Food Anal.
Methods
DOI 10.1007/s12161-017-0875-5
Detection of Organophosphorus Pesticide Residues in Leaf
Lettuce and Cucumber Through Molecularly
Imprinted Solid-Phase Extraction Coupled to Gas
Chromatography
Jian Chen 1 & Wen-ting Zhang 1 & Yang Shu 1 & Xin-hui Ma 1 & Xian-ying Cao 1
Received: 28 December 2016 / Accepted: 15 March 2017
# Springer Science+Business Media New York 2017
Abstract How to determine multipesticide residues in vege-
tables is important for the modern society. The development
of a simple, rapid, and sensitive method to determine pesticide
residues is significant. Our study used common vegetables—
leaf lettuce and cucumber, and a new molecularly imprinted
polymer (MIP) was synthesized using O,O′-
dimethylthiophosphoryl chloride, an intermediate for the
manufacture of organophosphorus pesticides, as the template,
methacrylic acid as the functional monomer, and ethylene
glycol dimethacrylate as the cross-linker. The adsorption ca-
pacities of the prepared MIP toward five organophosphorus
pesticides (trichlorfon, dichlorvos, dimethoate, imidacloprid,
and methamidophos) were evaluated, and its potential as a
sorbent for solid-phase extraction was investigated. A novel
method for the simultaneous determination of multiple organ-
ophosphorus pesticide residues in vegetables was developed
by utilizing molecularly imprinted solid-phase extraction
(MISPE) coupled with gas chromatography. Under optimal
solid-phase extraction conditions, with a flow rate of
3.0 mL/min for loading 100 mL, the enrichment factor was
in the range of 36–452 for the five organophosphorus pesti-
cides. The peak area precision of the five organophosphorus
pesticides based on five replicates was from 1.59 to 4.07%.
The leaf lettuce spiked sample was used to verify the fortified
recovery for this method, and the results ranged from 87.48 to
97.85%. The MISPE–gas chromatography method could be
used to detect organophosphorus pesticide residues in lettuce
leaf and cucumber from the farmer’s market, and the detection
* Xian-ying Cao
cxying_02@126.com
1
College of Food Science and Technology, Hainan University,
Haikou 570228, China
limit had no obvious difference from the national standards.
This method can meet the requirements of the analysis of
pesticides in complex matrix.
Keywords Organophosphate pesticides . Molecularly
imprinted solid-phase extraction (MISPE) . Gas
chromatography
Introduction
Pollution caused by food organophosphorus pesticides has
become critical because of the broader area, higher frequency,
and wider usage of organic phosphorus (Chatterjee et al. 2016;
Shen et al. 2007). Traditional sample preparation technique
has been gradually replaced by new extraction technology.
However, even with constantly optimized extraction and elu-
tion conditions, some matrix components were still eluted
down the analyte, and the use of separation is difficult to
effectively remove analytes having similar properties, thereby
resulting in the failure of detection to meet the prescribed limit
(Chen et al. 2010; Savant et al. 2010). Furthermore, basing on
a molecularly imprinted polymer (MIP) as an adsorbent, the
researchers used molecularly imprinted solid-phase extraction
(MISPE) to introduce affinity and specificity of molecular
imprinting to solid-phase extraction technology, making it
one of the leading methods used for sample detection technol-
ogy (Farid et al. 2016; Mohamed et al. 2008).
In recent years, many pretreatment techniques have been
used to detect organophosphorus pesticide residue, such as dis-
persive liquid–liquid microextraction (DLLME), solid-phase
extraction (SPE), supercritical fluid extraction (SFE), solid-
phase microextraction (SPME), matrix solid-phase dispersion
extraction (MSPDE), microwave-aided extraction (MAE), su-
percritical fluid extraction (SFE), and stir bar sorptive extraction

Fig. 1 Schematic representation of the molecularly imprinted polymer
preparation and MISPE–GC procedure used in this study
(SBSE). Among them, SPE is the most widely used and com-
mercialized technique because it is simple, stable, and easily
automated. Currently, C18- and C8-bonded silica are commonly
used as solid-phase sorbents. However, the interaction between
the adsorbent and target in SPE is non-specific, which leads to
poor purification efficiency. Sometimes, it is also difficult to
remove matrix interference, which is unfavorable for the deter-
mination of target analytes. Molecular imprinting technology
possesses the advantages of scheduled choice of specific recog-
nition, practicability, reusability, and long service life. The en-
zyme and substrate used in this process are similar to the anti-
gens and antibodies of natural biological recognition system.
Food Anal. Methods
To achieve efficient enrichment of organophosphorus
pesticide, the use of MIP as a solid-phase extraction
adsorbent has been reported. Chen used MIP as a dis-
persed adsorbent, selectively extracting imidacloprid in
rice and effectively eliminating the interference from
samples matrix. The extraction and clean up were fin-
ished in one step, without the purification step. Under
optimized conditions, the linear range was 0.01 to
1 mg/kg; the low limit of detection (LOD) was
0.024 mg/kg. Wen synthesized atrazine as template mol-
ecule absorbents, successfully extracting atrazine and its
three analogs (simazine, ametryn, and propazine) from
three different models, thereby obtaining good linear
range. The detection limit of the sample was 0.13 to
0.32 mg/kg. Our research synthesizes novel MIP as
solid-phase extraction adsorbents, implying the efficient
enrichment of organophosphorus pesticides in the sam-
ple preparation, significantly reducing the detection limit
of gas chromatography (less than 1 mg/kg).
Imprinted adsorption has attracted considerable interest in
domestic and foreign research. MIP solid-phase extraction is
widely used for actual determination of pesticide residues.
Compared with traditional methods, this technology exhibits
improved selectivity and specificity but retains the advan-
tages of simple operation and minimal time and effort re-
quirement. MIP is used for pretreatment of pesticide residues
to efficiently enrich components. When combined with chro-
matographic detection, MIP can significantly improve the
accuracy and precision of detection and decrease the detec-
tion limit of the instrument (Chatterjee et al. 2016; Jenkins
et al. 2001). However, MIP possesses certain limitation. The
biggest drawback is that the traditional method of the prep-
aration of MIP is highly specific in the detection of the
template molecule. However, with the structural analogs
and other target objects, adsorption quantity is lower with
no selectivity (Guo et al. 2015; Xu et al. 2014). As func-
tional adsorption materials are applied in the sample prepa-
ration and detection of organophosphorus pesticide residues,
the method exhibits selective separation, enrichment, and
highly sensitive detection only to organophosphorus pesti-
cide. However, residual organophosphorus pesticides are of-
ten mixed in various species in agricultural products
(Armenta et al. 2015; Liu et al. 2016; Qi et al. 2014).
Table 1 The effect of the ratio of functional monomer and cross-linker
Imprinted Functional Crosslinking Template Mole
polymer monomers /mol agent ⁄mol molecules ⁄mol ratio
Polymer1 4 8 2 2:4:1
Polymer2 6 16 2 3:8:1
Polymer3 8 20 2 4:10:1
Food Anal. Methods
Fig. 2 FT-IR spectra of a trichlorfon, b imprinted polymer (MIP) after
extraction of the template, and c non-imprinted polymer (NIP)
Therefore, the crux of the problem is how to selectively
prepare multicomponent recognition MIPs (organophospho-
rus pesticides), which is the bottleneck for the development
of bionic adsorption method technology in the field of or-
ganophosphorus pesticide residues. Additionally, as separa-
tion and adsorption of some organophosphorus pesticide
residues occurred in water environment, and most of the
traditional molecular MIPs can only be identified in the or-
ganic phase, the preparation of hydrophilic MIPs with high
selectivity is challenging (John et al. 2010; Satunkhe et al.
2015). Our study synthesized MIPs with selective adsorption
performance for common organophosphorus pesticide, and
its adsorption properties have been characterized. This poly-
mer was used as an adsorbent for solid-phase extraction. We
then established a solid-phase extraction and gas
chromatography-coupled method to conduct rapid detection
of trace organophosphorus pesticide residue in vegetables,
thereby significantly reducing the detection limit of instru-
ments and improving detection sensitivity.
Fig. 3 Scanning electron
micrographs of imprinted
polymer (MIP) (a, ×15,000) and
non-imprinted polymer (NIP) (b,
×15,000)
Materials and Method
Materials
Leaf lettuce and cucumber were purchased from Haidian
farmers’ market of Haikou, Hainan Province, China.
Organophosphorus pesticide standard (trichlorfon, dichlorvos,
dimethoate, imidacloprid, and methamidophos) was bought
from the Drug Inspection of Agriculture Ministry (purity
>99%); O,O′-dimethylthiophosphoryl chloride, ethylene gly-
col dimethacrylate, 2-methylpropionitrile, and
azodiisobutyronitrile were bought from Sigma USA, purity
>99%. Glacial acetic acid, methanol, acetone, chloroform
(analysis grade), and methanol (chromatography grade) were
bought from Beijing Chemical Reagent Co. (Beijing, China).
All other reagents were of analytical grade. Double deionized
water was used throughout the experiment.
Preparation of Universal Organophosphorus Pesticides
MIP
Five kinds of organophosphorus pesticides (0.05 g) as tem-
plate molecules were separately sampled and dissolved in
5 mL O,O′-dimethylthiophosphoryl chloride, adding 0.689 g
methylacrylic acid and oscillating for 30 min to allow the
template molecule and functional monomer fully react. The
crosslinking agent, ethylene glycol dimethacrylate
(EGDMA), 5.946 g and the initiator 2-methylpropionitrile
(AIBN) 100 mg were added, undergoing ultrasound for
20 min after fully immiscible and ventilating with N2 for
20 min. Then, three sealed flasks were placed in a 60 °C water
bath tank, heating for 24 h to obtain MIP. Then, the polymers
were ground with a mortar before passing through a 300-mesh
sieve and placed in a Soxhlet extractor with filter paper.
Glacial acetic acid (50 mL) and methanol (150 mL) were used
as solvent extractors. Furthermore, the sample was subjected
to 24 h continuous extraction until the template molecule was
 Food Anal. Methods

35
b a (dissolved by chloroform) were added, oscillated for 4 h,
adsorption capacity (mg/g)

30 and centrifuged at 4000×g for 30 min. The supernatant was

c filtered through a 0.22-μm membrane. The sample was then
25
measured using gas chromatography (GC) to calculate the
b a
20 combined capacity (Cao et al. 2009; Tiwari et al. 2008).
15 d
c
MIP
e d
10 NIP Adsorption Choice Experiment of Five
e
5 Organophosphorus Pesticides MIP

0 To study the adsorption choice of polymer for five organo-

100 200 300 400 500
phosphorus pesticides, 15 mg MIP was accurately weighed in
organophosphorus pesticide concentration ˄mg/L˅
a 50-mL volumetric flask; 10 mL and 200 mg/L of five organ-
Fig. 4 Adsorption isotherms of the MIP and NIP. Different letters ophosphorus pesticides solution (dissolved by chloroform)
indicated significant difference (P < 0.05)
was added. Oscillation was performed at room temperature
for 240 min. Centrifugation was performed at 4000×g for
produced, employing methanol washing to neutralize the sam-
30 min. Supernatant was filtered using a 0.22-μm membrane.
ple and vacuum drying for 12 h at 60 °C to obtain the polymer
The sample was measured using GC. The combined percent-
we wanted (Fig. 1). The synthesis of non-imprinted polymer
ages of MIP were calculated according to the changes of the
was in accordance with the abovementioned method, except
organophosphorus pesticide concentration before and after
without the template molecules (Chen and Li 2012; Jenkins
adsorption (Tiwari et al. 2008).
et al. 2001; Yang et al. 2016).

Infrared Spectrum Characterization of Trichlorfon MIP
The samples’ infrared spectrum analysis was performed using
the KBr method, with a wave number in the range of 400 to
4000 cm−1. Scanning was conducted for 32 times at a resolu-
tion of 4 cm−1 (Mohamed et al. 2008). Infrared spectrum anal-
ysis of trichlorfon, MIP, and non-imprinted polymer (NIP)
was performed using infrared spectrogram of different groups
of stretching vibration peak to determine whether the template
molecule and functional monomer reacted.
Scanning Electron Microscopy Analysis of MIP
for Universal Organophosphorus Pesticides
MIP and NIP for universal organophosphorus pesticides
underwent scanning electron microscopy analysis. A drop of
nanometer compound was taken from the copper-covered net-
work with membrane, subjected to natural air and 2% phos-
photungstic acid dye for 30 min in the copper network carry-
MISPE–GC Method to Detect Five Organophosphorus
Pesticides
(1) Gas chromatographic conditions
GC-2010 FPD detector; Chromatographic column: RTX-
1701 (30.0 m × 0.25 mm × 0.25 μm). Temperature program
was as follows: maintained at 60 °C at 2.0 min, up to 160 °C at
a rate of 10 °C min−1; maintained at 1.0 min, then increased to
170 °C at a rate of 1 °C min−1; maintained at 2.0 min, up to
265 °C at the rate of 20 °C min−1; and maintained at 5.0 min,
for a total of 35.0 min. Injection port temperature was 240 °C,
and the detector temperature was 270 °C. The carrier was high
purity nitrogen (99.999%) with a total flow of 4.0 mL min−1.
Sample quantity was 1 μL, and the split ratio was 10:1
(Chatterjee et al. 2016; Shen et al. 2007).
(2) Formulation of organophosphorus pesticides standard
curve
40
b
adsorption capacity (mg/g)

ing the nanometer compound. A filter paper was used to ab- 35

sorb the excess dye. Then, the sample was scanned with a 30
a
transmission electron microscope using an acceleration volt- 25 b
age of 80 kV (Chen et al. 2010; Jenkins et al. 2001). 20 a d a
15
MIP
d NIP
Balance Combination Experiment of Five 10 c c c
Organophosphorus Pesticides MIP 5
0
trichlorphon dichlorvos dimethoate imidacloprid methamidophos
Accurately weighed samples of 15 mg MIP and NIP were pesticides
placed in a 50-mL volumetric flask; 10 mL and 100 mg/mL Fig. 5 The selectivity of MIP and NIP for five organophosphorus
to 500 mg/L of five organophosphorus pesticides solution pesticides
Food Anal. Methods

Table 2 Linear equations and

correlations of five Organophosphorus pesticides Linear range (mg/L) Linear regression equation Correlation coefficient
organophosphorus pesticides
Trichlorfon 0.05–10.0 Y = 6335X − 1722.4 0.9983 ± 0.0011
Dichlorvos 0.05–10.0 Y = 196,134X − 7856.9 0.9944 ± 0.0009
Dimethoate 0.05–10.0 Y = 365,581X − 2271 0.9938 ± 0.0008
Imidacloprid 0.05–10.0 Y = 386,681X + 11,074 0.9968 ± 0.0015
Methamidophos 0.05–10.0 Y = 6287X − 1897.5 0.9979 ± 0.0006

A preparation of 50 mg/L of the five organophosphorus (4) Samples preparation

pesticides (trichlorfon, dichlorvos, dimethoate, imidacloprid,
and methamidophos) was mixed (dissolved by chloroform)
and diluted to the corresponding concentration gradients.
GC was used to analyze the sample, with the concentrations
as the abscissa and the chromatographic peak area as the or-
dinate to plot the standard curve.
(3) Determination of the MISPE–GC process
Imprinted materials (50 mg) were placed in an empty
MISPE pre-enrichment column. First, 5 mL methanol and
double-distilled water were used to embellish the MISPE
column. Then, the velocity of 3.0 mL/min was controlled
for enrichment at 100 mL. Five standard organophosphorus
pesticides (0.1 mg/L) were mixed in aqueous solutions.
Organophosphorus pesticide molecules were selectively
enriched to adsorb functional materials, and those that were
not enriched were discharged as liquid. The MISPE column
was eluted with 5 mL methanol/water/glacial acetic acid
(92:6:2, v/v/v) after preliminary enrichment. Nitrogen was
used to dry the eluents, and 0.5 mL methanol was the sol-
vent. Filtration was performed by using a 0.22-μm mem-
brane, and 1 μL solution was used for gas chromatography
analysis. A solution ratio of 9:1, v/v 10 mL methanol and
glacial acetic acid was used to rinse the solid-phase extrac-
tion column after the experiment for the next sample prelim-
inary enrichment processing. To compare the selective en-
richment effect of MIP for five standard organophosphorus
pesticides, the NIP combination with C18 SPE column was
employed using the same experimental process (Brust et al.
2014; Su et al. 2011).
To establish the accuracy of the MISPE–GC method, our
experiment used leaf lettuce spike sample to verify the recov-
ery. Leaf lettuce (1.0 g) was accurately weighed in a beaker,
5 mL of five standard organophosphorus pesticides (5.0 mg/L
and 10.0 mg/L) in mixed standard solution were sprayed
evenly on the samples for 1 h, and 50 mL double-distilled
water was mixed in the samples and ultrasonically extracted
for 20 min. The extraction was repeated thrice, with the ca-
pacity of extracted solution at 100 mL. Detection was per-
formed by the MISPE–GC process (Jenkins et al. 2001;
Savant et al. 2010).
To show the practical application value for this method,
leaf lettuce and cucumber were bought from the farmer’s mar-
ket for measurement. Weights of 1.0 g leaf lettuce and cucum-
ber were prepared, with 30 mL double deionized water used
for ultrasonic extraction three times and mixed until the ca-
pacity reached 100 mL. The MISPE–GC method was used for
detection.
(5) Description of QA-QC procedures
Standard addition recovery test was performed to verify the
accuracy of the proposed method. In China, the maximum
limit standard of organophosphorus pesticides in vegetables
is 0.005 mg/g. In the present study, 0.005 and 0.01 mg/g
organophosphorus pesticides were detected. Samples of leaf
lettuce were added to the MISPE column under the optimized
conditions for gas phase detection. The leaf lettuce sample
contained 0.005 mg/g organophosphorus pesticides under
the optimized column conditions. Seven root MISPEs were
used to determine repeatability among different columns.

Table 3 Analytical parameters of the developed MISPE–GC method

Organophosphorus pesticides Linear range (mg/L) LODs (μg/L) LOQs (μg/L) RSD (n = 5) (%) Enrichment of multiple MRLs (μg/kg)

China EU

Trichlorfon 0.05–10.0 0.15 ± 0.01 0.50 ± 0.03 1.59 ± 0.08 452 ± 34 100 10
Dichlorvos 0.05–10.0 0.76 ± 0.07 2.53 ± 0.23 4.07 ± 0.16 75 ± 12 100–200 20
Dimethoate 0.05–10.0 0.81 ± 0.09 2.70 ± 0.29 2.21% ± 0.10 52 ± 10 200–1000 20
Imidacloprid 0.05–10.0 0.89 ± 0.09 2.97 ± 0.31 3.86 ± 0.15 36 ± 5 100–500 30
Methamidophos 0.05–10.0 0.63 ± 0.05 2.10 ± 0.17 3.03% ± 0.13 109 ± 18 20–500 20

LODs limit of detections, LOQs limit of quantitations, MRLs maximum residue limits, EU European Union
 Food Anal. Methods

Table 4 Recoveries of five

organophosphorus pesticides in Organophosphorus pesticides Standard addition concentration
spike leaf lettuce cucumber
(mean ± RSD, n = 3) (0.005 m g/g) (0.01 m g/g)

Recovery rate (%) RSD (%) Recovery rate (%) RSD (%)

Trichlorfon 95.84 ± 0.35 2.54 ± 0.06 97.85 ± 0.45 2.89 ± 0.05

Dichlorvos 92.25 ± 0.39 4.54 ± 0.08 87.48 ± 0.27 3.78 ± 0.09
Dimethoate 88.25 ± 0.28 2.87 ± 0.05 90.89 ± 0.31 3.41 ± 0.08
Imidacloprid 94.58 ± 0.24 4.28 ± 0.05 89.57 ± 0.31 3.47 ± 0.07
Methamidophos 96.87 ± 0.43 3.48 ± 0.08 92.17 ± 0.35 2.94 ± 0.05

Statistical Analysis The suitable template molecule, functional monomer, and

Final data were expressed as the mean ± standard deviation
̄ s). ANOVAwas employed to assess the differences among
(x±
groups. Statistical analysis was performed using SPSS 21.0.
Significant differences were denoted by P < 0.05.
Results and Discussion
Factors of Organophosphorus Pesticide Template MIP
Preparation
Organophosphorus pesticides are water- and fat-soluble, but
the crosslinking agent and initiator are only fat-soluble. By
screening experiment, we determine that chloroform, as sol-
vent, can completely dissolve all reagents. Methylacrylic acid
as functional monomer is a kind of acidic functional mono-
mer. The hydroxyl groups can bond with the phosphorus ox-
ygen of the template molecule, and the non-covalent binding
forces are reversible (Tan et al. 2014; Wen et al. 2012). The
template molecules were removed after extraction with meth-
anol and glacial acetic acid, and the left cavity with recogni-
tion sites in the polymer.
ratio between crosslinking agents could improve the effect of
MIP selective adsorption. If functional monomers were taken
in excess, the polymer would have a large surplus of function-
al groups. Non-specific adsorption of MIP was greatly in-
creased and the selectivity was reduced. If the crosslinking
agent was in excess, which would lead to the solid for synthe-
sis polymer, molecular mass transfer was not used (Chen and
Li 2012; Hamdan et al. 2016).
Ratios of the three polymers are shown in Table 1. Under
the condition of the above ratio, polymers1, 2, and 3 were all
aggregated. Adsorption capacity determination of the experi-
ment results showed that 0.25 g organophosphorus pesticide
was dissolved in 5 mL chloroform; 0.689 g functional
monomer-methyl acrylic acid and 5.946 g crosslinking
agent–glycol dimethyl acrylate were added. Synthesis of ma-
terial (polymer3) had the best selectivity and the ability to
recognize organophosphorus pesticide under the 1:4:10 poly-
merization ratio. The synthesis experiment was performed un-
der these polymerization conditions.
Eluent extraction generally involved a solvent with tem-
plate molecular solubility. To elute the template molecules as
much as possible, the use of a solvent that could not damage
the structure of the polymer is required (Lin et al. 2011; Xu
et al. 2014). Our study chose methanol and glacial acetic acid

Table 5 Results of five organophosphorus pesticides in leaf lettuce and cucumber (mean ± RSD, n = 3)

Organophosphorus pesticides Detection content/(mg/kg)

Leaf lettuce Cucumber

National standard of China MISPE National standard of China MISPE

Trichlorfon 0.1(GB16329–1996) 0.06 ± 0.01 0.1(GB16329–1996) 0.08 ± 0.01

Dichlorvos 0.2(GB5127–1998) 0.11 ± 0.01 0.2(GB5127–1998) 0.19 ± 0.02
Dimethoate 0.8(GB5127–1998) 0.61 ± 0.03 0.8(GB5127–1998) 0.78 ± 0.04
Imidacloprid 0.3(GB1633–1996) 0.22 ± 0.02 0.3(GB1633–1996) 0.18 ± 0.02
Methamidophos 0.5(GB1633–1996) 0.38 ± 0.02 0.5(GB1633–1996) 0.43 ± 0.02
Food Anal. Methods

Table 6 Comparison of the

proposed method with other References Pretreatment Detection method Analytes LODs
methods for determining
organophosphorus pesticide Farajzadeh et al. 2015 DLLME GC-NPD 4 12–56 μg/L
residues in vegetable samples Wang et al. 2015 MISPE HPLC-UV 3 8.73–19.78 μg/kg
Liu et al. 2014 SPE LC-MS 3 1–10 μg/L
Pirsaheb et al. 2013 DLLME HPLC-UV 4 50–180 μg/kg
Chen and Fung 2010 CE 4 0.01–0.03 μg/L
Our work MISPE GC-FPD 4 0.15–0.89 μg/L

LODs limit of detections, DLLME dispersive liquid–liquid microextraction, GC-NPD gas chromatography with
nitrogen–phosphorus detection, MISPE molecularly imprinted solid-phase extraction, HPLC-UV high-
performance liquid chromatography-ultra violet, SPE solid-phase extraction, LC-MS-MS liquid
chromatography-mass spectrometry-mass spectrometry, CE electrophoresis, GC-FPD gas chromatography with
flame photometric detection

mixture (3:1, v/v) as eluent. The effect was good in removing similar to the micro ball. The MIP specific surface area was
template molecules, and no template molecules in extraction larger, which was helpful in increasing the adsorption capacity
solution were checked out. of the polymer. Particle size of universal organophosphorus
pesticide MIP was approximately 25 μm, meeting the appli-
cation in solid-phase extraction (filler particle size range from
Infrared Spectrum Analysis
2 to 200 μm) (Lin et al. 2011; Wen et al. 2012). The polymer
was porous, had a loose texture, and a convex surface figure,
Infrared spectrum analysis of MIP and NIP of trichlorfon and the
which was advantageous to the increasing of polymer adsorp-
experiment results are shown in Fig. 2. The C = O groups are at
tion rate and adsorption capacity (Fig. 3). This finding may be
1729 cm−1 (Fig. 2b). The −CH2 group was at 2938 cm−1
due to the process of MIP and the interaction of template
(Fig. 2b), whereas the −NH group was at 3392 cm−1 (Fig. 2c).
molecule and functional monomer. Numerous cavities that
The vibration of −OH group was 3578 cm−1 (Fig. 2b). These
matched with the template molecules were formed, but the
data suggested that the P = O group in organophosphorus pesti-
template molecules were not joined in non-MIP, thereby
cides combined with −OH group in methyl acrylic acid, thereby
resulting in a low number of cavities (Farid et al. 2016; Xu
indicating that a hybrid template was in reaction with functional
et al. 2014).
monomer-methyl acrylic acid (Savant et al. 2010; Shen et al.
2007). MIP was prepared successfully. Figure 2b, c showed that
MIP had similar infrared spectrogram with NIP, thereby suggest-
Adsorption Equilibrium Experiment
ing that the template molecules had been removed from the MIP.
of Organophosphorus Pesticide MIP

Scanning Electron Microscopy Analysis To evaluate the synthetic ability of MIP for the combination of
organophosphorus pesticides, adsorption equilibrium experi-
The precipitation polymerization method was used to prepare ment measured the adsorption capacity of polymer for five
the universal organophosphorus pesticide MIP and was organophosphorus pesticides, along with the change of 100

Table 7 Advantages and limitations of methods for detecting organophosphorus pesticide residues

Detection methods Advantages Limitations

GC, HPLC-MS High detection limit sensitivity The accuracy of the result is relatively low
because organophosphorus pesticides are
unstable and degrade easily at high temperatures
CE Less sample consumption and low cost of reagents Low sensitivity to UV detectors
ELISA Sensitive, accurate, and specific Time consuming, expensive, and has difficulties
associated with antibody
production
Enzyme inhibition Operation is simple, fast, and does not require expensive equipment Poor sensitivity and low repetition and recovery rates
MISPE–GC The LODs of the organophosphorus pesticides were Selection of functional monomer is still less,
lower than the MRLs of China and EU; the prepared difficulty in identifying and preparing aqueous
MISPE column is stable; the cost per analysis is low solutions or polar solvents

to 500 mg/L concentration at room temperature. The experi-
mental results are shown in Fig. 4.
The adsorption capacity of MIP for organophosphorus pes-
ticides was higher than non-MIP at the same concentration.
The difference is more obvious when the concentration was
higher; this result was caused by different spatial structures
between these two kinds of polymer. The NIP of organophos-
phorus pesticides was mainly the specificity adsorption. MIP
space structure matched cavities with the functional groups of
organophosphorus pesticides. Given the existence of the cav-
ities, the polymer had good selective binding ability to tem-
plate molecules, which led to stronger adsorption (Tan et al.
2014; Wen et al. 2012).
Selective Experiments of MIP for Organophosphorus
Pesticides
Specific selectivity is one of the main characteristics of the
MIP, leaving the cavities to correspond to the shape and com-
bination position after the template was removed. The cavities
can be a selective recognition of template molecules (Guo
et al. 2015; Li et al. 2015). Figure 5 showed that MIP was
greater than the non-MIP in terms of adsorption quantity of
five organophosphorus pesticides, thereby suggesting that
MIP had specific adsorption on these five organophosphorus
pesticides. Among them, trichlorfon and methamidophos
showed larger specific adsorption values. All these indicated
that the cavity structure and recognition site of trichlorfon and
methamidophos could be recognized in the form of MIP.
MISPE–GC Detection Methods for Five
Organophosphorus Pesticides
(1) Standard curve of gas chromatography
Five concentrations of organophosphorus pesticide solu-
tion (dissolved by chloroform) were analyzed by GC chroma-
tography, with concentrations at the abscissa and peak area as
ordinate to plot the standard curve (Table 2). The table showed
that five organophosphorus pesticides had good linear
relationship.
(2) Analytical parameters of the MISPE–GC method
The analytical parameters of the MISPE–GC method for
simultaneous separation and determination of the five organ-
ophosphorus pesticides were evaluated under the best solid-
phase extraction. The low limit of detections (LODs) (S/N = 3)
of the five organophosphorus pesticides were from 0.15 to
0.89 μg/L, which were lower than the maximum residue limits
(MRLs) in both China and the European Union (EU)
(Table 3). The linear ranges of the calibration graph were all
between 0.05 and 10.0 mg/L. The enrichment ratio was from
36 to 452. The range of five enrichment consecutive precision
(RSD) was from 1.59 to 4.07%, which showed that the
Food Anal. Methods
method significantly reduced the LOD and improved detec-
tion sensitivity.
(3)The detection accuracy of MISPE–GC method
Standard addition recovery rate is an accuracy and feasibil-
ity index used to analyze pesticide residues. Generally, the
amount of pesticide residues is one to two times higher than
the maximum allowable limit. Therefore, 0.005 and 0.01 mg/g
organophosphorus pesticides were used in the present study.
Leaf lettuce was used as spike samples to calculate the recov-
ery rate of MISPE–GC method. As shown in Table 4, adding
the two concentrations exhibited high recovery rate, ranging
from 87.48 to 97.85%; this value meets the requirements for
detecting pesticide residues (recovery of 70–110%).
Therefore, this method had high accuracy and could be ap-
plied to detection in actual vegetables. Our experiment used
seven MISPE pillars to determine the repeatability. The results
of Table 4 showed that the RSD between the columns was less
than 5%.
(4)The vegetables testing experiment for MISPE–GC
method
Five organophosphorus pesticides in leaf lettuce and cu-
cumber from the farmer’s market were detected by the general
MISPE method; each sample was determined thrice in paral-
lel. According to Table 5, the organophosphorus pesticide
detection limit of our method had no obvious difference from
those of the national standard. Therefore, general MISPE can
satisfy a variety of organophosphorus pesticides under com-
plex matrix.
According to the source of uncertainty in the chromato-
graphic analysis results, the uncertainty of the present exper-
imental results could be mainly attributed to standard solution
preparation, method recovery, constant volume, and sample
repeat measured four aspects. The substance peak area be-
tween the sample and reference and the accuracy of weighing
the sample minimally contributed to the uncertainty of the
results.
Merits of the MISPE–GC Method
A lot of methods including GC (Farajzadeh et al. 2014),
HPLC (Wang et al. 2015), LC-MS (Liu et al. 2014), and CE
(Chen and Fung 2010) have been reported for the detection of
organophosphorus pesticide residues, and they are summa-
rized in Table 6. In comparison, each method has its advan-
tages and drawbacks in terms of sensitivity and interference of
matrix compounds, which can be seen from Table 7.
Conclusions
High selectivity of MIP was achieved by the precipitation
polymerization method, with a particle size of approximately
25 μm. The method was applied in solid-phase extraction. The
Food Anal. Methods
adsorption ability of universal organophosphorus pesticide
MIP for the target was greater than those of NIP, which proves
the existence of the hole of polymer surface specific recogni-
tion ability. With increasing adsorption time, the universal
organophosphorus pesticide MIP for target adsorption quanti-
ty increased gradually and was eventually balanced. Universal
organophosphorus pesticide MIP had the largest adsorption
specificity for trichlorfon and methamidophos. The recovery
rate of leaf lettuce spike samples was between 87.48 and
97.85%, thereby conforming to the requirements of the pesti-
cide residue detection. Repetitive experiments showed that the
RSD was less than 5%. MISPE can detect the organophospho-
rus pesticide concentration range from 0.05 to 10.0 mg/L.
Leaf lettuce and cucumber were bought from the farmer’s
market as actual samples. The detection results of the
MISPE method had no obvious difference from the national
standard. Therefore, MISPE–GC method can be used to rap-
idly detect trace organophosphorus pesticide residues in veg-
etables, thereby significantly decreasing the detection limit of
the instrument and improving the detection sensitivity.
Compliance with Ethical Standards
Funding This study was funded by BHainan province production pro-
ject (grant number CXY20140012)^, BHainan university social service
projects (grant number SHFW201605),^ BHainan province innovation
research project of graduate students(Hys2016–47),^ andBHainan
University graduate student outstanding paper cultivation plan.^
Conflict of Interest Jian Chen declares that he has no conflict of inter-
est. Wen-ting Zhang declares that she has no conflict of interest. Yang Shu
declares that she has no conflict of interest. Xin-hui Ma declares that he
has no conflict of interest. Xian-ying Cao declares that she has no conflict
of interest.
Ethical Approval This article does not contain any studies with human
or animal subjects.
Informed Consent Informed consent is not applicable for this study.
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