Microchim Acta
DOI 10.1007/s00604-014-1230-9
ORIGINAL PAPER
Magnetic molecularly imprinted polymer nanoparticles
for the solid-phase extraction of paracetamol from plasma
samples, followed its determination by HPLC
Saman Azodi-Deilami & Alireza Hassani Najafabadi &
Ebadullah Asadi & Majid Abdouss & Davood Kordestani
Received: 5 January 2014 / Accepted: 7 March 2014
# Springer-Verlag Wien 2014
Abstract We are presenting magnetic molecularly imprinted
polymer nanoparticles (m-MIPs) for solid-phase extraction and
sample clean-up of paracetamol. The m-MIPs were prepared
from magnetite (Fe3O4) as the magnetic component, paraceta-
mol as the template, methacrylic acid as a functional monomer,
and 2-(methacrylamido) ethyl methacrylate as a cross-linker.
The m-MIPs were then characterized by transmission electron
microscopy, FT-IR spectroscopy, X-ray diffraction and vibrat-
ing sample magnetometry. The m-MIPs were applied to the
extraction of paracetamol from human blood plasma samples.
Following its elution from the column loaded with the m-MIPs
with an acetonitrile-buffer (9:1) mixture, it was submitted to
HPLC analysis. Paracetamol can be quantified by this method
in the 1 μg L−1 to 300 μg L−1 concentration range. The limit of
detection and limit of quantification in plasma samples are 0.17
and 0.4 μg L−1. The preconcentration factor of the m-MIPs is
40. The HPLC method shows good precision (4.5 % at
50 μg L−1 levels) and recoveries (between 83 and 91 %) from
spiked plasma samples.
Keywords Molecularly imprinted polymer nanoparticles .
Magnetic solid phase extraction . Human plasma samples .
Cross-link agent . HPLC
Electronic supplementary material The online version of this article
(doi:10.1007/s00604-014-1230-9) contains supplementary material,
which is available to authorized users.
S. Azodi-Deilami (*) : A. H. Najafabadi : E. Asadi :
M. Abdouss (*)
Department of Chemistry, Amirkabir University of Technology, P. O.
Box: 15875–4413, Tehran, Iran
e-mail: azodi@aut.ac.ir
e-mail: phdabdouss44@aut.ac.ir
D. Kordestani
Faculty of Chemistry, Department of Organic Chemistry, Razi
University, Kermanshah, Iran
Introduction
Biological fluids such as blood, plasma or urine are referred to
as complex matrices and normally contain a number of en-
dogenous components. These matrix components may inter-
fere with and may adversely affect the subsequent separation
and identification of analyte(s) of interest if they are not
removed. Therefore, two-thirds of the total analysis time in
chromatographic methods is typically due to the sample prep-
aration steps and these are often the major source of error in
the overall analytical process.
A few types of extraction method are used routinely in
analytical toxicology such as Liquid-liquid Extraction (LLE)
[1], Liquid-liquid Micro-Extraction (LLE) [2], Solid Phase
Extraction (SPE) [3], Solid-Phase Micro-Extraction (SPME)
[4], and Molecularly Imprinted Solid Phase Extraction
(MISPE) [5, 6]. Simplicity and versatility of SPE rather than
LLE for intensive labor and solvent consuming, has been
made it most convenient method at biological fluids such as
drug extractions. MISPE is currently a growing field in clean
up techniques for the analysis of biological samples.
SPE is a simple, fast, portable sampling and sample prep-
aration technique using small volumes of solvent and is
solvent-free in several cases. SPE is a very common type of
sample extraction and clean up technique for biological
fluids in analytical chemistry and a well-accepted sam-
ple preparation technique in the analytical chemistry
community [3–6].
A Molecularly Imprinted Polymer (MIP) is a synthetic
polymer bearing on its surface the molecular imprint of a
specific target molecule (the template). Because of its imprint-
ing according to the size, shape and functional groups of a
template molecule, MIP acts as an artificial specific receptor.
In fact, the trace analysis of bio-fluids and other complex
matrices are usually based on selective sample pre-treatment
[7]. However, when a selective extraction from a complex

sample has to be performed, the typical SPE adsorbents lack
sufficient selectivity. Therefore, seeking more selective SPE
adsorbents is important. The SPE approaches using highly
selective adsorbents such as MIPs have the potential to over-
come this problem [8]. Besides the high selectivity of MIPs,
their simplicity of synthesis and less strict operation condi-
tions compared with immune sorbents have led to their out-
standing widespread applications [9]. The main advantage of
molecular imprinting is the possibility of preparing a selective
sorbent pre-determined for a particular substance or a group of
structurally related substances. The polymer has a permanent
memory of the template and is capable of selectively
rebinding the template or structurally similar molecules such
as the metabolites.
The application of MIPs as sorbents in SPE has been found
very useful for selective extraction for the reason that MIPs
offer higher selectivity than conventional SPE. MIPs also
reduce the influence of the matrix on the resulting chromato-
gram and high sample enrichment factors are achieved. The
MIPs are extensively employed in SPE and they found to be
quite effective and selective adsorbents [10].
Currently, magnetic nanoparticles have found numerous
biological and environmental applications owing to their
unique magnetic properties enabling them to be used by the
magnetic field in order to separate the target from the samples
[11]. Thus, it can be anticipated that the cooperation of the
magnetic nanoparticles with MIPs will offer a new approach
for separation applications and extend employing of MIPs. In
addition, the combination of magnetically susceptible proper-
ties of magnetic nanoparticles and the advantage of MIP not
only affords the selectivity for the target molecule but also
provides the ability of one-step separation. The magnetic
molecularly imprinted polymers (m-MIPs) were used for sep-
aration of dibenzothiophene [12]. The extraction of melamine
from milk followed by liquid chromatography-tandem mass
spectrometry was performed by m-MIPs [13]. The extraction
of chloramphenicol from honey using m-MIPs was also car-
ried out [14]. Moreover, the solid phase extractions of ceph-
alosporin from milk [15], synephrine from methanol-water
media [16], emodin from kiwi fruit root [17], five
sulphonylurea herbicides [18], organophosphorus pesticides
in fruit samples [19] and epinephrine in real samples [20] have
been conducted.
Herein, we successfully synthesized 2-(methacrylamido)
ethyl methacrylate that was used as a new cross linker to
prepare magnetic molecularly imprinted nanoparticles for
screening of paracetamol in human plasma using combination
of solid phase extraction and HPLC method. All magnetic
nanoparticles were characterized by TEM, XRD, FT-IR and
VSM analyses. The resulting magnetic molecularly imprinted
polymers (m-MIPs) could be quickly collected and regenerat-
ed by an external magnetic field without tedious centrifuga-
tion or filtration.
S. Azodi-Deilami et al.
Experimental
Materials
Iron (III) chloride hexahydrate (FeCl3.6H2O), iron (II) sul-
phate heptahydrate (FeSO4.7H2O), ammonia solution (28 %,
w/w) for synthesis of Fe3O4 nanoparticles, ethanol, tetraethyl
orthosilicate (TEOS) and methacryloxypropyl
trimethoxysilane (MPS) were obtained from Merck (http://
www.merckmillipore.com, Darmstadt, Germany). For
preparation of the cross linker, methacrylic acid (MAA), thi-
onyl chloride (SOCl2) and ethanol amine were obtained from
Merck (Darmstadt, Germany), Acros organics (http://www.
acros.com, New Jersey, US) and Sigma companies (http://
www.sigmaaldrich.com, US). Paracetamol and other drugs
obtained from the Ministry of Health and Medical Education
(Tehran, Iran). 2,2-Azobisisobutyronitrile (AIBN) and sol-
vents were also obtained from Merck.
Instruments and chromatographic conditions
1
H NMR and 13C NMR spectra were recorded in a Bruker
250 (250 MHz). Sample size was characterized by trans-
mission electron microscopy (TEM, Philips CM30) oper-
ating at an acceleration voltage of 150 kV. The samples
for TEM measurements were prepared by placing one
drop of the sample on copper grids coated with carbon.
The structure of the powder samples was characterized by
X-ray powder diffraction (XRD, INEL EQuinox 3000,
http://www.inel.fr/, France). The XRD patterns were
taken from 10° to 80° (2θvalue) using a Cu Kα radiation
source (λ=1.542 Å) and resolution below 0.1°. All FT-IR
spectroscopic measurements were performed on a Bruker
EQuinox 55 (http://www.bruker.com/, Germany). The
magnetic measurements were carried out in a vibrating
sample magnetometer (VSM, BHV-55, Riken Denshi,
h t t p : / / w w w. r i k e n d e n s h i . c o . j p / , J a p a n ) a t r o o m
temperature. The concentrations of template in the
magnetic polymers were detected on a UV-vis JEVWAY
6505 spectrophotometer (http://www.jenway.com/, US).
The particle size and size distribution of polymer particles
were measured by dynamic light scattering (DLS, Malvern
Zetasizer ZS, http://www.malvern.com, Malvern, UK). The
polymer particles were suspended in acetonitrile and
measured at a fixed scattering angle of 90°. The HPLC
system consist a Waters 515 pump, a 486Waters UV/Vis
detector, a model 7725i Rehodyne injector with a 20 μL
sample loop. Chromatographic separation was achieved on
an ACE 5 μm, C18 column of 4.6 mm×150 mm column.
HPLC data was acquired and processed using a PC and
Millennium 2010 Chromatogram Manager software (Version
2.1 Waters).
Extraction of paracetamol by magnetic molecularly imprinted polymers in plasma
MISPE conditions
m-MIP and m-NIP columns were prepared by packing
100 mg of the polymers into 2 mL empty cartridge. The
cartridge was conditioned sequentially with 1 mL of metha-
nol, 1 mL of deionized water and 1 mL ammonium phosphate
(20 mmol L−1) at pH 3.0. Extraction experiments involved
loading the column with 5 mL, 50 μg L−1 paracetamol
(pH 9.0) at a flow rate of 1 mL min−1. After loading, the
column was washed with 1 mL mixture of acetone:acetonitrile
(3:1v/v) and then 1 mL dicholomethan. After washes, SPE
cartridge was dried thoroughly by vacuum application. Final-
ly, the elution was performed by passing 3 × 1 mL of
acetonitrile:phosphate buffer (90:10, pH 9.0) through the car-
tridge. All fractions were evaporated to dryness at 20 °C under
a stream of nitrogen and finally recovered in 1 mL of mobile
phase. Then, 25 μL of each sample was injected onto the
analytical column of HPLC.
Synthesis procedures
Synthesis of 2-(methacrylamido) ethyl methacrylate as a cross
linker
Methacryloyl chloride was synthesized by reacting
methacrylic acid with thionyl chloride. Thionyl chloride
(78 mmol, 5.7 mL) was added dropwise to a mixture of
methacrylic acid (60 mmol, 5 mL) along with 3 mL of dichlo-
romethane, over a period of 1 h and the container was sealed
under N2 atmosphere. After complete addition of thionyl
chloride, the reaction mixture was maintained at 45 °C for
12 h with continuous stirring. Pure methacryloyl chloride was
distilled out at 55 °C.
Afterwards, based on a procedure by Spivak et al. [21], a
solution of methacryloyl chloride (81.6 mmol, 8 mL) in di-
chloromethane (20 mL) was added dropwise to a mixture of
ethanolamine (40 mmol, 2.35 mL), triethylamine (81.6 mmol,
11.3 mL) in dichloromethane (25 mL), in an ice bath for 1 h.
After the addition was complete, the reaction mixture was
stirred at 40 °C for a further 24 h.
The reaction mixture was filtered out and the precipitate
(Et3NHCl) discarded. The filtrate was washed with 0.5 M
NaHCO3 (3×15 mL) and 0.5 M Sodium Citrate (3×15 mL).
The organic phase was extracted and evaporated under vacu-
um. The pale yellow oily product was reached in a 55 % yield.
IR (cm−1): 3,300, 1,720, 1,665, 1,631, 1,450, 1,333, 1,158,
1,085 and 940. 1H NMR (CDCl3, 250 MHz) δ/ppm 7.0 (1H,
bs), NH; 5.91 (1H, m), RO2C(CH3)C=CHaHb; 5.73 (1H, t),
RHNCOC(CH3)C=CHcHd; 5.56 (1H, t),
RO2C(CH3)C=CHaHb; 5.40 (1H, t),
RHNCOC(CH3)C=CHcHd; 4.26 (2H, t) OCH2; 3.63 (2H,
quad) NHCH2; 1.9 (3H, s) CH3; 1.8 (3H, s) CH3. 13C NMR
(CDCl3, 62.5 MHz) δ/ppm 169.1, RNHCOR; 168.1, RCO2R;
141.2, RHNCO(CH3)C=CH2; 135.7, RCO2(CH3)C=CH2;
126.1, RCO2(CH3)C=CH2; 121.0, RHNCO(CH3)C=CH2;
64.0, OCH2; 39.7, NHCH2; 19.2, RHNCO(CH3)C=CH2;
18.7, RCO2(CH3)C=CH2.
Synthesis and modification of Fe3O4 MNPs
Iron oxide magnetic nanoparticles (MNPs) were prepared
according to a reported procedure by Yang et al. [22]. Briefly,
FeCl3.6H2O (5.4 g, 20 mmol) was dissolved in 140 mL ultra-
pure water, followed by adding FeSO4.7H2O (2.7 g, 10 mmol)
in N2 atmosphere. After being absolutely dissolved, 11 mL of
analytical grade ammonia solution (28 %, w/w) was quickly
added into the solution under rapid mechanical stirring
(900 rpm), and then the mixture was heated up to 60 °C, while
vigorously stirred by a mechanical stirrer for 1 hr under N2
atmosphere. Finally, the precipitates were collected using an
external magnet and rinsed with ultra-pure water, and dried to
powder in a vacuum.
Then, the MNPs (1 g) were dispersed in 50 mL of distilled
water by the ultrasonic treatment for 20 min to form a ferro
fluid. 2 mL of the ferro fluid was first diluted with water
(40 mL), and then the resultant suspension and 5 mL of
NH3.H2O were poured into 140 mL of ethanol with vigorous
stirring at 40 °C. Finally, under continuous mechanical stir-
ring, 1 mL of TEOS diluted in ethanol (20 mL) was added
dropwise to this dispersion. The resulting dispersion was
continuously stirred mechanically for 14 h at room tempera-
ture. The magnetic Fe3O4@SiO2 nanoparticles were collected
by magnetic separation and washed with ethanol and deion-
ized water in sequence.
Finally, Fe3O4@SiO2 MNPs were modified by MPS.
Briefly, 250 mg of Fe3O4@SiO2 MNPs was dispersed in
50 mL of anhydrous toluene containing 3 mL of MPS, and
the mixture was allowed to react at 70ºC for 12 h under
nitrogen. After magnetic separation, washing by water and
drying in vacuum, the products (Fe3O4@SiO2-CH=CH2)
were obtained.
Synthesis of magnetic MIP nanoparticles
The schematic diagram for the preparation of m-MIPs is
presented in Scheme 1. A solution of 4 mmol MAA as a
monomer and 1 mmol paracetamol as a template in 30 mL
acetonitrile/chloroform (5:1, v/v) was shaken at room temper-
ature for 5 h to form a template-monomer complex. After-
wards, 0.5 g of double-bond-functionalized Fe3O4 magnetic
nanoparticles that were dispersed in acetonitrile/chloroform
(5:1, v/v), 10 mmol 2-(methacrylamido) ethyl methacrylate
and 0.1 mmol AIBN were added to the solution and the
mixture was sonicated in an ultrasonic bath. After sonication
(5 min), it was purged with N2 and the flask was sealed under
N2 atmosphere. The magnetic MIPs were synthesized in a

Scheme 1 Schematic illustration of the synthesis of magnetic MIPs
100 mL flask equipped with a mechanical stirrer in an oil bath
kept at 60 °C for 20 h. The resultant polymeric nanoparticles
were collected by the external magnetic field and eluted by a
mixture solvent of methanol and acetic acid (9:1, v/v) for
several times to extract the template molecules until the eluent
was free from paracetamol as detected by UV-vis spectrome-
try (at 257 nm). The obtained polymers were finally rinsed
with ethanol to remove the remaining acetic acid and dried in
the vacuum for 24 h before use. For a comparison, magnetic
non-molecularly imprinted polymers (m-NIPs) were prepared
in the absence of the template during the polymerization
process and treated in an identical manner.
Binding experiment
For measuring of template binding, 50 mg of polymer nano-
particles were added to 10 mL paracetamol solution (pH 7.0)
of various initial concentrations in a conical centrifugation
tube and sealed. The mixtures were thermo stated at 25 °C
for 24 h under continuous stirring and then the polymer
nanoparticles were collected by the external magnetic field.
Then, the residual concentration of paracetamolin solution
was established using HPLC-UV at 257 nm. The quantity
(Q) of the template bound to m-MIP or m-NIP was calculated
according to the following equation [23]:
Q ¼ ½ðC0 −Ct Þ  VŠ=W
Where C0 and Ct (mg L−1) are the initial concentration and
the residual concentration of aspirin, respectively; V (L) is the
initial volume of the solution, and W (g) is the weight of the
m-MIPs or m-NIPs.
Extraction procedure for human plasma samples
Drug-free human plasma was obtained from the Iranian blood
transfusion service (Tehran, Iran) and stored at −20 °C until
use after gentle thawing. Due to possibility of protein-binding
for paracetamol and reducing the recoveries processes, it is
S. Azodi-Deilami et al.
necessary to have some treatments with plasma before extrac-
tion with m-MIP particles. So, the plasma samples was diluted
with 25 mM ammonium acetate (pH 9.0), then centrifuge
30 min at 6,000 rpm to remove excess of proteins. Then the
supernatant was filtered through a cellulose acetate filter
(0.2 μm pore size, Advantec MFS Inc., CA, USA). The filtrate
was collected in glass containers and stored at −20 °C until the
analysis was performed. 2 mL of the filtered supernatant was
collected to be directly percolated through the m-MIP or the
m-NIP.
Results and discussion
As illustrated in Scheme 1, the synthesis of the m-MIPs is a
multistep procedure, which involves synthesis of Fe3O4
MNPs, silica-shell deposition (Fe3O4@SiO2), modification
of Fe3O4@SiO2 with MPS, preparation of a cross linker
(2-(methacrylamido) ethyl methacrylate) and m-MIPs. At
first, Fe3O4 MNPs were prepared by the co-precipitation
method. Then, the surface of Fe3O4 MNPs was coated with
silica by the TEOS. The SiO2 shell provided a biocompatible
and hydrophilic surface, and prevented oxidation of Fe3O4.
Furthermore, silanol groups were beneficial to chemical mod-
ification on the surface of Fe3O4@SiO2. Thus, double bonds
were introduced onto Fe3O4@SiO2 using MPS to ensure tight
growth of the imprinted layer. We have prepared
2-(methacrylamido) ethyl methacrylate by from the reaction
of ethanol amine with methacryloyl chloride in a 1:2 mole
ratio in dichloromethane. Finally, m-MIPs were synthesized
using modified Fe3O4 MNPs, MAA, cross linker, paracetamol
and AIBN in acetonitrile/chloroform (5:1v/v). The resultant
polymers were used to study the in vitro controlled release of
drug and human plasma assay.
Optimal MIP formulation
Various amounts of monomer and cross link agent were used
for optimization of different formulations of MIPs with en-
hanced molecular recognition capabilities. Generally, for
Extraction of paracetamol by magnetic molecularly imprinted polymers in plasma

Fig. 1 FT-IR spectra of Fe3O4

(a); Fe3O4@SiO2 (b);
Fe3O4@SiO2–CH=CH2 (c) and
m-MIPs (d)

preparation of enhance specific polymers and formation of magnetically to an external magnetic field and this response
MIP recognition sites, several molar ratios of the functional vanished upon removal of the magnetic field. The saturation
monomer MAA and cross link agent to template were used in magnetizations of Fe3O4, Fe3O4@SiO2−CH=CH2 and m-
this study. As Table S1 (ESM) depicts, the optimum ratio of
functional monomer and cross link agent to template for the
specific rebinding of paracetamol was 3:10:1 (m-MIP 7),
which had the best extraction of 89 %. The extraction of the
corresponding NIP was low as 16 %.

Characterization of magnetic MIPs

FT-IR

The products of Fe 3 O 4 , Fe 3 O 4 @SiO 2 , Fe 3 O 4 @SiO 2 –

CH=CH2, and m-MIPs, were investigated by FT-IR spectros-
copy (Fig. 1). The absorption band at 573 cm−1 is attributed to
the Fe-O stretching bond for Fe3O4 nanoparticles. The peaks
of Si-O-Si group at 1,100 cm−1 and −OH group at about
1,623 cm−1 and 3,400 cm−1 indicated the formation of
Fe3O4@SiO 2. Also, we can conclude that the peak at
1,621 cm−1 in Fe3O4@SiO2−CH=CH2 was caused by C=C
stretching vibration and the peak at 1,100 cm −1 in
Fe3O4@SiO2−CH=CH2 was evidently stronger than that in
Fe3O4@SiO2, both showed that the surface of Fe3O4@SiO2
has been successfully modified with the functional vinyl
group. The FT-IR spectra of m-MIPs and m-NIPs were almost
the same, because after removing the template molecule, the
chemical composition of the m-MIPs was similar to that of the
m-NIPs. A very intense band of C=O stretching is seen at
about 1,720 cm−1 and C−H group of methyl at 2,990 cm−1
indicating that MIP layers have been successfully formed on
the surface of Fe3O4@SiO2−CH=CH2.

VSM

The magnetic properties of the Fe 3 O 4 , Fe 3 O 4 @SiO 2 ,

Fe3O4@SiO2−CH=CH2 and magnetic MIP nanoparticles were
characterized by VSM at room temperature, and the results are
displayed in Fig. S2 (Electronic Supplementary Material, Fig. 2 TEM images of Fe3O4 nanoparticles (a); Fe3O4@SiO2@MPS
ESM). This feature illustrated that the materials respond (b); m-MIPs (c)
 S. Azodi-Deilami et al.

Fig. 3 Effect of pH on rebinding

efficiency of paracetamol. 50 mg
of the polymers; sample volume:
5 mL; paracetamol concentration:
50 mM; room temperature (mean
± S.D., n=3)

MIPs were 61.36, 4.58 and 2.7 emu g−1, respectively. The
magnetic MIP achieved lower saturation magnetization value
than magnetic nanoparticles. This was expected because the
polymeric coating has effectively shielded the magnetite. How-
ever, the magnetic MIPs possess enough magnetic response to
meet the need of magnetic separation within a short time.
magnetic MIPs. This result suggested that the Fe3O4 nanopar-
ticles were indeed incorporated into MIPs and the structure of
Fe3O4 nanoparticles was not changed during the polymeriza-
tion process.
TEM

XRD The morphological structure of Fe 3 O 4 nanoparticles,

The X-ray powder diffraction patterns of Fe 3 O 4 ,
Fe3O4@SiO2, Fe3O4@SiO2−CH=CH2, and polymer-coated
Fe3O4 nanoparticles are indicated in Fig. S3 (ESM). Five
characteristic peaks for Fe3O4 marked by their indices (220),
(311), (400), (422), (511) and (440) were observed for all the
samples to reveal pure Fe3O4 in the resultant particles [24].
The peak positions of the Fe3O4 nanoparticles are all consis-
tent with the lower diffraction intensity before and after the
polymerization, and it is found that the peaks of magnetic
MIPs are obviously broadened due to lower Fe3O4 content in
Fe3O4@SiO2−CH=CH2 and m-MIPs were detected by TEM
as given in Fig. 2. It can be observed that Fe3O4@SiO2@MPS
cores were surrounded by polymer layers.
EDX
Figure S4 (ESM) shows the patterns of EDX analysis to
confirm the existence of magnetic imprinted polymers. In
Fig. S4(a), Fe and O signals are related to the elemental
composition of Fe3O4 nanoparticles. Besides peaks of O and
Fe, the presence of Si and C peaks in Fig. S4(b) clearly

Fig. 4 Effect of paracetamol

concentration on the adsorption
capacity on m-MIP and m-NIP
nanoparticles at pH 9.0
Extraction of paracetamol by magnetic molecularly imprinted polymers in plasma

Table 1 Distribution ratio (KD) and selectivity coefficient (α) values for Table 2 Assay of paracetamol in human plasma by SPE-HPLC
magnetic imprinted and non-imprinted polymers procedure

Compound m-MIP m-NIP Sample Spiked value (μg L−1) Recovery% ± SDa

KD α KD α MIP NIP

Paracetamol 900 22 Human serum 5 83±1.1 11±1.0

Bromhexine 51 17.65 24 0.92 10 90±1.2 13±1.7
Aspirin 85 10.59 20 1.10 25 87±1.3 10±1.4
Meloxicam 25 36.00 19 1.16 50 91±1.6 10±1.5
Diphenhydramine 37 24.32 21 1.05 a
Average of three determinations

indicates the modification of Fe3O4 nanoparticles surface with
MPS. Higher percentage of C and lower percentage of Si and
Fe in Fig. S4(c) validates successfully synthesis of magnetic
molecularly imprinted polymer nanoparticles.
Effect of pH on drug loading
Lesser effects were observed at lower pH values and which
may have been attributed to the protonation of the function-
al group of paracetamol and consequence breakdown of the
hydrogen bonds at the pH<7.0. Finally, as the recovery rate
of paracetamol flattens at around the pH 9.0, therefore, this
pH value was chosen for later SPE experiments.

The effect of pH on the sorption of paracetamol was exam-
ined by varying the pH of solutions from 4.0 to 10.0. Several
batch experiments were performed by equilibrating 50 mg
of the imprinted particles with 5 mL of the solutions con-
taining 0.05 mM of paracetamol under the desired levels of
pH. The results for different polymers (Fig. 3) displayed that
pH have great effects on loading. The binding behavior of
paracetamol was not greatly affected at pH>7. Although,
Adsorption capacity of polymers and preconcentration factor
One of the important factors we studied was the capacity of a
sorbent to quantitatively remove a specific amount of template
from the solution. Once the system has come to equilibrium,
the amount of free template in the solution is measured to
determine the amount of adsorbed template. In the measure-
ment of adsorption capacity of m-MIP and m-NIP absorbents,

Fig. 5 HPLC chromatograms

obtained after percolation of 2 mL
human serum spiked with
50.0 μgL−1 of paracetamol with a
cleanup step comprising the a m-
MIP and b m-NIP monitored at
257 nm; conditions: column ACE
5 μm, C18 4.6 mm×250 mm at +
40 °C, eluent acetonitrile:
phosphate buffer (0.01 mol L−1,
pH 3.0) 90:10 at flow rate of
1.0 mL min−1
 S. Azodi-Deilami et al.

Table 3 Figures of merit of comparable methods for determination of paracetamol

Method Analytical range Detection limit Comments Reference

HPLC 1.5–200 μg mL−1 50 ng mL−1 Simple, selective and sensitive [25]

Flow-through UV 0.5–0.8 μg mL−1 0.022 μg mL−1 A simple device based on a suitable active solid [26]
optosensing device support
HPCE 1–100 μM 0.5 μM A modification of CE combining the advantages [27]
of CE and chromatography for separation
from plasma proteins
HPTLC 120–360 μg mL−1 1.8 μg mL−1 On silica gel 60F254 HPTLC plates with [28]
toluene-acetone-methanol (8:1:1, v/v/v) as
mobile phase; detection at 263 nm
MEKC 260–520 μg mL−1 0.5 μg mL−1 Uncoated fused silica capillary for the separation [29]
at an applied voltage of 20 kV. A phosphate
run buffer (pH 9, 0.05 M) containing 0.05 M
sodium dodecyl sulphate was used for
analysis.
Flow solid-phase UV 3–30 μg mL−1 0.104 μg mL−1 A continuous and simple multiparameter sensor [30]
spectrophotometry for the sequential determination of
salicylamide and paracetamol by solid-phase
UV spectrophotometry
HPLC and SFC 0.3–20 μg mL−1 0.1–0.2 μg mL−1 Using packed column SFC employing internal [31]
standard method. The analytes were
monitored spectrophotometrically at 225 nm.
SFC method was compared to an HPLC assay
IC 0.5–7 mg L−1 0.06 mg L−1 A simple technique for the measurement of [32]
paracetamol and its degradation product
FTIR spectrometry 2–10 mg L−1 0.34 mg L−1 FTIR technique for the simultaneous [33]
determination of ibuprofen and paracetamol.
Quantification was carried out by measuring
the absorbances using the baseline for
measurement correction.
CE 0.5–100 μM 0.25 μM Developed CE method using paracetamol [34]
glucuronide as a novel probe for human ß-
glucuronidase activity
MIP-BAW sensor 0.005–0.1 μM 0.005 μM A piezoelectric quartz crystal modified with MIP [35]
to obtain selectivity for a bulk acoustic wave
sensor. Stable, selective and facile
m-MIP and SPE 1–300 μg L−1 0.17 μg L−1 Magnetic molecularly imprinted polymer This work
coupled with HPLC nanoparticles for solid-phase extraction and
sample clean-up of paracetamol simply and
precisely. Avoiding time consuming
separation operation and reducing the loss of
both liquid and solid during the separation
process. Having good precision and
recoveries.

HPCE high performance capillary electrophoresis, HPTLC high performance thin layer chromatography, MEKC micellar electrokinetic chromatogra-
phy, SFC supercritical fluid chromatography, IC ion chromatography, BAW bulk acoustic wave

50 mg samples of the absorbents were added to 100 mL
paracetamol solutions at concentrations of 10–500 mg L−1.
The suspensions were mechanically shaken at room tempera-
ture, followed by centrifuging and removal of absorbents. The
remaining paracetamol in the supernatant was measured by
HPLC-UV. The adsorption isotherm, which is the number of
milligram adsorbed per gram of adsorbent (Q) versus the
equilibrium concentration of paracetamol, is shown in
Fig. 4. According to these results, the maximum amount of
paracetamol that can be absorbed by m-MIP is 84 mg g−1 at
pH 9.0. As all the accessible specific cavities of the m-MIP
particles are saturated, the retention of the analyte is mainly
due to non-specific interactions which can be identical for m-
MIP and m-NIP polymers. In order to obtain preconcentration
factor, the effect of sample concentration on the sorption
behavior of analyte on m-MIPs was investigated. Amount of
paracetamol in supernatant solution determined by HPLC-
UV. The sorption of drug was quantitatively recovered at the
range of 10–600 mg L−1. At the higher concentration of
400 mg L−1, the extractions for analytes were not quantitative.
Extraction of paracetamol by magnetic molecularly imprinted polymers in plasma
The preconcentration factor of the m-MIPs is calculated by the
ratio of the highest sample concentration for analyte
(400 mg L−1) and the lowest concentration (10 mg L−1). The
preconcentration factor was 40.
Study of MIP selectivity
In order to evaluate the selectivity of the synthesized m-MIP,
Paracetamol, and several drugs with the ability to form hydro-
gen bonds with the m-MIP nanoparticles were selected in this
section. Their molecular structures are shown in Fig. S1
(ESM). The initial concentration of drugs (5 mL, 0.05 mM)
was extracted by 50 mg of m-MIPs and m-NIPs at pH 9.0. The
distribution ratio (KD) was calculated using the equation:

quantified in the 1 μg L−1 to 300 μg L−1 concentration range.
C i −C f V
KD ¼
Cf m
Where V, Ci, Cf, and m represent the volume of the solution
(mL), drug concentration before and after adsorption (mM)
and mass of the polymer, respectively. The selectivity coeffi-
cient (α) is defined as:
K D ðparacetamolÞ
a¼
K D ðforeigncompound Þ
The results are listed in Table 1. The higher KD obtained for
paracetamol strongly confirmed a higher selectivity of m-
MIPs this template and demonstrated the possibility of m-
MIP as a selective adsorbent for the extraction of paracetamol
from complex matrixes such as biological fluids.
Real sample analysis
To demonstrate the potential of MIP for the sample clean-up,
the MIP was applied to the purification of spiked paracetamol
in human plasma. Aqueous media was employed for the
loading solution, and the wash procedure was assessed for
obtaining maximum recovery of the analytes using acetone.
The chromatograms obtained for plasma samples are shown in
Fig. 5. This efficient method allowed cleaner extracts to be
obtained and interfering peaks arising from the complex bio-
logical matrices to be suppressed. Results from the HPLC
analyses in Tables 2 showed that the MIP extraction of para-
cetamol for plasma samples is linear in the ranges of 1–
300 μg L−1 with good precision (4.5 % for 50.0 μg L−1) and
recoveries (between 83 and 91). Typical chromatograms (pre-
sented in Fig. 5) reveal that the MIP can be used for the sample
clean up and when MIP sorbent was used, a board peak in
chromatogram was omitted. The limit of detection (LOD) and
limit of quantification (LOQ) for paracetamol in plasma sam-
ples were 0.17 and 0.4 μg L−1. The number of replicate for
experiment was been three times.
Conclusions
In this study, magnetic molecularly imprinted nanoparticles
were prepared using a novel cross-link agent.
Superparamagnetic Fe3O4 particles were synthesized and the
surfaces of magnetic Fe3O4 particles were successfully mod-
ified and functionalized in order to disperse in organic sol-
vents and take part in the reaction of molecular imprinted
polymerization. Then, m-MIPs were prepared by using para-
cetamol as the template, MAA as the functional monomer,
2-(methacrylamido) ethyl methacrylate as the cross linker and
double-bond-functionalized Fe3O4 nanoparticles as the sta-
tionary phase by the molecular imprinting technique. The
obtained m-MIPs were characterized by TEM, FT-IR, XRD,
and VSM analyses. By this method, paracetamol can be
The recoveries for the spiked human plasma samples were
between 83 and 91 % in 5–50 μgL−1 respectively. It could be
concluded that the technique has great potential in developing
selective extraction method for other compounds. Compari-
son of the results of the determination of paracetamol obtained
by several methods is shown in Table 3. Compared with other
techniques, the proposed method has impressive results and
the detection limit is much lower than other works.
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