the
performed DLLME and theoretical concentrations, correlation
Table 1 Approximate sedimented volume (in μL) of tested extractive
solvents (100 μL added) as a function of the volume of added water
Water added, mL BzCl CHCl3 CCl4
6 – 190 195
7 200 Not done Not done
8 170 140 90
10 100 80 85
coeffecient and two tailed t test in pairs, using EXCEL 2013,
were performed. Correlation coeffecients were 0.9997 and
0.9998 for ethion and bifenthrin, respectively indicating high
similarity of measured areas in DLLME and expected concen-
trations’ standards. Also, t test statistical analysis showed
no significant difference between the measured areas at
95% confidence level (Ps value 0.05). P values were higher
than 0.05. P value was ≤ 0.55 for ethion and ≤ 0.26 for
bifenthrin. The obtained results prove the ability of BzCl
to extract both compounds from MeCN extract over the
tested range. Further, they confirm the consistency of the
20-fold enrichment factor.
Recovery and Repeatability
Recovery experiments were performed at three levels for each
method. The blank samples, checked for the presence of
bifenthrin and ethion, were spiked within the corresponding
amount of working standard solution. Recovery percentages
(Table 2) were within the range recommended by the
European Commission (SANTE Document 2017). In case of
QuEChERS, the minimum recovery was 86.04% for
bifenthrin and maximum was 113.31% for ethion, while it
was 94.22% for bifenthrin and 112.47% for ethion for
QuEChERS-DLLME method. Repeatability (RSD %) was
in acceptable range for both methods and compounds. It
ranged from 4.0 to 11% for QuEChERS and from 1.6 to 8%
for QuEChERS-DLLME method. However, it reached 16%
for ethion at 1 μg/kg level only.
Intra-day pecision expressed as average RSDs % of all
spiked levels (Table 3) was 9.1 and 7.7 (QuEChERS) and
8.4 and 4.3 (QuEChERS-DLLME) for ethion and bifenthrin,
respectively (Table 3). The inter-day precision, as average
RSDs % for all tested levels were 11.1 and 7.9 (ethion) and
9.3 and 5.1 (bifenthrin) for QuEChERS and QuEChERS-
DLLME, respectively.
Linearity, LODs, and LOQs
Calibration curves were constructed using matrix-matched
(dates) standards in order to compensate for the matrix effect.
Coefficients of determination R2 of calibration curves for both
studied methods using 6 points of concentrations are
 Food Anal. Methods

Fig. 2 Enrichment factor for

ethion and bifenthrin at 10 μg/L
level in DLLME using various
extractive solvents (CHCl3, CCl4,
and BzCl)

summarized in Table 3. R2 values were very close to the value
of unity B1^ showing good fit of the regression line with the
actual measurements’ data.
LODs and LOQs were calculated by the MS software from
the signal to noise ratio (S/N) measured at the LCL (lowest
calibration level) (six replicates). S/N values of three and ten
were used for the calculation of LOD and LOQ, respectively.
For very low concentrations, newly developed method
emplying DLLME provides superior results in terms of
LODs and LOQs (Table 3).
Matrix Effect
A crucial problem in determination of pesticide residues in
complex fruit samples is matrix effect. Matrix effect (ME)
was evaluated at a level of 10 μg/kg of both compounds in
date sample. Matrix effect was calculated using the following
Eq. (1):
and the improvement of overall validation parameters. In
order to determine the need for the dispersive solid phase
extraction (dSPE) clean-up step in the combined
QuEChERS-DLLME method, the effect of primary sec-
ondary amine PSA sorbent as a clean-up agent on matrix
effect was assessed. A mixture of both studied pesticides
at 10 μg/L each was made up in date matrix extract with
and without PSA clean-up. The effect was determined by
comparing the measured areas for the following: standards
in matrix+DLLME vs. standard in matrix+PSA treatment
by dSPE+DLLME. The experiment was performed in
triplicate. Results showed (Fig. 3) that addition of dSPE
cleaning did not increase significantly the peak areas.
Therefore, the analyses by DLLME-QuEChERS method
were realized without additional clean-up step.
Application of the Developed Methods to Real-Life
Samples

ðX 2−X 1Þ Field Samples

ME% ¼  100
X1
where X1 is the measured area in matrix-free standard and X2
is the measured area in matrix-matched standard. Obtained
values of matrix effect are summarized in Table 3.
Although the matrix effects are quite low for both treat-
ments, at ultratrace concentration region, additional
cleaning step could bring higher degree of selectivity
ð1Þ
Data in Table 4 were obtained by QuEChERS analysis of field
samples. QuEChERS-DLLME was only utilized to analyze
samples on the 28th and 35th days. Results using
QuEChERS and QuEChERS-DLLME methods for the two
samples were close within ± 3.65%.
Initial deposit of ethion was about six times that of
bifenthrin, which is in good agreement with the sprayed rate

Table 2 Recovery (%) ± RSD

(%) (n = 5) at spiked level (μg/kg) QuEChERS QuEChERS-DLLME
for QuEChERS and QuEChERS-
DLLME methods 10a 100 1000 1 5 10a

Ethion 113.3 ± 6.5 92 ± 10 96 ± 10 103 ± 16 112.5 ± 2.3 104.3 ± 5.2

Bifenthrin 101.3 ± 4.0 86.0 ± 8.1 96 ± 11 94.2 ± 8.0 102.4 ± 1.6 97.2 ± 3.3
a
MRL level
Food Anal. Methods

Table 3 Validation parameters of QuEChERS and QuEChERS- Table 4 Residues (μg/kg) and dissipation parameters of ethion and
DLLME methods bifenthrin in field samples

QuEChERS QuEChERS-DLLME Days Ethion Bifenthrin

Ethion Bifenthrin Ethion Bifenthrin 0 3238.4 526.5

1 982.8 227.6
Accuracy/% 100.5 94 102.2 96.1
3 754.3 195.0
Precision (intra-day)/% 9.1 7.7 8.4 4.3
7 328.9 119.1
Precision (inter-day)/% 11.1 9.3 7.9 5.1
11 292.0 57.4
LCL/μg/kg 5 5 0.25 0.25
14 167.2 42.1
Calibration data points 6 6 6 6
21 88.0 21.7
Range/μg/kg 5–500 5–500 0.25–50 0.25–50
28 63.1 (65.4)b 20.6 (19.9)b
Linearity in matrix 0.9995 0.9991 0.9988 0.9999
35 46.3 (47.5)b 17.7 (17.3)b
LOD/μg/kg 1.2 0.24 0.056 0.011
k (days−1) 0.1213 0.0969
LOQ/μg/kg 4.1 0.79 0.19 0.036
Intercept 7.0634 5.4968
Matrix effect/%a 6.61 12.27 11.21 10.99
r − 0.9354 − 0.9292
a
At the concentration level of 10 μg/kg R2 0.8751 0.8635
t ½ (days) 3.34 0.827
PHI class (days)a 49 42
of both compounds. Dissipation rate k was calculated from
Eq. (2) (California Department of Pesticide Regulation 2012). a
According to EC guidelines (Document No. 7039/VI/95 EN 1997)
b
ln Y ¼ ln A−kt ð2Þ QuEChERS-DLLME results

Where ln Y is the natural logarithm of residue amount in
mg/kg at time (t), ln A is the natural logarithm of the initial
deposit in mg/kg, k is the dissipation rate constant in days−1
and t is time in days. Regression lines were plotted using
natural logarithm of residues in dates against time, from which
the intercepts, r, R 2, and t ½ were calculated (Table 4).
Dissipation behavior of both compounds followed first order
kinetics. The dissipation rate of ethion was 1.25 times faster
than that of bifenthrin. Correlation coefficient r showed strong
negative relation of residues with time. Determination
coefficient R2 was around 0.87 (ethion) and 0.86 for bifenthrin
indicating good fit of the regression lines to field data. The
MRL of both compounds is 10 μg/kg (EU MRL 2018).
Because of practical limitations, 35 days was the final interval
for sampling. After this time, date fruits began to soften, which
affects matrix properties and at this stage this date cultivar
(Barhi) loses its commercial value. PHIs were estimated using
Eq. (2) for the abovementioned EU MRL (as t when Y equals
10 μg/kg). PHI was 47.6 days for ethion and 40.9 days for
bifenthrin. PHI values can be rounded-up to be 49 and 42 days

Fig. 3 Comparison of the peak

area of analytes with and without
the extract cleaning by PSA
 Food Anal. Methods

for ethion and bifenthrin, respectively (Table 4) according to Conclusions

European Commission guidelines for pre-harvest interval
classes (Document No. 7039/VI/95 EN 1997).
Market Samples
Twenty two samples of different types of dates were collected
from supermarkets in Buraidah, Qassim, Saudi Arabia during
June 2017 season.
Residues of ethion and bifenthrin in market samples are
presented in Table 5. Analysis of market samples showed that
residues of both compounds determined using both methods
were closely matching.
MRL for both compounds in dates are 10 μg/kg. For
QuEChERS, only one sample contained quantifiable residues
of ethion (25.3 μg/kg). In case of QuEChERS-DLLME, two
samples, No. 1 and No. 3, contained 0.073 and 0.092 μg/kg of
bifenthrin, respectively. Samples 1–3, 12, and 14–22
contained ethion residues (Table 5). Only one sample (sample
20) exceeded MRL as it contained 25.3 (QuEChERS) or
27.4 μg/kg (QuEChERS-DLLME) of ethion. Utilizing
DLLME step enhanced the ability of the analytical method
to detect lower residue levels of both compounds. This clearly
demonstrates the advantage of the new developed method to
detect pesticide residues at ultra-trace level concentrations,
which are lower than levels checked by conventionally
adopted methods such as QuEChERS. Although concentra-
tion levels of residues were lower than MRLs, ultrasensitive
method such as QuEChERS-DLLME may shed light on the
history of ethion and bifenthrin usage on the produce and
might help in detection of compounds in organic farm
products.
The combination of QuEChERS-DLLME followed by GC-
MS was introduced and compared to the original QuEChERS
with GC-MS. It was shown that the new method is simple and
fast and enables a rapid and inexpensive sample pretreatment
with up to 20-fold higher enrichment factor and lower detec-
tion limits in comparison to the original one. The introduced
extraction procedure combined with GC-MS method provid-
ed sufficient accuracy and repeatability.
The applicability of the developed and validated
methods was demonstrated by real-life sample analyses
showing that the developed GC-MS methods in both,
QuEChERS and QuEChERS-DLLME sample preparation
mode, are suitable for the analysis of ethion and bifenthrin
at levels lower than MRL in palm date samples. For very
low concentrations, the newly developed method emplying
DLLME provides superior results in terms of LOQ. PHI
and dissipation rate of both compounds in dates were
investigated and discussed. One market sample was found
to exceed the MRL for ethion.
Funding Information Authors thank Saleh Kamel Chair for Date Palm
Research (SKCDR), Qassim University for funding this work.
Compliance with Ethical Standards
Conflict of Interest Sherif B. Abdel Ghani declares that he has no con-
flict of interests. Saleh S Alhewairini declares that he has no conflict of
interests. Svetlana Hrouzková declares that she has no conflict of
interests.
Ethical Approval This article does not contain any studies with human
participants or animals performed by any of the authors.

Table 5 Residue levels (μg/kg) of ethion and bifenthrin in marketed Informed Consent Not applicable.
positive samples using QuEChERS and QuEChERS-DLLME

QuEChERS DLLME-QuEChERS
References
Ethion Bifenthrin Ethion Bifenthrin
Anastassiades M, Lehotay SJ, Štajnbaher D, Schenck FJ (2003) Fast and
1 0.19 0.073 easy multiresidue method employing acetonitrile extraction/
2 3.70 partitioning and Bdispersive solid-phase extraction^ for the determi-
nation of pesticide residues in produce. J AOAC Int 86:412–431
3 3.43 0.092 Andraščíková M, Hrouzková S (2016) Fast preconcentration of pesticide
12 0.58 residues in oilseeds by combination of QuEChERS with dispersive
14 1.83 liquid–liquid microextraction followed by gas chromatography-
15 0.19 mass spectrometry. Food Anal Methods 9:2182–2193
Andraščíková M, Hrouzková S, Cunha SC (2013) Combination of
16 0.19 QuEChERS and DLLME for GC-MS determination of pesticide
17 0.29 residues in orange samples. Food Addit Contam Part A 30(2):
18 0.22 286–297
19 0.31 Andraščíková M, Matisová E, Hrouzková S (2015) Liquid phase
microextraction techniques as a sample preparation step for analysis
20 25.3 27.4 of pesticide residues in food. Sep Purif Rev 44(1):1–18
21 3.18 Berijani S, Assadi Y, Anbia M, Milani Hosseini MR, Aghaee E (2006)
22 2.13 Dispersive liquid–liquid microextraction combined with gas
chromatography-flame photometric detection: very simple, rapid
Food Anal. Methods
and sensitive method for the determination of organophosphorus
pesticides in water. J Chromatogr A 1123:1–9
Blumberg D (2008) REVIEW: date palm arthropod pests and their man-
agement in Israel. Phytoparasitica 36(5):411–448
California Department of Pesticide Regulation, SOP METH009.01.
Calculation of Pesticide Half-life from a Terrestrial Field
Dissipation Study, 2012. http://www.cdpr.ca.gov/docs/emon/pubs/
sops/meth009_01.pdf. Accessed 25 Sept 2017
Commission Regulation (EC) No 178/2006 of 1 February 2006
amending Regulation (EC) No 396/2005 of the European
Parliament and of the Council to establish Annex I listing the food
and feed products to which maximum levels for pesticide residues
apply. Official Journal of the European Union (2006), L29, 3–24,
available: https://www.fsai.ie/uploadedFiles/Legislation/Food_
Legisation_Links/Pesticides_Residues_in_food/Commission_
Regulation_EC_No_178_2006.pdf
Document No. 7039/VI/95 EN of 22 July 1997 Appendix I, calculation of
maximum residue levels and safety intervals, e.g. pre-harvest inter-
vals. p 8, available: https://ec.europa.eu/food/sites/food/files/plant/
docs/pesticides_mrl_guidelines_app-i.pdf
Du JJ, Yan HY, She DD, Liu BM, Yang GL (2010) Simultaneous deter-
mination of cypermethrin and permethrin in pear juice by
ultrasound-assisted dispersive liquid–liquid microextraction com-
bined with gas chromatography. Talanta 82:698–703
El-Shafie HAF (2012) Review: list of arthropod pests and their natural
enemies identified worldwide on date palm, Phoenix dactylifera L.
Agric Biol J N Am 3(12):516–524
El-Shafie HAF, Abdel-Banat BMA, Al-Hajhoj MR (2017) Arthropod
pests of date palm and their management. CAB Rev 12(049):1–18
EU pesticides database (2018) http://ec.europa.eu/food/plant/pesticides/
eu-pesticides-database/public/?event=homepage&language=EN .
Accessed 25 Jul 2018
European Commission Directive (2002/63/EC). Establishing Community
Methods of Sampling for the Official Control of Pesticide Residues
in and on Products of Plant and Animal Origin (2002) Official
Journal of the European Communities, p 40
FAO, Food and Agriculture Organization, FAOSTAT, 2014. http://www.
fao.org/faostat/en/#data. Accessed 25 Sept 2017
Farahani H, Yamini Y, Shariati S, Khalili-Zanjani MR, Mansour-Baghahi
S (2008) Development of liquid phase microextraction method
based on solidification of floated organic drop for extraction and
preconcentration of organochlorine pesticides in water samples.
Anal Chim Acta 626:166–173
GEMS/Food. Global Environment Monitoring System/Food
Contamination Monitoring and Assessment Programme Regional
per Capita Consumptionof Raw and Semi-processed Agricultural
Commodities, WHO: Geneva, 2003, 18 p. http://www.who.int/
foodsafety/chem/gems_regional_diet.pdf
Hrouzková S (2017) Analytical methods for pesticides detection in food-
stuffs. In: Spizzirri UG, Cirillo G (Eds) Food analysis: innovative
analytical tools for safety and quality assessment. John Wiley &
Sons, Hoboken, New Jersey and Scrivener Publishing, Beverly,
MA, 221–269
Hrouzková S, Matisová E (2012) Endocrine disrupting pesticides. In:
Soundararajan RP (ed) Pesticides- advances in chemical and botan-
ical pesticides. InTech, Rijeka, pp 99–126
Khalili-Zanjani MR, Yamini Y, Shariati S, Jönsson JA (2007) A new
liquid-phase microextraction method based on solidification of
floating organic drop. Anal Chim Acta 585:286–293
Leong M-I, Fuh M-R, Huang S-D (2014) Beyond dispersive liquid–liq-
uid microextraction. J Chromatogr A 1335:2–14
Melwanki MB, Fuh MR (2008) Dispersive liquid–liquid microextraction
combined with semi-automated in-syringe back extraction as a new
approach for the sample preparation of ionizable organic com-
pounds prior to liquid chromatography. J Chromatogr A 1198–
1199:1–6
Panagiotou AN, Sakkas VA, Albanis TA (2009) Application of chemo-
metric assisted dispersive liquid–liquid microextraction to the deter-
mination of personal care products in natural waters. Anal Chim
Acta 649:135–140
Pang L, Yang H, Yang P, Zhang H, Zhao J (2017) Trace determination of
organophosphate esters in white wine, red wine, and beer samples
using dispersive liquid-liquid microextraction combined with ultra-
high-performance liquid chromatography–tandem mass spectrome-
try. Food Chem 229:445–451
Peng GL, He Q, Mmereki D, Zhou GM, Pan WL, Gu L, Fan LL, Tang
XH, Chen JH, Mao YF (2015) Vortex-assisted liquid–liquid
microextraction using a low-toxicity solvent for the determination
of five organophosphorus pesticides in water samples by high-
performance liquid chromatography. J Sep Sci 38:3487–3493
Picó Y, El-Sheikh MA, Alfarhan AH, Barceló D (2018) Target vs non-
target analysis to determine pesticide residues in fruits from Saudi
Arabia and influence in potential risk associated with exposure.
Food Chem Toxicol 111:53–63
Rezaee M, Assadi Y, Hosseini MRM, Aghaee E, Ahmadi F, Berijani S
(2006) Determination of organic compounds in water using disper-
sive liquid–liquid microextraction. J Chromatogr A 1116:1–9
SANTE Document 2017. Guidance document on analytical quality con-
trol and validation procedures for pesticide residues analysis in food
and feed. Document No. SANTE/11813/2017 Available from:
https://ec.europa.eu/food/sites/food/files/plant/docs/pesticides_mrl_
guidelines_wrkdoc_2017-11813.pdf
Xu XY, Jing-Qing Y, Jing N, Zu-Guang L, Maw-Rong L (2015) A new
liquid–liquid microextraction method by ultrasound assisted salting-
out for determination of triazole pesticides in water samples coupled
by gas chromatography-mass spectrometry. Anal Methods 7:1194–
1199
Yang ZH, Liu Y, Lu YL, Wu T, Zhou ZQ, Liu DH (2011) Dispersive
suspended microextraction. Anal Chim Acta 706:268–274
Zhao E, Wenting Z, Lijun H, Shuren J, Zhiqiang Z (2007) Application of
dispersive liquid–liquid microextraction for the analysis of organo-
phosphorus pesticides in watermelon and cucumber. J Chromatogr
A 1175:137–140