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Journal of Chromatography A, xxx (2018) xxx–xxx

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Selective recognition and fast enrichment of anthocyanins by dummy

molecularly imprinted magnetic nanoparticles
Qian-Yu Zhao a,1 , Hai-Tian Zhao a,1 , Xin Yang a,∗ , Hua Zhang a , Ai-Jun Dong a , Jing Wang b,∗ ,
Bin Li c,∗
a
Department of Food Sciences and Engineering, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin, 150001, PR China
b
Key Laboratory of Agro-Product Quality and Safety, Institute of Quality Standard & Testing Technology for Agro-Product, Chinese Academy of Agricultural
Sciences, Beijing, 100081, PR China
c
School of Pharmacy, Jiangxi University of Traditional Chinese Medicine, 818 Meiling Avenue, Nanchang, 330004, Jiangxi, PR China

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

Article history: In this work, a novel synthetic strategy for separation media which quickly specific recognize antho-
Received 22 May 2018 cyanins was developed by dummy molecular imprinting technology and magnetic separation technology.
Received in revised form 25 July 2018 Rutin, similar to cyanidin-3-O-rutinoside structure, was selected as a virtual template. The appropriate
Accepted 12 August 2018
molecular imprinting system was selected by NIP library screening method, 4-vinyl pyridine and ace-
Available online xxx
tonitrile as functional monomer and solvent respectively. The molecularly imprinted layer was formed
on the surface of the magnetic carrier to prepare dummy magnetic molecularly imprinted polymers.
Keywords:
The microstructures of prepared composites were characterized by scanning electron microscopy (SEM),
Magnetic molecularly imprinted polymers
Surface imprinting method
Fourier transform infrared spectroscopy (FT-IR), thermal gravimetric analysis (TGA) and X-ray diffrac-
Dummy template tion (XRD). Adsorption kinetics, isothermal adsorption curves and selective specificity were investigated
Cyanidin-3-O-rutinoside to further reveal the specific recognition mechanism of the adsorbents on anthocyanins. The dummy
molecularly imprinted polymers showed a short kinetic equilibrium time, high selectivity (comparing
with quercetin and naringin), and satisfying adsorption capacity for anthocyanins. The binding capac-
ity and the imprinting factor of dummy molecularly imprinted polymers can reach 15.69 mg g−1 and
2.05, respectively. In addition, the purity of cyanidin-3-O-rutinoside in the crude extract from Lonicera
caerulea increased to 89% after the adsorption of dummy magnetic molecular imprinted polymers. Many
favorable capabilities of the prepared molecularly imprinted polymers also provided the basis for further
application for separation and purification of anthocyanins in the future.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction According to the reported researches recently, more than 500

Anthocyanins belong to flavonoid polyphenols, and anthocyani-
din forms glycosides with various sugars under natural conditions.
Anthocyanins, as an important class of water-soluble natural food
colorings, are safe, non-toxic, rich in resources, and have some
nutrition and pharmacological effects, such as the removal of free
radicals [1], anti-cancer [2], anti-inflammatory [3], inhibition of
lipid peroxidation [4], protection liver [5], prevention diabetes [6],
protection vision [7] and more.
∗ Corresponding authors.
E-mail addresses: yangxin@hit.edu.cn (X. Yang), w jing2001@126.com
(J. Wang), lbin@jzjt.com (B. Li).
1
Qianyu Zhao and Haitian Zhao contributed equally to this work and should be
considered co-first authors.
anthocyanins have been found from plants [8,9]. However, unsta-
ble structures as well as various analogues leads to very difficult
isolation and purification of anthocyanins. Therefore, the rapid iso-
lation and purification of anthocyanin monomer has always been
a difficult problem to be solved. The traditional separation and
purification methods including column chromatography [10], high
performance liquid [11], membrane separation [12], high-speed
countercurrent chromatography [13] and so on, cannot be accom-
plished in one go and requires multiple methods to work with each
other in order to obtain a relatively single anthocyanin component,
and have the following problems exist: the using of a large amount
of organic solvents, very low yield and the structure changed.
Since anthocyanins are difficult to purify, many pharmacological
experiments only use anthocyanin crude extracts as experimental
materials, which greatly hinder the anthocyanin structure-activity
relationship research process [14]. Therefore, there is an urgent

https://doi.org/10.1016/j.chroma.2018.08.029
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Fig. 1. The chemical structure of cyanidin-3-O-rutinoside (A) and rutin (B).
requirment to develop a new and effective separation and purifi-
cation method for anthocyanins.
Molecular imprinting technology (MIT) is a technology for the
synthesis of molecular imprinting polymers (MIPs), a synthetic
receptor with special molecular recognition function [15]. The pre-
pared polymers form a special “memory” to the template molecule
both in the spatial structure and the chemical bond which can
identify the target compound from the complex system in a short
period of time. Because molecularly imprinted polymers have the
properties of special selectivity, easy preparation, simple operation
and low solvent consumption, MIPs can replace of the traditional
chromatography stationary phases for the purification of active
ingredients.
Currently, very little work has been reported on antho-
cyanins molecularly imprinted polymers because anthocyanins are
highly unstable and susceptible to pH, oxygen, temperature, light,
enzymes and other factors. Similarly, under the existing synthetic
molecular imprinting conditions (UV light, radiation or heat), it is
difficult to ensure that the structure of anthocyanins is not dam-
aged. In addition, the expensive price of anthocyanin monomers
increases the cost of MIPs.
Dummy template molecular imprinting technology is the use
of close structural analogs to replace the real template molecules
[16], which is developed to avoid template leak in trace detection.
It was reported that dummy templates based on MIPs for nons-
teroidal anti-inflammatory [17], bisphenol A [18], triazophos [19]
have been reported. The resultant MIPs demonstrated highly selec-
tive and sensitive recognition and determination abilities, which
have great application potential in real sample. In recent years, it
is also applied to the imprinting of a class of template molecules
that are expensive, unsafe, unstable and low solubility in synthe-
sis system [20,21]. Therefore, we plan to use the dummy template
molecular imprinting synthesis strategy to solve this problem. In
dummy template molecular imprinting technology, how to choose
a virtual template is crucial.
In this study, we selected cyanidin-3-O-rutinoside (C3R) as a
research target, and rutin as a dummy template for synthesis of
MIPs (Fig.1) because they are all flavonoids and have similar C6 C3 C6
carbon skeletons and the number of hydroxyl groups on the car-
bon skeleton and substitution position of these two substances are
the same. In addition, they have uniform type of linked sugar and
glycosidic bond.
The type of functional monomer plays a crucial role in the
imprinting process. The interaction between the template and the
functional monomer directly determine the recognition perfor-
mance and adsorption capacity of the polymers. The selection of the
best functional monomer is a key step in the preparation of MIPs.
The usual methods for screening functional monomers including
UV spectroscopy [22], nuclear magnetic resonance [23], fluores-
cence spectroscopy [24] and computer simulation [25] and so on,
only can judge the strength of the interaction of the template and
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the functional monomer, however the actual synthesis process of
molecularly imprinted polymers is also affected by many factors.
A new method, NIP screening library, is put forward which use
non-imprinted polymers (NIPs) to replace corresponding MIPs for
screening functional monomers and porogens [26]. NIPs and MIPs
have the same adsorption behavior, if the NIPs have interaction
with the template molecule, and the corresponding MIPs will show
a very good imprinting effect. This method simulates the forma-
tion of MIPs more accurately. The synthesis of NIP screening library
skips the process of template elution and reduces costs. NIP Screen-
ing Library one-time high-throughput screened multiple factors
that affect the adsorption quantity of MIPs. In this paper, functional
monomers and solvents were screened by NIP screening library
simultaneously.
In this paper, a novel adsorbent that can quickly isolate antho-
cyanins is prepared by the combination of surface molecular
imprinting technique and dummy template molecular imprint-
ing strategy. Surface molecular imprinting technology makes the
imprinting sites located in or near the surface, which increases the
effective imprinting sites, improves the imprinting capacity, accel-
erates the mass transfer rate and facilitates the diffusion and elution
of the template molecules. We select 4-vinyl pyridine as func-
tional monomer and acetonitrile as solvent according to NIP library
screening. 4-VP can form electrostatic attraction and ␲–␲* inter-
action with the dummy template rutin. The structure of C3R was
similar to rutin. DMMIPs also recognized C3R by electrostatic inter-
action and ␲–␲* interaction. These two forces were not disturbed
in water, which was a guarantee to recognize anthocyanins in
water. The optimized molar ratio of template-functional monomer-
crosslinker were 1:8:40 (shown in Fig. S1). Magnetic nanoparticles
are selected as the supporter because of its large specific surface
area, good compatibility and superparamagnetic. In the separation
process magnetic molecularly imprinted polymers (MMIPs) can be
quickly separated from liquid, which simplify the separation pro-
cess. The synthesis route of dummy magnetic molecular imprinted
polymers (DMMIPs) is represented in Fig. 2.
2. Experimental
2.1. Instrumentation
A TU-1900 Double-beam UV–vis spectrophotometer (Persee,
China) was used to measure the absorbance of rutin and C3R.
Scanning electron micros-copy(SEM) was observed using a Quanta
200FEG (FEI, U. S.) which was employed to examine the size and
morphology of the nanoparticles. Fourier transform infrared spec-
tra (FT-IR) were perform on an Avatar 360 (Nicolet, U. S.) instrument
to verify whether the molecularly imprinted film was coated onto
the surface of vinyl-modified magnetic nanoparticles. The crystal
structure of the nanoparticles was characterized by a JCXA 733
X-ray diffractometer (Shimadzu, Japan). Thermal Gravity Analysis
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Fig. 2. The synthesis route of dummy magnetic molecular imprinted polymers (DMMIPs).

(TGA) was obtained from room temperature to 800 ◦ with a heating
rate of 10 ◦ C min−1 under a nitrogen atmosphere by a Netzsch STA
449C (Germany) thermogravimetric analyzer. The magnetic prop-
erties were made using a LakeShore 7404 (USA) Vibrating sample
magnetometer (VSM). Nitrogen sorption isotherms were obtained
at −196 ◦ C on a 3H-2000PS1 Gas Sorption and Porosimetry system.
Samples were normally prepared for measurement after degassing
at 150 ◦ C under vacuum until a final pressure of 1 × 10-3 Torr was
reached. High-performance liquid chromatography (HPLC) analysis
was carried out using an Agilent 1200 series liquid chromatogra-
phy (U. S.), equipped with an Agilent G1315B UV–vis Detector and a
TC-C18 column (4.6 mm i.d. × 250 mm, Agilent Technologies, USA).
2.2. Chemicals and materials
All commercial reagents were analytical grade and used
directly. Double distilled water was used in this study. Iron(III)
chloride hexahydrate (FeCl3 ·6H2 O, 99%) and methacrylic acid
(MAA, 99.5%) were purchased from Tianli Chemical Reagent Co.
(Tianjing, China). Tetraethyl orthosilicate (TEOS, 99.5%), tetrahy-
drofuran (THF, 99.5%), acetonitrile (MeCN, %) and acetone (AC,
99%) were purchased from Kemiou Chemical Reagent Co. (Tian-
jing, China). Rutin (Ru, 98%), naringin (Nar, 95%), qurecetin (Qu,
97%), 2-propenoic acid, 2-methyl-, 3-(trimethoxysilyl)propyl ester
(MPS, 97%), methyl-2-propen-1-ol (MAOH, 99%), 4-vinylpyridine
(4-VP, 96%), 4- vinylbenzoic acid (4-VBA, 97%) and ethy-
lene glycol dimethacrylate (EGDMA, 98%) were purchased from
Aladdin (Shanghai, China). Acrylamide (AM, 99.9%) was pur-
chased from Amresco Chemical Reagent Co. (Shanghai, China).
2,2-Azoisobutyronitrile (AIBN, 98%) was purchased from Xiya
Chemical Reagent Co. (Shandong, China). Acrylic acid (AA, 99%)
was purchased from Basf Chemical Reagent Co. (Tianjing, China).
Methacrylic acid (MAA, 98%) was purchased from Sinopharm
Chemical Reagent Co. (Shanghai, China). Cyanidin-3-O-rutinoside
(97%) was purchased from Seebio Chemical Reagent Co. (Shanghai,
China).
2.3. Synthesis of vinyl-functionalized magnetic nanoparticles
An aqueous suspension of superparamagnetic magnetite
nanoparticles was synthesized by a hydrothermal method. Briefly,
13.5 g of FeCl3 ·6H2 O and 36 g of NaAc were dissolved in 500 mL of
ethylene glycol, then the solution was transferred into an autoclave.
The reaction maintained 6 h at 200 ◦ C. After reaction finished, we
obtained black product by a magnet, used ethyl alcohol and water
to wash product several times to remove unreacted reagent, dried
under vacuum at 60 ◦ C for 12 h. The Fe3 O4 @SiO2 magnetic nanopar-
ticles were prepared according to a sol-gel method. 0.4 g of Fe3 O4
were first dissolved in 200 mL of the mixture of isopropanol and
water (4:1, v/v). Followed by ultrasonic treatment for 15 min. Sub-
sequently, 6 mL ammonium hydroxide and 3 mL TEOS were added
drop by drop, and the mixture was mechanically stirred at room
temperature for 12 h. After the resultant particles were isolated by
a magnet, washed three times with ethyl alcohol and water respec-
tively, dried under vacuum at 60 ◦ C for 12 h. Then used MPS to
introduce vinyl groups on the surface of Fe3 O4 @SiO2 . Took 0.2 g
of Fe3 O4 @SiO2 into a three-neck flask which contained 200 mL
toluene. The mixture with ultrasonic treatment maintained 15 min
to make nanoparticles disperse in toluene evenly. 4 mL MPS were
added dropwise into flask under successive mechanical stirring.
The reaction continued 12 h at room temperature. After reaction,
the mixture was collected by an external magnetic field. The pro-
cess of wash and dry were consistent with the above.

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imprinted magnetic nanoparticles, J. Chromatogr. A (2018), https://doi.org/10.1016/j.chroma.2018.08.029
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2.4. Non-imprinted polymers combinatorial libraries
Non-imprinted polymers combinatorial libraries included 24
different polymers prepared by bulk polymerization which com-
binations were reported in Table S1. In a glass vial, 0.6 mmol
functional monomer was dissolved in the solvent, and then added
3 mmol of cross-linker EGDMA and 0.1 mmol of initiator AIBN into
solution. Subsequently deoxygenated via nitrogen for 10 min. The
polymerization was at 60 ◦ C for 24 h. Used ethyl alcohol to wash
product three times to remove unreacted reagent and dried under
vacuum at 60 ◦ C for 12 h. The capacity of non-imprinted polymers
was evaluated by the adsorption of rutin.
2.5. Preparation of DMMIPs and DMNIPs
In this work, we used the surface molecular imprinted method
to synthesize DMMIPs with rutin as dummy template and 4-VP as
functional monomer. The pre-polymerization solutions were pre-
pared by adding 0.075 mmol of dummy template in 15 mL of MeCN
in a glass vial, with a molar ratio template: functional monomer
1:8. Pre-polymerization continued 6 h for sufficient self-assembly
between rutin and 4-VP. And then 40 mg of vinyl-functionalized
magnetic nanoparticles were added into pre-polymerization solu-
tions. After 2 h, put 3 mmol of EGDMA and 0.1 mmol of AIBN
into mixture. The mixture was purged with nitrogen for 10 min.
Then the polymerization process was performed at 60 ◦ C for 24 h,
imprinted polymers were collected through magnet separation. To
remove the template, the obtained product was washed repeatedly
with the mixture of methanol and acetic acid (9:1, v/v) until no rutin
detected by UV–vis spectrophotometer. For comparison, the mag-
netic non-imprinted polymers (DMNIPs) were prepared with the
same condition without adding dummy template.
2.6. Adsorption experiments
The experiment which studied the property of DMMIPs included
four parts, adsorption isotherm, adsorption kinetic, specificity and
reusability. The first part, the measurement of adsorption isotherm,
was that added 5 mg of absorbent (i.e. DMMIPs and DMNIPs, respec-
tively) into solutions of C3R with different concentrations ranging
from 5 mg L−1 to 50 mg L−1 (The solvent was 0.1 mol L−1 of citrate
buffer solution, pH = 4, temperature was 35 ◦ ), respectively. When
reached the adsorption equilibrium, the concentration of C3R in
the supernatant solution was measured by a UV–vis Spectropho-
tometer at 520 nm detection wavelength. The adsorption quantum
capacity [27] was calculated according to Eq. (1), thus we can fab-
ricate adsorption isotherm curves.
Qe = (C0 − Ce )V/m
Where Qe (mg g−1 ) is the equilibrium adsorbed amount of
cyanidin-3-O-rutinoside, C0 (mg L−1 ) and Ce (mg L−1 ) are the initial
and equilibrium concentration of cyanidin-3-O-rutinoside, respec-
tively. V (L) is the solution volume, m (g) is the weight of absorbent.
Secondly, adsorption kinetic, 5 mg of absorbent and 5 mL
50 mg L−1 C3R solution (The solvent was 0.1 mol L−1 of citrate buffer
solution, pH = 4, temperature was 35 ◦ ) were mixed together. The
content of C3R in supernatant solution was detected at differ-
ent time intervals (5–180 nm). And then corresponding adsorption
kinetic curves were fabricated.
Thirdly, adsorption specificity studies were used to evaluated
the specificity of synthetic adsorbents. Quercetin (Que), naringin
(Nar), cyanidin-3-O-rutinoside (C3G), cyanidin (Cy), delphinidin
(Dp), pelargonidin (Pg), malvidin (Mv), peonidin (Pn), petunidin
(Pt), rutinose (Ruo), lactose (Lac), sucrose (Suc), glucose (Glu),
rhamnose (Rha), raffinose (Raf) were selected as structural analogs.
5 mg of DMMIPs and DMNIPs were placed in a solution of analogs
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at a concentration of 40 mg L−1 , respectively, at room tempera-
ture for 2 h. The samples were separated by external magnetic
field. The equilibrium concentrations of anthocyanins, quercetin
and naringin in the solution were measured at 520 nm, 375 nm
and 282 nm using a two-beam UV–vis spectrophotometer. The con-
centration of the carbohydrates was measured by phenol-sulphate
colorimetry at 490 nm. The imprinting factor (IF) [28] was calcu-
lated according to Eq. (2).
IF = QMIPs /QNIPs (2)
Where IF was the imprinting factor of molecularly imprinted, QMIPs
was the adsorption amount of molecularly imprinted polymers
(mg g−1 ), QNIPs was the adsorption amount of non-molecularly
imprinted polymers (mg g−1 ).
The selectivity coefficient (SC) [29]were calculated by the fol-
lowing Eq. (3):
SC = IF t /IF c (3)
Where IFt and IFc were the imprinting factor for template and its
structure analogues.
Finally, the reusability and stability of DMMIPs were investi-
gated by measuring the adsorption capacity for target molecule
in the adsorption-desorption cycles. Took 5 mg DMMIPs to 5 mL
50 mg L−1 of C3R aqueous solution, placed on a thermostatic shaker
for 2 h. Under the action of an applied magnetic field to solid-liquid
separation, the cyanidin-3-O-rutinoside concentration in super-
natant was measured by UV spectrophotometry. The collected
DMMIPs were eluted with the mixture of methanol / acetic acid
(9:1, v/v) until C3R were not detected, and then the sample was
vacuum dried at 40 ◦ C. Repeated the above steps 10 times.
2.7. Analytical applications in purification of crude anthocyanin
200 mg MIPs were added to a 100 mL centrifuge tube followed
by the addition of 20 mL of 100 mg L−1 40% crude anthocyanin prod-
uct from the lonicera caerulea. After adsorption for 2 h, the MIPs
with magnetic separation were washed three times with 5 mL of
double distilled water and then were eluted with 1 mL of 1% acetic
acid/ methanol for 30 min. After magnetic separation, the eluted
solution was filtered through a 0.22 microfiltration membrane for
HPLC-UV detection. HPLC conditions and the preparation methods
for crude anthocyanins were described in detail in the Supporting
information.
3. Results and discussion
3.1. Characterization of DMMIPs
(1)
The morphology of Fe3 O4 , Fe3 O4 @SiO2 , DMMIPs, DMNIPs were
characterized by SEM. It can be seen from the Fig. 3A that the mean
diameter of the superparamagnetic Fe3 O4 nanoparticles prepared
by the hydrothermal method was about 170 nm and the particle
size distribution was uniform. The particle size of the microspheres
was increased to about 400 nm (Fe3 O4 @SiO2 ), which indicated that
hydrolytic TEOS coated on the surface of the Fe3 O4 particles suc-
cessful to form microspheres with smooth surface, uniform size
dispersion. Compared with Fe3 O4 @SiO2 , the average size of the
magnetic molecularly imprinted particles (DMMIPs and DMNIPs)
did not change significantly, but the surface was rough. Because
the reaction was carried out on the surface of the support, and the
molecularly imprinted layer was porous structure.
FT-IR spectra of Fe3 O4 , Fe3 O4 @SiO2 , Fe3 O4 @SiO2 @C = C,
DMMIPs, DMNIPs were shown in Fig. 3B. The peak at 575 cm−1 was
attributed to the Fe O that was the characteristic absorption peak
of Fe3 O4 , which proved the successful synthesis of Fe3 O4 magnetic
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Fig. 3. (A)SEM images, (B) FT-IR spectra, (C) TGA curves, (D) XRD patterns, (E) magnetization curves at 300 K.

particles (curve of Fe3 O4 ). In comparison with the curve of Fe3 O4 ,
the new characteristic peaks at 951 cm−1 , 799 cm−1 and 1091 cm−1
(curve of Fe3 O4 @SiO2 ) resulted from the stretching of Si OH, Si O,
and Si O Si, respectively, which proved that SiO2 layer was
synthesized on the surface of Fe3 O4 . The typical peak at 1637 cm-1
for C C groups showed that MPS was grafted on the surface of
Fe3 O4 @ SiO2 (curve of Fe3 O4 @SiO2 @C C). The spectra of DMMIPs
and DMNIPs (curve DMMIPs and DMNIPs) all showed typical
stretching vibration of C O (1736 cm−1 ), CH3 (2987 cm−1 ), CH2
(2956 cm−1 ) and aromatic ring (1599 cm−1 ) which demonstrated
the cross-linker EGDMA and the functional monomer 4-VP were
grafted successfully. Each of the above characteristic absorption
peaks indicated the successful synthesis of each reaction in the
preparation of DMMIPs and DMNIPs.
The thermogravimetric analysis was used to determine the
composition of magnetic microspheres coated with the polymers.
Fig. 3C gave the TGA curve of four kinds of particles, Fe3 O4 ,
Fe3 O4 @SiO2 @C C and DMMIPs. The weight loss at the first 100 ◦
was mainly due to the desorption of organic solvent and the evap-
oration of water in the polymers structure. We mainly analyzed
the data between 200 ◦ and 900 ◦ . In curve of Fe3 O4 , the weight loss
of Fe3 O4 was 2.34% (does not contain water) when the tempera-
ture reached 900 ◦ because the part of Fe3 O4 microspheres were
pyrolyzed into Fe2 O3 . From the curve of Fe3 O4 @SiO2 , the mass loss
of Fe3 O4 @SiO2 was 5.59% in the temperature range of 200–900 ◦ ,
due to the loss of silanol groups on the surface of Fe3 O4 @ SiO2 . The
high melting point of SiO2 made it keep steady in high temperature.
Therefore, the mass loss of SiO2 during the whole heating process
was very low. In contrast to curve of Fe3 O4 @SiO2 , the thermal loss
of Fe3 O4 @SiO2 @C C was 6.16%, which was 0.56% more than that
of Fe3 O4 @ SiO2 . The reason was that Fe3 O4 @SiO2 was coated with
MPS. The weight of DMMIPs decreased fast from 250 ◦ to 400 ◦ , and
the temperature was higher than 400 ◦ , the weight loss of DMMIPs
was not obvious, which originated from the imprinted polymers
on the surface of magnetic support. It improved that the layer of
molecular polymers was synthesized successfully on the surface of
the alkylated magnetic nanocomposite that occupied 28.89% of the
magnetic molecularly imprinted particles approximately.
Fig. 3D showed the characterization of the Fe3 O4 , Fe3 O4 @SiO2 ,
and DMMIPs crystal forms used X-ray diffraction patterns. The six
obvious diffraction peaks of 30.14◦ , 35.51◦ , 43.08◦ , 53.57◦ , 56.96◦
and 62.62◦ shown in curve (a) correspond to the indices of crystal

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Table 1
Data of nitrogen adsorption of nanoparticles.
Polymers SBET (m2 g−1 ) Vp (cm3 g−1 )
Fe3 O4 40.13 0.19
Fe3 O4 @SiO2 15.88 0.047
DMMIPs 65.62 0.12
DMNIPs 30.42 0.084
face of (220), (311), (400), (422), (511), and (440), respectively, was
in agreement with standard Fe3 O4 XRD data [30]. After coated with
the polymer layer, the characteristic peaks position did not change
which proved Fe3 O4 crystal structure unchanged.
The magnetic properties of nanoparticles were studied using
VSM. The hysteresis loop of Fe3 O4 and Fe3 O4 @SiO2 , DMMIPs at
300 K were shown in Fig. 3E. The saturation magnetization values
of the prepared Fe3 O4 , Fe3 O4 @SiO2 , DMMIPs were 67.34, 28.84 and
20.01 emu g−1 , respectively. It can be seen that an increase in the
number of non-magnetic surface coatings caused a decrease in the
magnetization value of nanoparticles. Three magnetic hysteresis
loops are all symmetrical to the origin, indicating the existence of
superparamagnetic behavior [31].
Data of nitrogen adsorption of nanoparticles were shown in
Table 1. After coating silica on the surface of Fe3 O4 , the spe-
cific surface area, pore volume, and average pore diameter of the
nanoparticles were significantly reduced because of increased the
particle size and the silica layer was relatively dense. However, the
specific surface area, pore volume and average pore diameter of the
DMMIPs were increased significantly, indicating that the imprinted
membrane formed on the surface of the carrier with porous struc-
ture, which mainly because the imprinting holes formed after
template molecules eluted, which also ensured a large adsorption
of DMMIPs to the target molecule.
3.2. Non-imprinted polymers combinatorial libraries
Fig. S2A showed the adsorption capacity of 24 different poly-
mers in the NIP screening library. Comparison of the congener
functional monomer, the adsorption capacity of polymers with
acetonitrile as the porogenic agent were significantly higher than
others (Fig. S2B). In the experiment, we found an interesting phe-
nomenon that the morphology of polymers formed in different
solvent were differently. The polymers’ morphological properties
(porosity and surface area) were determined by the type of solvent
during the polymerization. That was also the factor affected the
adsorption amount. The non-molecularly imprinted polymers syn-
thesized in acetonitrile were dispersed in the solution uniformly
as a white solution without precipitation. The NIPs synthesized
with acetone were dispersed partially in the solution. Synthesized
with methanol, polymers’ morphology liked residue which were of
the worst dispersion in the solution and all precipitated. Synthe-
sized in tetrahydrofuran, NIPs were translucent viscous colloidal,
good affinity for solvents. The difference of microstructure between
these polymers were observed by the scanning electron microscopy
for further study. In Fig. S3, the polymers formed in acetonitrile
was monodisperse sphericity which size was smallest. But the
polymers were formed in other solvents with varying degrees of
cross-linking. Because the force between the solvent molecules and
the polymer chains in a good solvent was stronger than the force
between the polymer chains. In poor solvents, the force between
the polymer chains increased, formed a larger aggregation. The
phase separation of porogen and growing polymer occurred dur-
ing the polymerization thereby forming pores. The separation of
porogen and low solubility phase happened in the early period
of polymerization which tended to form larger pores and lower
surface area. In contrast, porogen and higher solubility separated
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imprinted magnetic nanoparticles, J. Chromatogr. A (2018), https://doi.org/10.1016/j.chroma.2018.08.029
slowly in the polymerization to provide a material with smaller
pore size and larger surface area [32]. Large surface area of the poly-
Dmean (nm) mers meant the more recognition sites and the higher adsorption
10.51 of the target. Therefore, we choosed acetonitrile as porogen.
6.28 Comparing the same solvents in the Fig. S7C, it can be obviously
5.19 observed that the adsorption capacity of polymers with 4-VP and
4.27 MA as functional monomers were significantly higher. 4-VP inter-
acted with the template molecule strongly that was mainly due
to its structural characteristics of molecules. 4-VP was a nitrogen-
containing heterocyclic compound with strong basicity which can
be used as a hydrogen bond acceptor and also formed ␲-␲* conju-
gation with the benzene ring. These features allowed it to interact
with template molecules to form a stable host-guest complex. In
addition, the strength of the hydrogen bond was decided to the
electronegativity of the acceptor and donor. The higher the elec-
tron density of the acceptor, the stronger the hydrogen bond. It also
was affected by the steric hindrance of the acceptor, the smaller the
steric hindrance, the stronger the hydrogen bonding. Therefore, MA
formed stronger interaction with template molecules than other
functional monomers.
The first five groups with good effects were selected from the
24 combinations and the corresponding MIPs were synthesized to
verify the accuracy of the NIP library screening method. The experi-
mental results were shown in Fig. S4. It verified that NIPs have some
interaction sites on the template molecule, and the correspond-
ing MIPs showed good imprinting effect on the template molecule,
which can be used to screen functional monomers and porogen
with NIP library. The imprinting factor was the index to evaluate
the molecular imprinting effect. The polymers with combination
of 4-VP-ACE had the best imprinting effect. The 4-VP molecular
structure is a rigid plane, and the recognition site formed is more
stable. However, the MA conformation is easily changed so that
the molecularly imprinted holes cannot well match with the tar-
get compound and the specificity was reduced. Therefore, 4-VP and
acetonitrile were chosen as the functional monomer and porogen
respectively.
3.3. The optimization of extraction conditions
3.3.1. The effect of pH
The extent of C3R extraction depended on its interaction with
the pyridine groups of DMMIPs. Anthocyanins existed in different
forms under different pH. To evaluate the effect of pH, working
solutions with different pH in the range of 2.0–7.0 were studied.
As shown in Fig. S5A, the highest adsorption capacity of C3R by
DMMIPs was at pH 4.0 and lesser adsorption was observed at lower
and higher pH values, which may be due to the fact that when
the pH of the extract solvent was close to pKa (The pKa of C3R is
4.7), the anthocyanin was the most acidic and had the strongest
electrostatic interaction with DMMIPs. Therefore, the pH 4.0 was
selected as optimized pH.
3.3.2. The effect of temperature
DMMIPs recognized anthocyanins through electrostatic inter-
actions, and temperature affected electrostatic forces. Therefore,
the effects of different temperatures (5 ◦ –35 ◦ ) on the adsorption
amount were investigated. It can be seen from the Fig. S5B that at
35 ◦ , the adsorption of C3R by DMMIPs was highest, because the
molecular thermal motion was slower at low temperatures, slow-
ing down the mass transfer rate. When the temperature increased,
the molecular thermal motion was intensified making the distance
between the molecules become large, and the electrostatic force
was weakened, so the adsorption amount decreased. Therefore, the
35 ◦ was selected as optimized temperature.
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Fig. 4. (A) Curve of adsorption kinetics, (B) the pseudo-first-order model, (C) the pseudo-second-order model, (D) segmented model.
3.3.3. The effect of solvent
Solvent was also an important factor affecting DMMIPs bind-
ing capacity. In order to study the effect of this factor, water and
ethanol were mixed in different proportions (1:4, 2:3, 3:2, 4:1)
as binding solvents, the results were shown in Fig. S5C. It could
be observed that the binding performance of DMMIPs was best in
pure water solution. Because water promoted the ionization of C3R
which enhanced the electrostatic force between C3R and DMMIPs.
In addition, the hydrophobic group will spontaneously aggregate
in water, shortening the distance between the pyridine functional
group of DMMIPs and the benzene ring of C3R, increasing the ␲–␲*
interaction. Therefore, water was selected as optimized solvent.
3.4. Binding properties of DMMIPs
3.4.1. Adsorption kinetics
Fig. 4A showed the adsorption kinetic curves of 50 mg L−1 C3R
solution onto DMMIPs and DMNIPs. At 40 min, DMNIPs almost
reached saturation, and the adsorption capacity was 7.28 mg g−1
at this time. The maximum saturated adsorption capacity was
7.58 mg g−1 at 180 min. Polypyridine compounds were formed on
the surface of DMNIPs, so DMNIPs can adsorb C3R. At 60 min,
the adsorption capacity of molecularly imprinted polymers came
near to saturation and the adsorption rate was fast compared with
the molecularly imprinted polymers that were synthesized with
bulk polymerization [33]. At this time, the adsorption amount of
DMMIPs was 15.59 mg g−1 , and the maximum saturated adsorption
capacity was 16.57 mg g−1 at 180 min. The experimental results
showed that the adsorption amount of C3R by DMMIPs was 2.18
times higher than that of DMNIPs. In the preparation of molecular
imprinting, DMMIPs added rutin as the dummy template, whereas
DMNIPs were polypyridines formed by the polymerization of 4-
Please cite this article in press as: Q.-Y. Zhao, et al., Selective recognition and fast enrichment of anthocyanins by dummy molecularly
imprinted magnetic nanoparticles, J. Chromatogr. A (2018), https://doi.org/10.1016/j.chroma.2018.08.029
7
VP on the support surface in the absence of template. The physical
structure, electrostatic interaction and ␲–␲* interaction of DMMIPs
imprinting cavities matched the target molecules, which identi-
fied C3R through physical and chemical forces and adsorbed C3R
in the imprinted pores. In order to further study the adsorption
mechanism of DMMIPs, the first-order adsorption kinetics model
(Fig. 4B) and the second-order adsorption kinetics model (Fig. 4C)
were used. The pseudo-first-order rate expression and the pseudo-
second-order rate expression were calculated according to Eqs. (4)
and (5), respectively [34].
 
log Q eq − Qt = logQ1cal − k1 t/2.303 (4)
2
t/Qt = 1/(k2 Q2cal ) + t/Q2cal (5)
Where t was the adsorption time (min), Qeq was the equilibrium
adsorption capacity (mg g−1 ), Qt was the adsorption capacity at
different time intervals (mg g−1 ), Qcal was the theoretical adsorp-
tion capacity, k1 was the first-order rate constant (min−1 ), k2 was
the rate constant of second-order adsorption (g mg−1 min−1 ).
The fitting effect of two models is not very ideal according to
coefficient of determination, R2 . We divided the data into two
sections for analysis (Fig. 4D) and found that the adsorption was
consistent with the pseudo- first-order model up to 40 min with
a fitting curve of Y = −0.018 X + 1.30 and R2 = 0.98. After 40 min,
the adsorption process was in accordance with the pseudo-second-
order model, and the fitting curve was Y = 0.059 X + 0.28, R2 = 0.99.
According to the slope and intercept of the linear plot, the adsorp-
tion rate constants k1 (0.041 min−1 ) and k2 (0.012 g mg−1 min−1 )
of two kinds of adsorption were calculated respectively. These
results suggested that the pseudo first order kinetic model pre-
dominated in the first 40 min and the pseudo second order kinetic
model dominated after 40 min in the adsorption of target molecules
by prepared DMMIPs. The whole process was that the adsorbate
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Fig. 5. (A) Curve of adsorption isotherms, (B) fitted Langmuir equation of DMMIPs, (C) fitted Freundlich equation of DMMIPs, (D) the Scatchard plot for the DMMIPs and
DMNIPs.
diffused and accumulated to the surface of the adsorbent firstly
through the physical interaction, and then the adsorbate and the
functional monomer in the cavity further occurred the electron and
the common electron transfer and was adsorbed to the recognition
hole.
3.4.2. Adsorption isotherm
Fig. 5A showed the adsorption isotherms of C3R solutions on
DMMIPs and DMNIPs. In the range of 0–40 mg L−1 C3R solutions, the
adsorption amount of DMMIPs increased with the increase of C3R
concentration. When the C3R concentration was over 40 mg L−1 ,
the increasing rate of DMMIPs adsorption tended to be balanced
gradually. Adsorption was saturated because when the C3R con-
centration reached a certain value, all the specific imprinting sites
on the surface of the DMMIPs were occupied by the C3R molecules.
Adsorption isotherms made clear how adsorbed molecules acted
on the sorbent surface. Therefore, it was very important to predict
the adsorption mode of the imprinted material surface by using
the theoretical formula to simulate adsorption isotherms data. The
Langmuir model and the Freundlich model were used. The empir-
ical linear equations for the Langmuir model and the Freundlich
model were shown in Eqs. (6) and (7), respectively [34].
1/Qeq = 1/Qm + 1/(bQm Ceq )
logQeq = logkf + (1/n)logCeq
Where Qeq was the equilibrium adsorption capacity (mg Qm g−1 ),
was the saturated adsorption capacity of polymers (mg g−1 ), Ceq
was cyanidin-3-O-rutinoside concentration (mg L−1 ), b was the
Langmuir adsorption equilibriumconstant (L mg−1 ), kf was the Fre-
undlich adsorption equilibrium constant (L mg−1 ), n was a constant.
Please cite this article in press as: Q.-Y. Zhao, et al., Selective recognition and fast enrichment of anthocyanins by dummy molecularly
imprinted magnetic nanoparticles, J. Chromatogr. A (2018), https://doi.org/10.1016/j.chroma.2018.08.029
Fig. 5B and C were the fit linear of the Langmuir model and Fre-
undlich model. Compared with the R2 of two fitting models, the
Langmuir model was suitable for the adsorption process for C3R.
The Langmuir model reflected the monolayer adsorption of C3R on
the surface of imprinting material, and all the adsorption sites had
the same adsorption energy and enthalpy change.
The Scatchard equation was adopted to evaluate the binding
ability of the DMMIPs and DMNIPs. The Scatchard equation [35]
was expressed as follows Eqs. (8) and (9), respectively.
Qeq /Ceq = Qmax /Kd − Qeq /Kd (8)
Ka = 1/Kd (9)
where Qeq was the equilibrium adsorption capacity (mg g−1 ), Qmax
was the apparent maximum binding amount (mg g−1 ), Ceq was the
cyanidin-3-O-rutinoside concentration at equilibrium (mg mL−1 ),
Kd was the equilibrium dissociation constant (g L−1 ), and Ka was
the equilibrium binding constant (L g −1 ).
As shown in Fig. 5D, the Scatchard plot for the DMMIPs were
one straight line, which indicated that there was only one type of
binding site in DMMIPs. The fitting result can be consistent with the
Langmuir plot results. According to the linear regression equation,
the Ka of DMMIPs and DMNIPs were calculated to be 0.057 L g −1
(6) and 0.037 L g −1 , respectively. The Ka of DMMIPs was higher than
that of DMNIPs, which confirm the high affinity of the DMMIPs to
(7)
C3R.
3.4.3. Selectivity of DMMIPs
In order to prove the high affinity and selectivity of DMMIPs for
cyanidin-3-O-rutinoside, flavonoids (Que and Nar), anthocyanins
(C3G, Cy, Dp, Pg, Mv, Pn, Pt), carbohydrates (Ruo, Lac, Suc, Glu,
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Fig. 6. (A) Selective recognition of DMMIPs and MNIPs for cyanidin-3-O-rutinoside. (B) Reusability of the DMMIPs for ten adsorption and desorption times.

Fig. 7. (A) The HPLC chromatograms of the extract of Lonicera caerulea. The standard of cyanidin-3-O-rutinoside (a), the chromatogram of initial sample of the extract
of Lonicera caerulea (b), the chromatogram of the extract after being adsorbed by DMMIPs (c), the chromatogram of obtained cyanidin-3-O-rutinoside after elution from
DMMIPs (d). (B) Comparison of C3R selectivity and saturation uptake amount for DMMIPs with that of other benchmark materials.

Rha, Raf) were chosen as competitive molecules in this study. The
selectivity of DMMIPs was evaluated by selective adsorption exper-
iments and the results were displayed in Fig. 6A and Table S2. The
chemical structure of analogue was shown in Fig. S6. The adsorption
amount of C3R (15.69 mg g−1 ) was significantly higher than other
structure analogues, which demonstrated that DMMIPs can recog-
nize C3R specifically. The reason why the adsorption quantity of
carbohydrates by DMMIPs was very low was that carbohydrates
were neutral and can only be combined with DMMIPs through
hydrogen bonds, but the interaction was diminished in water. The
remaining analogues were weakly acidic and had benzene rings,
which can bind with DMMIPs in water, but still showed low adsorp-
tion quantity. The size, shape, distribution of the characteristic
functional groups of molecules did not match with the molecu-
larly imprinted three-dimensional pores that resulted in the weak
adsorption ability of DMMIPs for them.
3.4.4. Reusability
The reusability of molecularly imprinted polymers was an
important property for application. Reusability of the DMMIPs and
DMNIPs were shown in Fig. 6B. The adsorption capacity of DMMIPs
only decreased by 15.3% after ten adsorption-elution cycles. The
experiment results showed that the three-dimensional pores of the
imprinted polymers were very stable which were not destroyed
during the process of elution. The slight decrease of adsorption
capacity was because that the three-dimensional cavities on the
surface of imprinted polymers were blocked partly after repeated
the template molecule binding and elution. Therefore, the effec-
tive elution process was particularly important in the application
of molecularly imprinted polymers. The results also proved that
DMMIPs still have good adsorption capacity for anthocyanins after
repeatedly used.
3.5. Analytical applications in purification of crude anthocyanin
Anthocyanins were used in many areas due to their special phys-
iological activity, especially antioxidation activities. However, the
complexity of natural substrates and the poor stability of antho-
cyanins limited the isolation and purification of anthocyanins.
Therefore, in this work we developed a novel adsorption mate-
rial that can rapidly and efficiently extract anthocyanins from the
extract of the Lonicera caerulea. The chromatograms of the extract
before and after being adsorbed by DMMIPs, and the eluent solu-
tion from DMMIPs were exhibited in Fig. 7A. Compared with the
chromatograms of the extract before (curve b) and after (curve c)
being adsorbed by DMMIPs, we can see that the adsorption of C3R in
DMMIPs is significantly higher than other components in the crude
extract. We achieved the expected goal of separating and purify-
ing anthocyanins. The purity of C3R in the eluate was increased to
89% (The content of anthocyanin in crude extract was only 40%).
It is indicated that the prepared DMMIPs had excellent selectiv-
ity for C3R and can be applied to separation and purification of
anthocyanins in real samples.
Three benchmark adsorbents (such as octa decylsilyl silicion
(ODS), polyamide resin (PAM), column chromatography silica gel
(SG)) were selected to compare with the prepared DMMIPs. The

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HPLC of the anthocyanin crude extract after their adsorption was
shown in Fig. S7. It can be seen that these adsorbents could also
absorbe other substances besides C3R. After calculation, Fig. 7B
was obtained, which can more directly reflect the adsorption and
selectivity of several materials for C3R. Among several materials,
DMMIPs had the best adsorption ability and selectivity for C3R
because of the spatial structure of the imprinted sites, which were
tailor-made based on the special coordination pattern of ␲–␲*
conjugation and electrostatic interaction. Notably, the adsorption
effect of SG and ODS on C3R was very low, this mainly because
ODS just adsorbed hydrophobic substances. Although SG was
hydrophilic, SG was weakly acidic, which was not conducive to
adsorb acidic substances. PAM and DMNIPs had similar adsorption
effects, and the amount of adsorption was relatively large because
their main functional groups were basic (amide groups and pyri-
dine groups), which had good adsorption to acidic substances, but
had low selectivity to C3R. All these phenomena indicated that
DMMIPs materials with a high selectivity and large adsorption
capacity were excellent adsorbents for C3R extraction.
4. Conclusions
In this study, the novel and efficient magnetic imprinted poly-
mers with core-shell structure were synthesized by coating dummy
molecularly (rutin), functional monomers (4-VP) and cross-linking
agent (EGDMA) on the surface of Fe3 O4 @SiO2 modified with dou-
ble bond in acetonitrile using surface imprinting technique. NIP
library screening method was a simple and effective method to
determine the “tailored” synthesis system according to the struc-
ture and properties of the template that verified by experiment.
Based on electrostatic attraction and ␲–␲* interaction, DMMIPs
processed strong adsorption affinity, specific recognition and excel-
lent selectivity for cyanidin-3-O-rutinoside. In addition, DMMIPs
had excellent elution ability and reusability. After purification by
DMMIPs, the purity of anthocyanin in the crude extract increased
from 40% to 89%. This work provided a new idea for the sepa-
ration and analysis of anthocyanins and has important practical
value for further research on the structure-function relationship of
anthocyanins.
Acknowledgements
This work was financially supported by National Natural Science
Foundation of China (No. 31571798), Harbin Special Foundation for
Excellent Academic Leader Project (No. 2014RFXXJ113), National
Natural Science Foundation of China (No. 81760708) and Jiangxi
Provincial Academic and Technology Project for Main Disciplines
Leader (No. 20162BCB22016).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in
the online version, at doi:https://doi.org/10.1016/j.chroma.2018.
08.029.
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Please cite this article in press as: Q.-Y. Zhao, et al., Selective recognition and fast enrichment of anthocyanins by dummy molecularly
imprinted magnetic nanoparticles, J. Chromatogr. A (2018), https://doi.org/10.1016/j.chroma.2018.08.029