Ionic Liquid-Hybrid Molecularly Imprinted Material-Filter Solid-Phase Extraction Coupled with HPLC for Determination of 6-Benzyladenine and 4-Chlorophenoxyacetic Acid in Bean Sprouts

Article abstract of DOI:10.1021/acs.jafc.6b03922

A new method involving ionic liquid-hybrid molecularly imprinted material-filter solid-phase extraction coupled to high-performance liquid chromatography (IL-HIM-FSPE-HPLC) was developed for the simultaneous isolation and determination of 6-benzyladenine (6-BA) and 4-chlorophenoxyacetic acid (4-CPA) in bean sprouts. Sample preconcentration was performed using a modified filter, with the new IL-HIM as the adsorbent, which shows double adsorption. The first adsorption involves special recognition of molecular imprinting, and the second involves ion exchange and electrostatic attraction caused by the ionic liquid. This method combines the advantages of ionic liquids, hybrid materials, and molecularly imprinted polymers and was successfully applied to determine 6-BA and 4-CPA in bean sprouts. The adsorption of 6-BA to IL-HIM is based on selective imprinted recognition, whereas the adsorption of 4-CPA is mainly dependent on ion-exchange interactions.

Full text of DOI:10.1021/acs.jafc.6b03922

Subscriber access provided by University of Newcastle, Australia
Article
Ionic Liquid - Hybrid Molecularly Imprinted Material-filter Solid
Phase Extraction coupled with HPLC for Determination of 6 -
Benzyladenine and 4-Chlorophenoxyacetic Acid in Bean Sprouts
Yehong Han, Chunliu Yang, Yang Zhou, Dandan Han, and Hongyuan Yan
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03922 • Publication Date (Web): 02 Feb 2017
Downloaded from http://pubs.acs.org on February 4, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 34
Journal of Agricultural and Food Chemistry
1
2
3
4
5
6
7
8
9
Ionic LiquidꢀHybrid Molecularly Imprinted Materialꢀfilter Solid Phase
Extraction coupled with HPLC for Determination of 6ꢀBenzyladenine and
4ꢀChlorophenoxyacetic Acid in Bean Sprouts
1,2
2
2
1
1,2
∗
Yehong Han , Chunliu Yang , Yang Zhou , Dandan Han , Hongyuan Yan
1
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, College of Public Health,
Hebei University, Baoding, 071002, China
2
Key Laboratory of Analytical Science and Technology of Hebei Province & College of Chemistry
and Environmental Science, Hebei University, Baoding, 071002, China
∗
Corresponding author. Tel.: +86ꢀ312ꢀ5079788
E-mail address: yanhongyuan@126.com
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 34
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
ABSTRACT: A new method involving ionic liquidꢀhybrid molecularly imprinted
materialꢀfilter solid phase extraction coupled to high performance liquid
chromatography (ILꢀHIMsꢀFSPEꢀHPLC) is developed for simultaneous isolation and
determination of 6ꢀbenzyladenine (6ꢀBA) and 4ꢀchlorophenoxyacetic acid (4ꢀCPA) in
bean sprouts. Sample preꢀconcentration is performed using a modified filter, with the
new ILꢀHIMs as the adsorbent, which shows double adsorption. The first adsorption
involves special recognition of molecular imprinting, and the second one involves
ionꢀexchange and electrostatic attraction caused by the ionic liquid. This method
combines the advantages of ionic liquids, hybrid materials, and molecularly imprinted
polymers, and is successfully applied to determine 6ꢀBA and 4ꢀCPA in bean sprouts.
The adsorption of ILꢀHIMs to 6ꢀBA is based on selective imprinted recognition, while
the adsorption of 4ꢀCPA is mainly dependent on ionꢀexchange interactions.
KEYWORDS: ionic liquid hybrid molecularly imprinted polymer, 6-benzyladenine,
4-chlorophenoxyacetic acid, filter solid phase extraction, bean sprouts
2
ACS Paragon Plus Environment

Page 3 of 34
Journal of Agricultural and Food Chemistry
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
INTRODUCTION
6ꢀBenzyladenine (6ꢀBA) is a common cytokinin, one of the important classes of plant
1
hormones, which is widely used to promote seed germination and nursery trees.
4ꢀChlorophenoxyacetic acid (4ꢀCPA) is a widely used chlorophenoxy herbicide and
plant growth regulator in agriculture and is considered as moderately toxic (class II or
2,3
III) by the World Health Organization. 6ꢀBA and 4ꢀCPA are widely applied to
promote stalk growth and restrict root growth in the production process of bean
sprouts. Thus, bean sprouts contain 6ꢀBA and 4ꢀCPA residues that could be hazardous
to human health in the long term. Therefore, the maximum residue limits (MRLs) for
4ꢀCPA (1.0 mg/kg) and 6ꢀBA (0.2 mg/kg) in bean sprouts have been established by
4
Chinese government. In recent years, numerous poisoning incidences caused by bean
sprouts containing 6ꢀBA and 4ꢀCPA have been reported in China. The quantitative
analysis of 6ꢀBA and 4ꢀCPA in bean sprouts is difficult because these phytohormones
are present in low concentrations and the tissues are complicated as they contain lots
of interfering substances. In order to monitor the trace levels of 6ꢀBA and 4ꢀCPA, a
sensitive, accurate, and robust analytical method is desired.
Up until now, the approaches for the determination of 6ꢀBA and 4ꢀCPA include gas
5
6,7
chromatography (GC), high performance liquid chromatography (HPLC), and
8
voltammetric methods. In order to isolate and concentrate the analytes prior to
instrumental analysis, a cleanꢀup and preꢀconcentration step is desired. Some routine
9
sample preꢀtreatment techniques such as liquidꢀliquid extraction (LLE), solidꢀphase
1
0
11
extraction (SPE), and dispersive liquidꢀliquid microextraction have been proposed
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 34
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
for extraction and isolation of 6ꢀBA and 4ꢀCPA. Recent trends in sample preꢀtreatment
technology are oriented toward high selectivity, miniaturization, and low consumption
of reagents and equipment. The extractant/adsorbent is an important factor in most
preꢀtreatment techniques, especially for SPE, which significantly affect the extraction
8
efficiency. However, common adsorbents (such as C18, C , silica, hydrophileꢀlipophile
balance, strong cation exchanger, etc) was lack of good selectivity and common
preꢀtreatment techniques were faced with the difficulty of extracting trace amounts of
the analytes from complicated sample matrices; therefore, development of selective
adsorbent materials for sample preꢀtreatment procedures is required.
Molecular imprinting is an attractive strategy for generating tailorꢀmade polymers,
containing molecular recognition sites with memory of the shape, size, and functional
12
groups of template molecules. Molecularly imprinted polymers (MIPs) have been
increasingly used for the isolation and efficient separation of trace compounds from
complex matrices due to their high selectivity, reusability, and physicochemical
1
3,14
stability in harsh chemical media.
However, these materials may shrink or swell
when exposed to different organic solvents and change the morphology of the
polymer network, causing considerable deformation of the MIP receptors and
1
5,16
decreasing the recognition ability toward the template.
Common MIPs are
normally compatible with organic solvents and fail to show specific binding in
17
aqueous solution, which significantly limits their practical application as adsorbents
1
8
and biomimetic sensors. Recently, organicꢀinorganic hybrid MIPs are developed,
which offer high mechanical strength, good solvent resistance, and high stability in
4
ACS Paragon Plus Environment

Page 5 of 34
Journal of Agricultural and Food Chemistry
7
7
7
7
7
7
7
7
7
7
8
8
8
8
8
8
8
8
8
8
9
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
different pH environments. They have been widely applied for environmental
19
monitoring and food analysis, because of their attractive properties. However, most
hybrid MIP bonding analytes depend on hydrogen bonding and other hydrophobic
interactions, which limit their further application in the fields of environmental
science and biology. Herein, extensive studies have been carried out on hybrid MIPs
with specific molecular recognition ability in aqueous solution.
Recently, ionic liquids (ILs) as alternative green solvents, have received a great
deal of attention as the surface modifier of polymers or functional monomers, because
they can provide ionꢀexchange interactions, electrostatic interactions, πꢀπ interactions,
etc. ILs maintain their properties, except outflow, when they immobilize on a silica
surface or an organic polymer; therefore, ILꢀmodified materials have been used in
separation owing to their particular characteristics such as their electronegative group
relay on ion exchange, electrostatic interaction, and πꢀπ interaction between the
20ꢀ24
analyte and functional groups of ILs.
In this study, a new type of ionic liquidꢀhybrid molecularly imprinted material
(ILꢀHIMs) was synthesized using 1ꢀallylꢀ3ꢀpropyltrimethoxysilane imidazole as both
the monomer and surface modifying agent. The synthesized ILꢀHIM is a double
adsorption mechanism material, which not only can interact with a template and its
structural analogs based on the special recognition of molecular imprinting, but also
absorbs analytes with electronegative groups by ionꢀexchange, electrostatic, and πꢀπ
interactions. Owing to their synergic effect, the novel ILꢀHIMs show good selectivity,
and they can be used for various purposes as they show a variety of adsorption
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 34
9
9
9
9
9
9
9
9
2
3
4
5
6
7
8
9
mechanisms, have no swelling and shrinking, and have a specific affinity for analytes
in aqueous solutions. ILꢀHIMs are successfully applied as the adsorbent using filter
solidꢀphase extraction, resulting in rapid extraction and isolation of 6ꢀBA and 4ꢀCPA
in bean sprouts.
MATERIALS AND METHODS
Materials. 6ꢀBenzyladenine (6ꢀBA), 2, 4ꢀchlorophenoxyacetic acid sodium (2,4ꢀD),
4ꢀchlorophenoxyacetic acid sodium (4ꢀCPA), acyclovir (ACV), kinetin (KT), and
indoleacetic acid (IAA) were purchased from Jingchun Co., Ltd. (Shanghai, China)
and their molecular structure were shown in Figure 1. 1ꢀVinylimidazole, ethylene
glycol dimethacrylate (EGDMA), 3ꢀchloropropyltrimethoxy silane (CPTMO),
tetraethyl orthosilicate (TEOS), and methacrylic acid (MAA) were purchased from
Aladdin reagent Co., Ltd. (Shanghai, China). MAA and EGDMA were used after
vacuum distillation. Azodiisobutyronitrile (AIBN) was purchased from Yuefeng
Chemical Co., Ltd. (Tianjin, China) and recrystallized before use. Methanol (MeOH),
ethanol (EtOH), acetone, acetonitrile (ACN), and acetic acid (AA) were purchased
from Guangfu chemical Co., Ltd. (Tianjin, China). Bean sprouts were bought in the
1
1
1
1
1
1
1
1
1
1
1
1
1
1
00
01
02
03
04
05
06
07
08
09
10
11
12
13
2
local markets of Baoding, China. The commercial C18 and amino (NH ) sorbent were
supplied by Varian Co. (Palo Alto, CA, USA). All the other reagents used in the
experiment were of the highest grade commercially available.
Instrumentations and Conditions. A FTIRꢀ8400S Fourier transform infrared
(FTIR) spectrometer (Shimadzu, Kyoto, Japan) was used to examine the infrared
−
1
spectra of the obtained materials in a range of 400ꢀ4000 cm . The morphological
6
ACS Paragon Plus Environment

Page 7 of 34
Journal of Agricultural and Food Chemistry
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
evaluation was performed by JSMꢀ7500F Scanning electron microscopy (JEOL Ltd,
Tokyo, Japan). Nitrogen adsorptionꢀdesorption measurements were performed on a
Tristar II 3020 Pore Size and Surface Area Analyzer (Micromeritics, Norcross, USA).
The surfaceꢀarea was measured on BrunauerꢀEmmettꢀTeller (BET), and the poreꢀsize
distribution was based on BarrettꢀJoynerꢀHalenda (BJH) formula. Chromatographic
analysis was carried out on a LCꢀ20A system equipped with two LCꢀ20AT Solvent
Delivery Units, a SUSꢀ20A gradient controller, and a SPDꢀ20A UVꢀVIS Detector
(Shimadzu, Kyoto, Japan). The Ultimate AQꢀC18 column (4.6×150 mm, 5 ꢁm) was
purchased from Welch Materials, Inc., and the mobile phase was methanolꢀwater (1:1,
v/v, containing 1% acetic acid) with a flow rate of 1.0 mL/min. The wavelength of the
detector was set at 267 nm (0ꢀ8 min) and 228 nm (8ꢀ12 min).
Synthesis of the ILꢀHIMs. Allylimidazole (3.5 mmol) was added dropwise to a
conical flask containing 3ꢀchloropropyltrimethoxysilane (CPTMO, 2.5 mmol),
sonicated for 10 min and purged with N for 20 min. Then, the flask was sealed and
2
heated at 80 °C using an oil bath for 24 h. The viscous liquid obtained was washed
with ether and dried under vacuum to obtain the desired IL (1ꢀallylꢀ3ꢀpropylꢀ
trimethoxysilane imidazolium chloride).
KT (0.5 mmol) and MAA (2.0 mmol) were dissolved in 10.0 mL of acetonitrile
(ACN) in a conical flask containing 1ꢀallylꢀ3ꢀpropylꢀtrimethoxysilane imidazolium
chloride. The mixture was sonicated for 10 min and stored at 4 °C in darkness.
EGDMA (25.0 mmol) and AIBN (40 mg) were added to the conical flask and
sonicated for 10.0 min. This was then added to a flask containing TEOS (4.48 mmol)
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 34
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
after alcoholysis. The mixture was degassed by sonication and subjected to thermal
initiation at 60 °C for 24 h in a water bath. After polymerization, the ILꢀHIMs were
washed with a methanolꢀacetic acid mixture (90:10, v/v) and methanol successively to
remove the template, and they were then dried under reduced pressure (Figure 2). The
nonꢀionic liquidꢀhybrid imprinted material (NILꢀHIMs) and nonꢀionic
liquidꢀhybridꢀnonꢀimprinted material (NILꢀHNIMs, without IL and template) were
prepared using an identical procedure.
Adsorption Capacity of ILꢀHIMs. Firstly, static adsorption experiments were
carried out, in the following steps. (1) Each ILꢀHIM, NILꢀHIM, and NILꢀHNIM
adsorbent (50 mg) was added into an empty cartridge and preꢀtreated first with 1.0 mL
of methanol and then with 1.0 mL of water. The solution containing 6ꢀBA and 4ꢀCPA
was loaded onto the cartridge discontinuously, at 1 mL intervals. The concentration of
the mixed solution was 10 (1ꢀ3 mL) and 30 µg/mL (4ꢀn mL). The concentration of
6ꢀBA and 4ꢀCPA in the effluent was determined by HPLC. (2) Each ILꢀHIM,
NILꢀHIM, and NILꢀHNIM filter was placed in 2.0 mL aqueous solutions containing
40 µg/mL of 6ꢀBA and 4ꢀCPA. The solutions were shaken in a water bath at 25 °C and
analyzed at 0.5, 1, 3, 5, 8, 9 and 15 h to measure the concentration of 6ꢀBA and 4ꢀCPA
using HPLC.
A competitive adsorption experiment was also performed by placing each ILꢀHIM,
NILꢀHIM, and NILꢀHNIM adsorbent into a 2.0 mL solution containing 0.2 mmol/mL
of 6ꢀBA, KT, 4ꢀCPA, ACV, 2,4ꢀD, and IAA. The concentrations of the different
compounds after adsorption were obtained using HPLC by means of standard curves.
8
ACS Paragon Plus Environment

Page 9 of 34
Journal of Agricultural and Food Chemistry
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
Analysis of Bean Sprouts. Bean sprouts were purchased from local supermarkets
in Baoding, China. Sprout samples (500 g) were compressed using a juice extractor,
and the juice was collected and centrifuged at 4000 rpm for 15 min. The residue was
reꢀextracted using a methanolꢀacetic acid mixture (9:1, v/v). After removing the
solvent by reduced pressure distillation, the extract was reꢀdissolved using the sprout
juice for filter solidꢀphase extraction (FSPE).
The ILꢀHIM adsorbent (40 mg) was placed into a replaceable film needle filter. The
process of the FSPE (Figure 3A) coupled to HPLC, for extraction and determination
of 6ꢀBA and 4ꢀCPA, was as follows: Firstly, the filter was conditioned using methanol
(1.0 mL) and water (1.0 mL) at a flow rate of 0.2 mL/min and then the sprout juice
sample was loaded onto the filter using a syringe at a flow rate of 0.2 mL/min. After
washing with 0.8 mL of water, the filter was subsequently flushed with 3.0 mL of
methanolꢀAA (9:1, v/v), at a flow rate of 0.2 mL/min. The eluted fractions were
collected, evaporated to dryness, and then reconstituted in 0.5 mL of the mobile phase
for HPLC analysis.
RESULTS AND DISCUSSION
Preparation of ILꢀHIMs. The aim of this work was to produce an adsorbent
material for the extraction and isolation of 6ꢀBA and 4ꢀCPA. The new IL was
2
5,26
synthesized following a reported method
with some modifications, making use of
both surfaceꢀmodifying agents and monomers in the polymerization process,
promoting stronger conglutination of the outer organic polymer onto the silica matrix.
The C=C of the IL can be crossꢀlinked with EGDMA or MAA, and the silicate ester
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 34
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
bond can be embedded in the silica matrix after condensation. MAA is also used as a
monomer in the polymerization, and the proportion of MAA and IL was investigated.
The adsorbent synthesized using IL without MAA showed 51% recovery of 6ꢀBA, the
adsorbent synthesized using MAA only gave a recovery of below 10% for 4ꢀCPA, and
MAA:IL (2:2, n/n) exhibited over 95% recovery for both 4ꢀCPA and 6ꢀBA. The
adsorption of 4ꢀCPA is mainly dependent on πꢀπ and ionꢀexchange interactions
between the IL and 4ꢀCPA, while the adsorption of 6ꢀBA is mainly dependent on
hydrogen bonding between ꢀCOOH and =Nꢀ or ꢀNHꢀ; therefore, the IL and MAA
were both chosen as monomers. EGDMA is the cross linker, and the ꢀC=Cꢀ of
EGDMA can interact with the ꢀC=Cꢀ of MAA and IL by atomꢀtransfer
3
radicalꢀpolymerization. TEOS can interact with the –Si(OH) of the IL by
polycondensation. For the synthesis of the outer organic polymer under thermal
initiation, acetonitrile is a good porogenic agent, and its use resulted in an adsorbent
with a satisfactory binding capacity. After optimization, 30 mL acetonitrile was added
to the reaction mixture.
Characterization of the Materials. The scanning electron microscopy (SEM)
images in Figure 3B revealed that the ILꢀHIM (a) has a rough surface and a loose,
porous structure, which leads to a higher surface area and is beneficial for the
adsorption rate. Figure 3C shows the infrared (IR) spectrum of the ILꢀHIMs. The band
ꢀ1
at 1160 cm is assigned to the asymmetric stretching vibration, SiꢀOꢀSi, and at 1650
ꢀ1
27
cm a band corresponding to the C=C and C=N vibrations is observd. A band
ꢀ1
observed at 1725 cm is attributed to the C=O vibration of the EGDMA. Absorption
1
0
ACS Paragon Plus Environment

Page 11 of 34
Journal of Agricultural and Food Chemistry
ꢀ
1
−1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
bands at 2995 cm and 960 cm show the presence of the –COOH moiety in this
ꢀ1
polymer. At the same time, the absorption band at 3400 cm shows that the materials
28
possess abundant –SiOH moieties to afford the adsorption capacity. The band at
−1
1459 cm corresponding to the vibration of imidazole ring indicated that the
vinylimidazolium IL had been incorporated into the polymers.
The results of BET showed that the specific surface area of the ILꢀHIMs (403.5
2
2
m /g) were larger than those of the NILꢀHIM (187.4 m /g) and NILꢀHNIMs (172.8
2
m /g), which resulted from more porous and rough surface of ILꢀHIMs caused by IL
(acting as porogenic solvent and stabilizers for designing nanoporous structures) and
template molecules (more pores remained after the removal of the template). The data
of the desorption pore size distribution of the ILꢀHIMs and NILꢀHNIMs show that the
pore diameter of the ILꢀHIMs is mainly distributed between 5 and 50 nm and the
NILꢀHNIMs between 5 and 30 nm.
To evaluate the binding property of the ILꢀHIMs, NILꢀHIMs, and NILꢀHNIMs,
static absorption and dynamic adsorption experiments were performed. The results
(Figure 4A) of the static adsorption experiment show that the adsorption of 4ꢀCPA
increased with an increase in concentration, and the adsorption capacities gradually
tended to be saturated when the concentration of 4ꢀCPA was more than 275 µg/mL.
Compared with NILꢀHIMs and NILꢀHNIMs, ILꢀHIMs revealed a higher adsorption
capacity owing to the electrostatic and ionꢀexchange interactions of the IL and πꢀπ
interactions between the imidazole ring of the IL and the benzene ring of 4ꢀCPA. Due
to the large difference in the molecular structure between the imprinted template and
11
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 34
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
4ꢀCPA, there are no specific imprinted recognition interactions between the materials
(ILꢀHIMs and NILꢀHIMs) and 4ꢀCPA. Figure 4B shows that the adsorption capacity
of the ILꢀHIMs and NILꢀHIMs to 6ꢀBA was significantly higher than that of the
NILꢀHNIMs, owing to specific imprinted recognition. The adsorption capacities of
the ILꢀHIMs for 6ꢀBA were higher than those of the NILꢀHIMs because of the
electrostatic and πꢀπ interactions between the IL and 6ꢀBA.
To investigate the equilibrium time, the effect of contact time was studied at 25 °C
in aqueous media with an initial 6ꢀBA and 4ꢀCPA concentration of 0.25 mmol/L. The
dynamic adsorption (Figure 4C) of 6ꢀBA shows that both the ILꢀHIMs and NILꢀHIMs
were balanced at nearly 60 min, which indicates that the interaction of 6ꢀBA and
ILꢀHIMs was similar to that of 6ꢀBA and NILꢀHIMs, mainly owing to imprinted
recognition. The adsorption equilibrium showed that the mass transfer rate of the
ILꢀHIMs and NILꢀHIMs became better than that of the NILꢀHNIMs. From the results
presented in Figure 4D, it can be deduced that the adsorption capacity of the ILꢀHIMs
for 4ꢀCPA increased with an increase in time and reached the equilibrium value at 90
min. The equilibrium value of the NILꢀHIMs and NILꢀHNIMs had a lower adsorption
capacity than the ILꢀHIMs for 4ꢀCPA under the same conditions. This partially
accounted for the higher surface area of the ILꢀHIMs compared with that of the
NILꢀHIMs and NILꢀHNIMs (as confirmed by BET analysis). Obviously, through
electrostatic, ionꢀexchange, and πꢀπ interactions, the adsorption of 4ꢀCPA was greatly
enhanced.
Selectivity and Competitive Experiments. A mixed solution containing 6ꢀBA,
1
2
ACS Paragon Plus Environment

Page 13 of 34
Journal of Agricultural and Food Chemistry
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
4ꢀCPA, KT, 2,4ꢀD, ACV, and IAA was analyzed to evaluate the selectivity and
competitive adsorption of the ILꢀHIMs, NILꢀHIMs, and NILꢀHNIMs. Figure 5 shows
that the adsorption capacities of 6ꢀBA for the ILꢀHIMs and NILꢀHIMs were
significantly higher than that of the NILꢀHNIMs. The ILꢀHIMs, NILꢀHIMs, and
NILꢀHNIMs had a low adsorption capacity toward ACV. It was further certified that
6ꢀBA was adsorbed onto the ILꢀHIMs by means of specified imprinted sites, but other
compounds were adsorbed mainly by nonꢀspecified sites on the ILꢀHIMs. However,
the ILꢀHIMs could adsorb much more 2,4ꢀD, 4ꢀCPA, and IAA than the NILꢀHIMs and
NILꢀHNIMs, mainly because the –COOH (2,4ꢀD, 4ꢀCPA, and IAA) and –Cl (2,4ꢀD
and 4ꢀCPA) moieties can effectively interact with the ILꢀHIMs by electrostatic and
ionꢀexchange interactions.
Optimization of ILꢀHIMsꢀFSPE Procedures. The developed filter solidꢀphase
extractio n（ FSPE), a miniaturized SPE method, uses a modified needle filter instead of
common SPE cartridges and has many advantages, mainly including small device,
rapid and facile operation, and low consumption of sorbent and solvent. To achieve
the highest recoveries of trace levels of 6ꢀBA and 4ꢀCPA from bean sprout samples,
several parameters including the type of washing solvent, volume of washing solvent,
and elution solvent for the ILꢀHIMsꢀFSPE system were investigated.
The washing step played a crucial role in removing interferences originating from
sample matrices, while maintaining specific interactions between the analytes and
ILꢀHIMs. Different washing solvents such as MeOHꢀH
2 2
O (1:9, v/v), ACNꢀH O (1:9,
v/v), acetoneꢀH O (1:9, v/v), MeOH, ACN, water and acetone were investigated (1.0
2
1
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 34
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
mL spiked sample (3.0 µg/mL), 1.0 mL of each solvent), and the results were shown
2 2
in Figure 6A. Using MeOHꢀH O (1:9, v/v), ACNꢀH O (1:9, v/v), and water as the
washing solvent, provided improved recoveries compared to MeOH, acetone,
acetoneꢀH O (1:9, v/v), and ACN. Meanwhile, the recoveries obtained when water
2
was used were slightly higher than those obtained when MeOHꢀH
ACNꢀH O (1:9, v/v) were used as the solvents. Considering that water is an
environmentally friendly solvent and its purification efficiency is similar to that of
MeOHꢀH O (1:9, v/v) and ACNꢀH O (1:9, v/v), water was chosen as the washing
2
O (1:9, v/v) and
2
2
2
solvent. Various volumes (0.5, 0.8, 1.0, 1.2, and 1.5 mL) of water were investigated to
improve the purification with minimal loss of 6ꢀBA and 4ꢀCPA. The results (Figure
6A) indicate that the recoveries and purification efficiency were satisfactory if the
volume of water was increased to 0.8 mL, whereas the recoveries gradually decreased
if the volume was more than 0.8 mL. Hence, 0.8 mL of water was selected as the
optimal volume.
Various types of elution solvents were investigated, including waterꢀAA (90:10,
v/v), methanolꢀAA (95:5, 90:10, 85:15, v/v), acetoneꢀAA (10:90, v/v), ethanolꢀAA
(10:90, v/v), and acetonitrileꢀAA (10:90, v/v), to ensure complete analyte desorption
from the ILꢀHIMs. Figure 6B shows the elution efficacy in terms of recoveries for
6ꢀBA and 4ꢀCPA. The highest elution efficiency was achieved using methanolꢀAA
(95:5, v/v) as the elution solvent, which suggested that 6ꢀBA and 4ꢀCPA were bound
to the binding sites of the ILꢀHIMs, possibly through hydrogen bonds and other
interactions, which are affected and altered by polar solvents and acid, allowing for
1
4
ACS Paragon Plus Environment

Page 15 of 34
Journal of Agricultural and Food Chemistry
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
rapid desorption. After optimization of the elution volume, 3.0 mL methanolꢀAA (95:5,
v/v) was selected as the elution volume for the ILꢀHIMsꢀFSPE.
Comparison of Different Adsorbents. To further demonstrate the characteristics
of the ILꢀHIMs, various adsorbents including ILꢀHIMs, NILꢀHNIMs, C , and NH
18
2
were investigated for the FSPE. The loading, washing, and elution conditions of C18
6
and NH
2
were modified from literature. The conditions of the ILꢀHIMs and
NILꢀHNIMs for FSPE were constant, based on two adsorbents prepared in an
identical manner. As shown in Figure 7, the recoveries of 6ꢀBA were less than 78.2%
when using C , NH , and NILꢀHNIMs as the adsorbents. Moreover, the recoveries of
1
8
2
4ꢀCPA using C18 and NILꢀHNIMs were 56.6% and 33.4%, respectively. Conversely,
the ILꢀHIMs yielded the highest recovery of 6ꢀBA (94.5%) and 4ꢀCPA (96.4%) with
obviously improved purification efficiency, which indicates a high selectivity and
affinity of the ILꢀHIMs for 6ꢀBA than that of the other adsorbents. Meanwhile, the
recovery (90.2%) from the NH adsorbent for 4ꢀCPA was a little lower than that of the
2
ILꢀHIMs, which indicates that the –NH
ionꢀexchange similar to the ILꢀHIMs.
2
group can interact with 4ꢀCPA by
Validation of the ILꢀHIMsꢀFSPEꢀHPLCꢀUV Method. The developed method
was evaluated for linearity, precision, accuracy, limits of detection (LOD) and limits
of quantification (LOQ) under the optimized conditions. The calibration curve was
constructed using spiked bean sprout samples with 6ꢀBA and 4ꢀCPA in the range of
0.2ꢀ4.8 µg/mL. The calibration equations (peak height vs. analyte concentration) for
ꢀ5
ꢀ5
6ꢀBA and 4ꢀCPA are Y = 2.8×10 X ꢀ 128.46 and Y = 1.7×10 X + 2464.7 with
1
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 34
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
correlation coefficients of r ≥ 0.9992 and 0.9994, respectively. The precision of the
method was evaluated with relative standard deviation (RSD) of reproducibility
(intraday and interday precision), and the reproducibility was determined every three
months. Adequate precision was found and intraday RSD values for 6ꢀBA and 4ꢀCPA
were less than 2.6% and 1.6%, respectively. The interday RSD values for 6ꢀBA and
4ꢀCPA were less than 7.6% and 3.9%, respectively. The LOD and LOQ values (n = 3)
(calculated at signalꢀtoꢀnoise ratios of 3 and 10, respectively) of 6ꢀBA were 0.04 and
0.13 µg/mL and those of 4ꢀCPA were 0.055 and 0.18 µg/mL, respectively. To
demonstrate the effect of the sample matrix, the accuracy of the proposed method was
assessed by recovery experiments at three spiking levels (0.2, 2.0, and 4.0 µg/mL) of
6ꢀBA and 4ꢀCPA. The recoveries of 6ꢀBA and 4ꢀCPA were in the range
88.6%ꢀ106.2% with RSD ≤7.6% (n=3) and 81.1%ꢀ106.1% with RSD ≤3.9%,
respectively, which indicated that the ILꢀHIMsꢀFSPEꢀHPLCꢀUV method was reliable
and could be used for simultaneous determination of 6ꢀBA and 4ꢀCPA in bean sprouts
(Table 1).
The comparison of the presented method with reported methods for the extraction
of 6ꢀBA and 4ꢀCPA has been shown in Table 2. Most reported methods involving the
determination of only one of the two target substances. Compared with these reported
methods, the developed ILꢀHIMsꢀFSPEꢀHPLC method can detect 6ꢀBA and 4ꢀCPA
simultaneously with the advantages of high recovery and low LOD.
Analysis of Bean Sprouts. Twenty kinds of bean sprout samples collected from the
local markets of Baoding were analyzed to evaluate the applicability of the proposed
1
6
ACS Paragon Plus Environment

Page 17 of 34
Journal of Agricultural and Food Chemistry
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
method. The results showed that one of the bean sprout samples contained 6ꢀBA at
levels of 0.18 µg/g (MRL < 0.2 µg/g). Another sample was observed to contain 4ꢀCPA
at levels of 0.36 µg/g (MRL < 1.0 µg/g). The chromatograms of the spiked bean
sprout were cleaner after the ILꢀHIMsꢀFSPE process, as seen in Figure 8, and there
were no interfering peaks originating from the bean sprout matrix. Hence, it can be
concluded that the ILꢀHIMsꢀFSPEꢀHPLCꢀUV method is reliable and can be used for
simultaneous determination of 6ꢀBA and 4ꢀCPA in bean sprouts.
AUTHOR INFORMATION
Corresponding Author
∗
Tel.: +86ꢀ312ꢀ5079788. Eꢀmail: yanhy@hbu.edu.cn.
Funding
The project is sponsored by National Natural Science Foundation of China (31301464,
21575033), Natural Science Foundation of Education Department of Hebei Province
(ZD2015036), and Natural Science Foundation of Hebei Province (B2015201132,
B2014201089).
Notes
The authors declare no competing financial interest.
REFERENCES
(1) Zhang, H.; Huang, Y.; Xue, S.F.; Zhu, T.; Zhu, Q.J. Hostꢀguest interactions of 6ꢀbenzyladenine
with normal and modified cucurbituril: 1H NMR, UV absorption spectroscopy and phase
solubility methods. Supramol. Chem. 2011, 23, 527ꢀ532.
1
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 34
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
(2) Bucheli, T.D.; Grüebler, F.C.; Müller, S.R.; Schwarzenbach, R.P. Simultaneous determination
of neutral and acidic pesticides in natural waters at the low nanogram per liter level. Anal.
Chem. 1997, 69, 1569ꢀ1576.
(3) Boye B.; Dieng M.M.; Brillas E. Degradation of herbicide 4ꢀchlorophenoxyacetic acid by
advanced electrochemical oxidation methods. Environ. Sci. & Technol. 2002, 36, 3030ꢀ3035.
(4) Zhang F.; Zhao P.; Shan W.; Gong Y.; Jian Q.; Pan C. Development of a Method for the
Analysis of Four Plant Growth Regulators (PGRs) Residues in Soybean Sprouts and Mung
Bean Sprouts by Liquid ChromatographyꢀTandem Mass Spectrometry. B. Environ Contam
Tox. 2012, 8, 674ꢀ679.
(5) Lee SM.; Kim JY.; Lee HJ.; Chang Ml.; Chae YS.; Rhee GS. Establishment of analytical
method for 6ꢀbenzylaminopurine residue, a plant growth regulator for brown rice, mandarin,
pepper, potato, and soybean by using GC/NPD. J. Korean Soc. Appl. Biol. Chem. 2014, 57,
83ꢀ89.
(6) Lin SL.; Fuh MR. Orthogonal array optimization of ultrasoundꢀassisted emulsificationꢀ
microextraction for the determination of chlorinated phenoxyacetic acids in river water. J.
Chromatogr. A 2010, 1217, 3467ꢀ3472.
(7) Ma Z.; Ge L.; Lee A.S.Y.; Yong J.W.H.; Tan S.N.; Ong E.S. Simultaneous analysis of different
classes of phytohormones in coconut (Cocos nucifera L.) water using highꢀperformance
liquid chromatography and liquid chromatographyꢀtandem mass spectrometry after
solidꢀphase extraction. Anal. Chim. Acta 2008, 610, 274ꢀ281.
(8) Sun D.; Zhang H. Voltammetric determination of 6ꢀbenzylaminopurine (6ꢀBAP) using an
acetylene blackꢀdihexadecyl hydrogen phosphate composite film coated glassy carbon
1
8
ACS Paragon Plus Environment

Page 19 of 34
Journal of Agricultural and Food Chemistry
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
electrode. Anal. Chim. Acta 2006, 557, 64ꢀ69.
(9) Jin M.; Ren Y.; Chen X. Determination of 6ꢀBenzyladenine in Bean Sprout by
LCꢀESIꢀITꢀMSꢀMS. Chromatographia 2007, 66, 407ꢀ410.
(10) Dobrev P. I.; Kamınek M. Fast and efficient separation of cytokinins from auxin and abscisic
acid and their purification using mixedꢀmode solidꢀphase extraction. J. Chromatogr. A 2002,
950, 21ꢀ29.
(11) Huang G.Q.; Dong S.Y.; Yang Z.; Huang T.L. Simultaneous determination of four plant
hormones in soil by ultrasoundꢀassisted ionic liquid based dispersive liquidꢀliquid
microextraction. Appl. Mech. Mater. 2014, 675, 181ꢀ184.
(12) Zhou H.; Xu Y.P.; Tong H.W.; Liu Y.X.; Han F.; Yan X.Y.; Liu S.M. Direct synthesis of
surface molecularly imprinted polymers based on vinylꢀSiO nanospheres for recognition of
2
bisphenol A. J. Appl. Polym. Sci. 2013, 128, 3846ꢀ3852.
(13) Tsermentseli S.K.; Manesiotis P.; Assimopoulou A.N.; Papageorgiou V.P. Molecularly
imprinted polymers for the isolation of bioactive naphthoquinones from plant extract. J.
Chromatogr. A 2013, 1315, 15ꢀ20.
(14) Zhao Q.; Li H.; Xu Y.; Zhang F.; Zhao J.; Wang L.; Hou J.; Ding H.; Li Y.; Jin H.; Ding L.
Determination triazine pesticides in cereal samples based on singleꢀhole hollow molecularly
imprinted microspheres. J. Chromatogr. A 2015, 1376, 26ꢀ34.
(15) He J.; Fang G.; Deng Q.; Wang S. Preparation, characterization and application of
organicꢀinorganic hybrid ractopamine multiꢀtemplate molecularly imprinted capillary
monolithic column. Anal. Chimica. Acta 2011, 692, 57ꢀ62.
(16) Wang H.F.; Zhu Y.Z.; Yan X.P.; Gao R.Y.; Zheng J.Y. A room temperature ionic liquid
1
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 34
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
(RTIL)ꢀmediated, nonꢀhydrolytic solꢀgel methodology to prepare molecularly imprinted,
silicaꢀbased hybrid monoliths for chiral separation. Adv. Mater. 2006, 18, 3266ꢀ3270.
(17) Nematollahzadeh A.; Sun W.; Aureliano C.S.A.; Lütkemeyer D.; Stute J.; Abdekhodaie M.J.;
Shojaei A.; Sellergren B. Highꢀcapacity hierarchically imprinted polymer beads for protein
recognition and capture. Angew. Chem. 2011, 123, 515ꢀ518.
(18) Fodey T.; Leonard P.; O’Mahony J.; O’Kennedy R.; Danaher M. Developments in the
production of biological and synthetic binders for immunoassay and sensorꢀbased detection
of small molecules. TrAC Trend. Anal. Chem. 2011, 30, 254ꢀ269.
(19) Yan H.; Wang M.; Han Y.; Qiao F.; Row K.H. Hybrid molecularly imprinted polymers
synthesized with 3ꢀaminopropyltriethoxysilaneꢀmethacrylic acid monomer for miniaturized
solidꢀphase extraction: A new and economical sample preparation strategy for determination
of acyclovir in urine. J. Chromatogr. A 2014, 1346, 16ꢀ24.
(20) Zhao L.; Zhao F.; Zeng B. Electrochemical determination of methyl parathion using a
molecularly imprinted polymerꢀionic liquidꢀgraphene composite film coated electrode.
Sensor. .Actuat. B 2013, 176, 818ꢀ824.
(21) Zhang Q.; Du Y.; Du S. Evaluation of ionic liquidsꢀcoated carbon nanotubes modified chiral
separation system with chondroitin sulfate E as chiral selector in capillary electrophoresis. J.
Chromatogr. A 2014, 1339, 185ꢀ191.
(22) Fan J.P.; Tian Z.Y.; Tong S.; Zhang X.H.; Xie Y. L.; Xu R.; Qin Y.; Li L.; Zhu J.H.; Ouyang
X.K. A novel molecularly imprinted polymer of the specific ionic liquid monomer for
selective separation of synephrine from methanolꢀwater media. Food Chem. 2013, 141,
3578ꢀ3585.
2
0
ACS Paragon Plus Environment

Page 21 of 34
Journal of Agricultural and Food Chemistry
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
(23) Zhao L.; Zeng B.; Zhao F. Electrochemical determination of tartrazine using a molecularly
imprinted polymerꢀmultiwalled carbon nanotubesꢀionic liquid supported Pt nanoparticles
composite film coated electrode. Electrochim. Acta 2014, 146, 611ꢀ617.
(24) Yan H.; Yang C.; Sun Y.; Row K.H. Ionic liquid molecularly imprinted polymers for
application in pipetteꢀtip solidꢀphase extraction coupled with gas chromatography for rapid
screening of dicofol in celery. J. Chromatogr. A 2014, 1361, 53ꢀ59.
(25) Kore R.; Srivastava R. Synthesis of triethoxysilane imidazolium based ionic liquids and their
application in the preparation of mesoporous ZSMꢀ5. Catal. Commun. 2012, 18, 11ꢀ15.
(26) Yang L.; Su P.; Chen X., Zhang R.; Yang Y. Microwaveꢀassisted synthesis of poly(ionic
liquid)ꢀcoated magnetic nanoparticles for the extraction of sulfonylurea herbicides from soil
for HPLC. Anal. Methods 2015, 7, 3246ꢀ3252.
(27) Zhou H.; Wang H.; Tian X.; Zheng K.; Cheng Q. Effect of 3ꢀAminopropyltriethoxysilane on
polycarbonate based waterborne polyurethane transparent coatings. Prog. Org. Coat. 2014,
77, 1073ꢀ1078.
(28) Rao F.; Deng S.; Chen C.; Zhang N. Graphite oxideꢀsupported Karstedt catalyst for the
hydrosilylation of olefins with triethoxysilane. Catal. Commun. 2014, 46, 1ꢀ5.
(29) Wang F.; Zhang W.; Chen Y.; Xu G.; Deng Z.; Du H.; Wang Y.; Zhang S.; Zhao W.
Simultaneous determination of six plant growth regulators in fruits using high performance
liquid chromatography based on solidꢀphase extraction and cleanup with a novel mixedꢀmode
functionalized calixarene sorbent. Anal. Methods 2015, 7, 6365ꢀ6371.
(30) Zhang W.; He L.; Zhang R.; Gao S.; Yue H.; Ning X.; Tan G.; Li Q.; Wang B. Development of
a monoclonal antibodyꢀbased enzymeꢀlinked immunosorbent assay for the analysis of
2
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 34
4
4
4
4
4
4
4
4
4
44
45
46
47
48
49
50
51
52
6ꢀbenzylaminopurine and its ribose adduct in bean sprouts. Food Chem. 2016, 207, 233ꢀ238.
(31) Zhang H.; Tan S.N.; Teo C.H.; Yew Y.R.; Ge L.; Chen X.; Yong J.W.H. Analysis of
phytohormones in vermicompost using a novel combinative sample preparation strategy of
ultrasoundꢀassisted extraction and solidꢀphase extraction coupled with liquid
chromatographyꢀtandem mass spectrometry. Talanta 2015, 139, 189ꢀ197.
(32) Zhang Y.; Jiao B. Dispersive liquidꢀliquid microextraction combined with online
preconcentration MEKC for the determination of some phenoxyacetic acids in drinking water.
J. Sep. Sci. 2013, 36, 3067ꢀ3074.
2
2
ACS Paragon Plus Environment

Page 23 of 34
Journal of Agricultural and Food Chemistry
4
4
4
4
4
4
4
4
4
4
4
53
54
55
56
57
58
59
60
61
62
63
FIGURE CAPTIONS
Figure 1. Molecular structure of 6ꢀBA, 4ꢀCPA and some analogues.
Figure 2. Schematic illustration of ILꢀHIMs preparation.
Figure 3. The process of FSPE (A) and the SEM (B) and FTIR (C) of ILꢀHIMs.
Figure 4. Loading isotherm of analytes on ILꢀHIMs, NILꢀHIMs, and NILꢀHNIMs.
(A and B: static adsorption; C and D: dynamic adsorption)
Figure 5. The selectivity of ILꢀHIMs, NILꢀHIMs, and NILꢀHNIMs.
Figure 6. Effect of washing and eluent solvents on the recovery of ILꢀHIMsꢀFSPE.
(A: washing solvents; B: eluent solvents)
Figure 7. The purification effect (A) and recovery (B) of different adsorbents.
Figure 8. Chromatogram of spiked sample (A, B) and real samples (C, D).
2
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 34
TABLES
Table 1. Recoveries of 6ꢀBA and 4ꢀCPA in spiked samples (n=3).
Spiked level
0.2 µg/mL
Recoveries
%)
2.0 µg/mL
RSD Recoveries
4.0 µg/mL
RSD Recoveries RSD
Analytes
(
(%)
3.5
4.0
7.6
2.1
2.4
1.2
(%)
(%)
1.5
2.3
1.1
1.7
1.3
1.2
(%)
95.4
(%)
1.4
6
6
6
ꢀBA
ꢀBA
ꢀBA
92.1
96.3
94.2
81.1
86.9
106.2
100.5
105.3
106.1
103.5
104.8
88.6
5.4
5.8
1.1
3.9
2.0
103.8
100.2
101.9
98.4
4
4
4
ꢀCPA
ꢀCPA
ꢀCPA
83.9
2
4
ACS Paragon Plus Environment

Page 25 of 34
Journal of Agricultural and Food Chemistry
Table 2 Comparison of the presented method with reported methods and standard methods.
Samples
Apple
Pretreatment
SPE
Detection
Linearity
LOD
Recovery (%)
77.2–89.4
RSD (%)
≤2.22
Ref.
29
HPLCꢀUV
0.1–200 ꢁg/g
10.6 ng/g
Bean sprouts
Vermicompost
Water
ꢀ
icELISA
HPLCꢀUV
CEꢀDAD
HPLCꢀUV
HPLCꢀUV
3.6–106 ng/mL
5–50 µg/mL
0.002–2 µg/mL
–
18.9 ng/mL
0.67 µg/mL
0.002 µg/mL
0.006 µg/g
75.7–88.9
≤ 13.0
–
30
SPE
–
31
32
DLLME
SPE
67.2–68.1
–
≤ 12.3
–
Food, vegetable
Bean sprouts
GB/T23381ꢀ2009
FSPE
0.2–4.8 µg/mL
0.055 µg/mL
81.1ꢀ106.2
≤7.6
Present work
(
DLLME: dispersive liquid–liquid microextraction; icELISA: indirect competitive enzymeꢀlinked immunosorbent
assay CEꢀDAD: capillary electrophoresisꢀdiode array detection; GB/T: national standard of China)
2
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 34
FIGURE GRAPHICS
Figure 1
2
6
ACS Paragon Plus Environment

Page 27 of 34
Journal of Agricultural and Food Chemistry
OH
Cl
O
O
HO
HO
O
Si
O
Si
O
O
NH
O
NH
O
OH
N
O
OH
OH
OH
H
N
N
N
O
N
N
N
O
O
H
O
N
N
H
N
H
N
N
H
N
O
N
O
O
N
OH
H
H
HO OH
HO Si
OH
O
O
N
N
Bonding
Analytes
OH
N
HO
HO
Si
N
O
Si
O
O
N
Cl
Cl
O
Si
O
O
NH
O
O
Si
H
O
O
O
N
N
H
O
O
H
HO OH
HO Si
EDMA
AIBN
OH
O
ACN
N
N
Washing template
OH
HO
Si
HO
HO
O
Si
O
ACN
Si
O
OH
O
O
O
Si
OH
O
O
H
NH3 H2O
OH
N
N
N
O
H
O
N
N
H
O
N
O
N
H
H
HO OH
HO Si
OH
O
N
N
Figure 2
2
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 34
B
9
8
7
6
5
4
0
0
0
0
0
ILꢀHIMs
C
vibration of imidazole ring
1
460
960
ꢀCOOH
2
995
1650
C=Cꢀ
C=Nꢀ
-
COOH
ꢀ
ꢀ
1
ꢀ
725
C=O
A
ꢀSiꢀOꢀSiꢀ
160
3
400
ꢀSiꢀOH
0
4
1
000 3500 3000 2500 2000 1500 1000 500
ꢀ
1
Wavenumber (cm )
Figure 3
2
8
ACS Paragon Plus Environment

Page 29 of 34
Journal of Agricultural and Food Chemistry
6
5
4
3
2
1
0
10
NILꢀHIMs
NILꢀHNIMs
ILꢀHIMs
A
NILꢀHIMs
NILꢀHNIMs
ILꢀHIMs
B
9
8
7
6
5
4
3
2
1
0
0
50
100
150
200
250
300
350
400
0
50
100
150
200
250
300
350
400
ꢀ1
ꢀ1
Initial concentration of 4ꢀCPAꢀNa (µg mL )
Initial concentration of 6ꢀBA(ꢁg mL )
1
0
9
8
7
6
5
4
3
2
1
NILꢀHIMs
NILꢀHNIMs
ILꢀHIMs
NILꢀHIMs
NILꢀHNIMs
ILꢀHIMs
C
D
6
4
2
0
0
100
200
300
400
500
600
0
200
400
600
800
1000
Time (min)
Time (min)
Figure 4
2
9
ACS Paragon Plus Environment