Ultrasonics Sonochemistry 33 (2016) 67–76

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Ultrasonics Sonochemistry
journal homepage: www.elsevier.com/locate/ultson

Synthesis and application of molecularly imprinted nanoparticles

combined ultrasonic assisted for highly selective solid phase extraction
trace amount of celecoxib from human plasma samples using design
expert (DXB) software
Maryam Arabi a, Mehrorang Ghaedi a,⇑, Abbas Ostovan b, Javad Tashkhourian c, Hamideh Asadallahzadeh b
a
Chemistry Department, Yasouj University, Yasouj 75918-74831, Iran
b
Department of Chemistry, Kerman Branch, Islamic Azad University, Kerman, Iran
c
Department of Chemistry, College of Science, Shiraz University, Shiraz 71454, Iran

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

Article history: In this work molecular imprinted nanoparticles (MINPs) was synthesized and applied for ultrasonic
Received 25 January 2016 assisted solid phase extraction of celecoxib (CEL) from human plasma sample following its combination
Received in revised form 14 April 2016 by HPLC–UV. The MINPs were prepared in a non-covalent approach using methacrylic acid as monomer,
Accepted 15 April 2016
CEL as template, ethylene glycol dimethacrylate as cross-linker, and 2,2-azobisisobutyronitrile (AIBN) as
Available online 18 April 2016
the initiator of polymerization. pH, volume of rinsing and eluent solvent and amount of sorbent influence
on response were investigated using factorial experimental design, while optimum point was achieved
Keywords:
and set as 250 mg sorbent, pH 7.0, 1.5 mL washing solvent and 2 mL eluent by analysis of results accord-
Celecoxib
Design expert
ing to design expert (DX) software. At above specified conditions, CEL in human plasma with complicated
HPLC–UV matrices with acceptable high recoveries (96%) and RSD% lower than 10% was quantified and estimated.
Human plasma The proposed MISPE-HPLC–UV method has linear responses among peak area and concentrations of
Molecular imprinted nanoparticles CEL in the range of 0.2–2000 lg L
1, with regression coefficient of 0.98. The limit of detection (LOD)
Solid phase extraction and quantification (LOQ) based on three and ten times of the noise of HPLC peaks correspond to blank
solution were 0.08 and 0.18 lg L
1, respectively.
Ó 2016 Elsevier B.V. All rights reserved.

1. Introduction spectrometry (LC–MS/MS) [6], liquid chromatography (LC) [1,7,8],

Celecoxib or 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-
pyrazol-1-yl] benzene sulfonamide (Fig. 1) is the first cyclo-
oxygenase selective inhibitor for treatment of osteoarthritis and
rheumatoid arthritis [1]. It is approved worldwide for the relief
of the signs and symptoms of osteoarthritis, rheumatoid arthritis,
and ankylosing spondylitis for the management of acute pain in
adults and primary dysmenorrhoea treatment [2]. CEL is easily
absorbed and extensively metabolized in humans with <3% of the
dose excreted unchanged and its further oxidization product
is carboxylic acid and partial amount of its changed product as
glucuronides excreted in the urine [3]. Various methods such as
liquid chromatography–tandem mass spectrometry (LC–MS) [4],
chemometric methods [5], LC coupled with tandem mass
⇑ Corresponding author.
E-mail addresses: m_ghaedi@mail.yu.ac.ir, m_ghaedi@yahoo.com (M. Ghaedi).
voltammetry [9], LC–inductively coupled plasma mass spectrome-
try (LC–ICPMS) [10], solid phase extraction [11,12] and liquid–
liquid extraction have been used for quantification and/or matrix
modification [10,13,14]. Some of these methods in despite of their
own advantages and remarks including simplicity and rapidity
suffer from low selectivity for celecoxib monitoring. Complicated
matrices of human plasma samples and also presence of celecoxib
in such samples at value lower than detection limit encourage the
researchers to design and develop effective sample preparation
prior to instrumental analysis.
Molecularly imprinted polymer (MIPs) is good and efficient
sorbent for separation and pre-concentration of analytes from
complex samples such as food, drugs, biological and environmental
samples [15]. Combination and application of such materials with
liquid chromatography, capillary electrophoresis, capillary electro-
chromatography, solid-phase extraction [16] lead to simplifying
and avoiding matrices effect [17,18] also significant improvement
in figures of merit correspond to under study method. MIPs are

http://dx.doi.org/10.1016/j.ultsonch.2016.04.022
1350-4177/Ó 2016 Elsevier B.V. All rights reserved.
68 M. Arabi et al. / Ultrasonics Sonochemistry 33 (2016) 67–76
Fig. 1. The structure of celecoxib.
superior to traditional sorbents in term of higher selectivity which
emerged from pre-determined synthetic pattern for particular
analyte [19]. MIPs are tailor-made polymers in which recognition
sites are imprinted in the polymer matrix according to the size,
shape and the functional groups of template molecule [20]. In
general, MIPs are synthesized through bulk polymerization and
subsequently are grinded and sieved to achieve irregularly shaped
particles for different applications. Unfortunately, in this method
deep imbedding of template molecules cause its incomplete
removal and cause lower binding capacity. Unlike the bulk mono-
liths, nanostructured imprinted materials have small dimension
with extremely higher surface-to-area ratio which provide
quantitative removal of templates, better site accessibility, lower
mass-transfer resistance and well defied material’s shape [21].
The molecularly imprinted nanoparticles (MINPs), in comparison
with the polymers obtained by bulk or precipitation polymeriza-
tion doesn’t need crushing and sieving steps and particles size
could be controlled by stirring speed during synthesis procedure.
Ultrasound irradiation accelerate chemical process and majority
of its contribution and influence in chemical reaction and activities
such as food chemistry, material extraction, nanotechnology,
different polymerization reactions and surface cleaning can be
attributed to the formation and collapse of microbubbles, which
are formed during the passage of the sound waves through the
solution, which namely as cavitation [22,23]. Application of
sonication for synthesis and preparation of MIPs lead to increasing
solubility of the template and monomers and also homogeneity of
the pre-polymerization solution [24,25] and also degassing
throughout the polymerization. In this procedure, the formation,
growth and collapse of micrometer-scale bubbles formed by the
promulgation of a pressure wave through a liquid cause accelera-
tion and enhancement of mass transfer process that may be
attributed to best immersion of sorbent in balk solution and also
strong enhancement of diffusion coefficient. This unique advantage
is emerged from physical phenomena such as micro-streaming,
micro-turbulence, shock waves and micro jets without significant
change in equilibrium characteristics of the adsorption/desorption
system which is a great key in solid phase extraction [26–28].
The selection of the optimum condition for MISPE based proce-
dure is complex task and require extensive amount of time and
analysis. Various parameters such as pH, volume of eluent and
washing solvent and amount of sorbent (MISPE) with at least
number of runs (cost & time) must be undertaken to simultaneously
give information about main contribution and interaction among
variables [29]. Simultaneous optimization of variables undertaken
by ‘‘design of experiments” (DOEs) based on well-known phenomena
and advantages which candidate it for successful parameter
screening and finding best operational condition [30–32].
In this study, MIPNs were synthesized based on best previously
optimized operational conditions (1:4:20 ratio of template:
monomer:cross linker molar ratio) [15,33] At this situation an
highly selective MIPNs as novel sorbent was prepared which lead
to increase in the selectivity and efficiency of under-developed
method for determination of trace amount of CEL in MISPE
technique. To the best of author’s knowledge, no study using ultra-
sonic assisted-MINPs-SPE method have been done for extraction
and pre-concentration of CEL from plasma samples.
2. Materials and methods
2.1. Materials
All chemicals including NaOH and HCl (with the highest purity
available) and methacrylic acid was obtained from Merck KGaA
(Darmstadt Germany). Ethylene glycol dimethacrylate (EDMA,
98%) was supplied by Aldrich (St. Louis, MO, USA). 2,2-Azobis
(2-isobutyronitrile) (AIBN) was obtained from Aldrich (St. Louis,
MO, USA). Ultrapure water was obtained from MilliQ gradient
ultrapure water system. Methanol (HPLC gradient grade) was
produced by J.T. Baker (Deventer, Holland). Acetonitrile, DMSO
and ethanol (puriss. p.a.) were obtained from Fluka (Buchs,
Switzerland). CEL pure powder was obtained from Ministry of
Health and Medical Education (Tehran, Iran). Human plasma
samples were obtained from Healthy volunteers and stored at

20 °C until use.
2.2. Instruments
High-performance liquid chromatography was performed on
Agilent 1100 liquid chromatography (Wilmington, DE, USA)
equipped with Micro Vacuum Degasser (model G1379A), a
Quaternary Pump (model G1311A), a Series Multiple Wavelength
Detector (model G13658: was set at 250 nm for CEL), a sample
injection valve with 20 lL sample loop, and Knauer C18 column
(4.6 mm i.d. 250 mm, 5 lm). Data were collected and analyzed
using the Agilent Chemstation software. The mobile phase was
acetonitrile–water (75/25, v/v) and the flow rate was 1.0 mL min
1.
The mobile phase was filtered through a 0.45 lm filter and
degassed under vacuum before use. The system was operated at
ambient temperature.
The shape and surface morphology of the polymers were
investigated by field emission scanning electron microscopy
(FE-SEM, Hitachi S4160, Japan) under an acceleration voltage of
26 kV. Fourier transform infrared (FT-IR) spectra were recorded
using KBr disks (FTIR-8300, Shimadzu, Japan). Absorption mea-
surements were carried out on a Jusco UV–Vis spectrophotometer
model V-530 (Jasco, Japan) using a quartz cell with an optical path
of 1 cm at 250 nm. A centrifuge instrument model 2010D centurion
scientific (west Sussex, UK) was used for centrifugation. An
ultrasonic bath with heating system (Tecno-GAZ SPA Ultra Sonic
System) at 40 kHz of frequency and 130 W of power was used
for the ultrasound-assisted adsorption. A pH/ion meter (691,
Metrohm, Herisau, Switzerland) with combined glass-calomel
electrode was used for adjustment of pH. A Windaus two-
channel peristaltic pump (model D-38678, Germany) was used to
pump solvents during MISPE experiments.
2.3. Synthesis of imprinted and non-imprinted nanoparticles
MINPs were prepared according to the following Procedure:
firstly the functional monomers methacrylic acid (4 mmol) and
template molecule (celecoxib, 1 mmol) were dissolved in 40 mL
acetonitrile and sonicated for 10 min. Then, EGDMA (20 mmol)
and 2,2-azobisisobutyronitrile (AIBN, 0.05 g) were added and the
mixture was purged with nitrogen under sonication for 20 min
and subsequently the reaction mixture was refluxed in an oil bath
(24 h at 60 °C) under sonication. Subsequently, the nanoparticles
were collected by centrifugation and washed with acetone and
 M. Arabi et al. / Ultrasonics Sonochemistry 33 (2016) 67–76 69

methanol, respectively to remove additional reagents and solvent.

The resultant nanoparticles were purified by several cycles of
centrifugation, decantation and re-suspended in methanol/acetic
acid (90/10 v/v) by sonication until the template was not detected
by UV–Vis spectrophotometry. Finally, the as-prepared nano-
particles were dried at room temperature under vacuum for
further use. Non imprinted nanoparticles (NINPs) were prepared
according to above mention polymerization reaction in absence
of template.

2.4. Preparation of stock solution and calibration standards of

celecoxib in plasma

CEL is soluble in organic solvent such as methanol, ethanol,

DMSO and acetonitrile, with high stability (two years) according to
storage at
20 °C. A standard stock solution of CEL (1.0 mg mL
1)
was prepared by dissolving 10 mg of CEL in 10 mL of methanol.
The working standard solution of CEL (100 lg mL
1) was prepared
by diluting the stock solution with methanol. The calibration
curves in plasma were constructed by spiking drug-free human
plasma with CEL to obtain desired concentrations of 20, 50, 100,
200, 500, 1000 and 2000 lg L
1. The standards of CEL were stored
at
20 °C for up to 70 days, +4 °C for at least 1 month and at
Fig. 2. FT-IR spectra (a) celecoxib (b) NINPs (c) MINPs after celecoxib extraction.
ambient temperature for 24 h.

2.5. Conditioning procedure of cartridge

The applicability of the sorbent for extraction of trace amount of
CEL was conducted as below: 250 mg of accurately weighted
polymers (MIPs or NIPs) was packed into an empty polypropylene
SPE cartridge. After the packing process the cartridge was firstly
rinsed with 10 mL methanol and then with 10 mL deionized water
to eliminate possible adsorbed materials.
2.6. Preparation of samples
5 mL of human plasma with known CEL concentration was
mixed completely with 10 mL of MeCN and centrifuged at
5000 rpm for 10 min to precipitate proteins. The clear supernatant
solution was diluted to 20 mL with distilled water at pH 7.0 to
obtain the same composition as loading solvent. Then, the solution
was passed through the conditioned cartridge by a flow rate of
1.0 mL min
1. The cartridge was washed with 1.5 mL of washing
solvent and in later stage, the CEL was eluted by 2.0 mL of elution
solution. The eluent (2.0 mL of MeOH/ACN/HOAC) in both ends of
the cartridge were closed with polypropylene caps and the column
was immersed in an ultrasonic bath (operating at 40 kHz). Accord-
ing to the design, the time of ultrasonic bath were set at 7.5 min.
After sonication, the caps were removed and the extract was
collected in a vial. Each elution fraction was concentrated to
dryness under nitrogen stream and dissolved in 50 lL of HPLC
mobile phase, while 20 lL of this solution was analyzed by HPLC
which permit achievement of pre-concentration factor of 100.
1637 cm
1 is corresponded to C@C and simultaneously suggests
that most MAAs were cross-linked and only a few ones remained.
The –COOH group of MAA and the O–H stretching are recognized
based on their bending vibrations at 3494 cm
1 and 1398 cm
1.
However, the features of the O–H group were 3494 cm
1 and
1398 cm
1 for the NIPNs. The major band spectra of NIPNs and
MIPNs have the same locations and appearances which could
prove that the template had been removed thoroughly from the
MIPNs after extraction.
The morphology of the nano-structure MIP by SEM (Fig. 3)
reveals that the synthesized nanoparticles have nodules size with
average size around 74 nm. The microspheres were obtained by
acetonitrile according to the selection of appropriate amount of
template/functional monomer/cross-linker agent (molar ratio of
1:4:20). These observations indicate that the formation of CEL
imprinted MIPs were performed successfully. The prepared nano-
structure MIP is superior to cluster form in term of representation
of high surface area, high capacity, fast extraction of analyte
without high mass-transfer and totally confirm high efficiency of
under study MIP for preconcentration and determination of CEL.

3. Results and discussion

3.1. Characterization of MINPs/NINPs

The FT-IR spectra of CEL, MIPNs after extraction and NIPNs

(Fig. 2) reveal characteristic S@O symmetric and asymmetric
stretching at 1164 and 1347 cm
1, respectively. Medium intensity
bands at 3338 and 3232 cm
1 were seen as double that probably is
attributed to the N–H stretching vibration of –SO2NH2 group. For
the MIPNs (b), the peak at 1732 cm
1 was attributed to C@O
stretching vibration absorbance. The weak absorbance peak at Fig. 3. Scanning electron micrographs (SEM) of MINPs in the optimum porogen.
70 M. Arabi et al. / Ultrasonics Sonochemistry 33 (2016) 67–76

3.2. Adsorption ability characterization Table 1

Compare capacity of MINPs with different porogen.

CEL has several functional groups and/or atoms in its structure MINPs Porogen solvent Capacity (mg absorbed of analyte/g MINPs)
like nitrogen, oxygen and sulfur that simply react with carboxylic 1 DMSO 74.39
acid group of MAA via strong electrostatic interaction and/or 2 Ethanol 51.33
hydrogen bond that possible accumulation and pre-concentration 3 Acetonitrile 85.47
of CEL. Solvents have an important role in polymerization process
in terms of their ability for dissolution of polymerization agents,
providing porous structures to imprinted polymers and promoting
their guest binding rates [34]. In non-covalent imprinting, the
choice of solvent is more critical in progress of formation of
non-covalent adducts between the functional monomer and the 120
template and thus improve the imprinting efficiency [35]. The
effect of solvent on polymer adsorption was studied using different 100

Extraction recovery (%)

solvents like DMSO, ethanol and acetonitrile in synthesis stage. The
80
adsorption capacity of MINPs for CEL was determined by following
equilibrium situation associated with chromatographic analysis
60
and the final adsorption capacity (Q) was calculated based on
Eq. (1): 40
ðC0
Cr ÞV
Q¼ ð1Þ 20
M
0
In (Eq. (1)), C0 and Cr are the CEL concentrations before and after MIPs NIPs
sorption, V is the volume of CEL solution (L), and M is the mass of
MINPs or NINPs (g). The results show that MINPs synthesized with Fig. 4. Adsorption capacities of the MINPs and NINPs.
acetonitrile as solvent has higher capacity than the nanoparticles
that was synthesized with DMSO or ethanol as solvent (Table 1).
Also it was found that the sorption capacity of MINPs toward CEL effect. During the synthesis process, removal template causes
was higher than NINPs at the same concentration (Fig. 4). It was formation of imprinted cavities and specific binding sites in
concluded that the prepared MINPs has higher sorption capacity predetermined orientation (Fig. 5). However, NINPs had no such
than NINPs in plasma samples that emerged from imprinting imprinted cavities and specific binding sites.

O
O
O
O
O
H
O
H
O
O O NH2 O
NH2 O H O
S NH2
S H S
F O
F O OH O N F
H Polymerization O
N O F N O N
F N O H H
O F N
F O H
F O
EGDMA F
H O
H O
O
CH3 O
Celecoxib CH3 CH3
O
O

O
O NH2
S O
O F O
N O
O F N
H O
H
F
O
O NH2 O
H S H
F Celecoxib Removal
O
O N CH3
H O
H F N H
O O
F O H O
Celecoxib Rebinding
H O
H O
O
CH3 O

O
O

Fig. 5. Schematic representation of the MINPs synthesis.

 M. Arabi et al. / Ultrasonics Sonochemistry 33 (2016) 67–76 71

3.3. Optimization of the MISPE procedure influence the extraction efficiency. In this model, 32 random
experiments were selected to minimize the effect of uncontrolled
Optimization was carried out by extracting aliquots of blank variables (Table 4). The influence of critical factors and the
plasma spiked with CEL at the level of 200 lg L
1. To ensure the model efficiency was checked by ANOVA according to Fisher’s
full description of the optimization approach, the types of eluent statistical analysis. As it shown in Table 5 criterion for significant
and washing solvent effect have been primarily investigated
through univariate method.
Table 4
3.3.1. Eluent composition The central composite design with experimental results.
An important parameter on SPE is eluent conditions (composition Run A: B: C: eluent D: washing E: sorbent ER%
and volume) and CEL was eluted by different kinds of organic sol- pH sonication volume volume amount
vents with various volumes of MeOH, MeOH:HOAc (90:10 v/v), Ace- time
tonitrile, MeOH/deionized water/ACN/HOAC (30:30:30:10 v/v), 1 6.00 12.50 2.00 2.00 250.00 93.14
MeOH:HOAc (85:15 v/v), MeOH:HOAc (80:20 v/v), and MeOH: 2 7.00 5.00 2.50 1.50 300.00 74.90
3 6.00 7.50 2.00 2.00 250.00 93.26
HOAc:TFA (79.9:20:0.1 v/v). As it is shown in Table 2, among
4 7.00 10.00 2.50 1.50 200.00 85.67
different eluents, the desorption ability of MeOH/deionized water 5 6.00 7.50 2.00 2.00 250.00 92.81
(DIW)/ACN/HOAC (30:30:30:10 v/v) was found higher than other 6 5.00 10.00 2.50 1.50 300.00 85.20
eluents. Acetic acid hinders the formation of hydrogen bonding 7 5.00 5.00 2.50 1.50 200.00 96.28
between CEL and functional monomer and lead to simple and more 8 6.00 7.50 2.00 2.00 150.00 81.95
9 6.00 7.50 3.00 2.00 250.00 89.53
facile elution of CEL. The effect of eluent volume on the recovery of
10 5.00 10.00 1.50 1.50 200.00 88.91
CEL was also studied using factorial design software. Experiment 11 5.00 5.00 1.50 1.50 300.00 88.86
showed that ultrasound irradiation extremely lowers the 12 7.00 5.00 1.50 1.50 200.00 81.17
consumption of eluent solvent. Decrease eluent solvent required with 13 8.00 7.50 2.00 2.00 250.00 91.16
14 5.00 10.00 1.50 2.50 300.00 94.73
ultrasound assisted is related to hydrogen bonds between MINPs and
15 7.00 5.00 2.50 2.50 200.00 88.07
analyte had been broken more convenient and mass transfer process 16 7.00 10.00 1.50 2.50 200.00 93.40
can be accelerated. The results reveal that the quantitative recovery 17 5.00 10.00 2.50 2.50 200.00 96.02
higher than 99% with good pre-concentration factor could be 18 6.00 7.50 2.00 2.00 250.00 93.80
obtained using 2 mL of MeOH/DIW/ACN/HOAC (30:30:30:10 v/v). 19 4.00 7.50 2.00 2.00 250.00 95.89
20 6.00 7.50 2.00 2.00 350.00 80.55
21 7.00 10.00 2.50 2.50 300.00 83.47
3.3.2. Effect of washing solvent 22 6.00 7.50 2.00 1.00 250.00 89.50
An appropriate washing solvent should elute accumulated 23 6.00 7.50 2.00 2.00 250.00 93.38
24 5.00 5.00 1.50 2.50 200.00 83.50
target compounds without affecting and removing matrix interfer-
25 5.00 5.00 2.50 2.50 300.00 91.67
ences from the sample as much as possible through at least volume 26 6.00 7.50 1.00 2.00 250.00 95.20
[28]. For this purpose, different washing solvents (acetone, tetra- 27 7.00 5.00 1.50 2.50 300.00 94.30
hydrofuran, dimethyl formamide (DMF), acetonitrile–acetone and 28 6.00 7.50 2.00 2.00 250.00 93.27
dichloromethane (DCM) were investigated. As shown in Table 3, 29 6.00 7.50 2.00 2.00 250.00 93.37
30 6.00 2.50 2.00 2.00 250.00 86.62
the cleanest extracts with the best recoveries were obtained using
31 6.00 7.50 2.00 3.00 250.00 95.03
1.5 mL of acetone as the washing solvent. 32 7.00 10.0 1.50 1.50 300.00 97.15

3.3.3. Screening of the effective parameters using central composite

design Table 5
Analysis of variance (ANOVA) for central composite design.
Central composite design was used to estimate the contribution
and coefficient of parameters and their interactions that screened Source Sun of Degree of Mean F-value P-value
by ANOVA at 95% confidence interval following conduction of squares freedom square

minimum number of runs and minimizing the cost and errors. Model 948.98 20 47.45 198.95 <0.0001
The amount of sorbent, pH, sonication time, volume of eluent A-pH 55.58 1 55.58 233.04 <0.0001
B-sonication time 62.93 1 62.93 263.78 <0.0001
and washing solvent are the significant factors that strongly
C-eluent volume 42.86 1 42.86 179.72 <0.0001
D-washing volume 60.36 1 60.36 253.07 <0.0001
Table 2 E-sorbent amount 1.29 1 1.29 5.39 0.0404
Recovery of celecoxib from MINPs cartridge using 2 mL different elution solvents.
AB 17.44 1 17.44 73.14 <0.0001
Eluent solvent Recovery% AC 138.57 1 138.57 581.01 <0.0001
AD 11.69 1 11.69 49.03 <0.0001
MeOH 73
AE 2.06 2.06 8.63 0.0135
MeOH:HOAc (90:10 v/v) 86
BC 45.33 1 45.33 190.06 <0.0001
MeOH/DIW/ACN/HOAC (30:30:30:10 v/v) 98
BD 1.97 1 1.97 8.24 0.0152
MeOH:HOAc (85:15 v/v) 80
BE 1.08 1.08 4.54 0.0566
MeOH:HOAc:TFA (79.9:20:0.1 v/v) 90
CD 3.39 1 3.39 14.21 0.0031
CE 216.47 1 216.47 907.65 <0.0001
DE 5.15 1 5.15 21.58 0.0007
Table 3
Celecoxib recoveries (%) on MINPs using 1.5 mL of different washing solvents. A2 0.062 1 0.062 0.26 0.6210
B2 22.04 1 22.04 92.40 <0.0001
Washing solvent Recovery (%) C2 1.76 1 1.76 7.38 0.0201
D2 2.11 1 2.11 8.86 <0.0001
Acetone 93
E2 268.09 1 268.09 1124.09
Tetrahydrofuran 70
Dimethyl formamide (DMF) 68 Lack of fit 2.12 6 0.35 3.55
Acetonitrile–acetone 82 Pure error 0.50 5 0.100
Dichloromethane (DCM) 59 Total error 951.60 31
72 M. Arabi et al. / Ultrasonics Sonochemistry 33 (2016) 67–76

contribution of each variable is P-value less than 0.05 and F value
more than 0.05 judged that all factors have significant contribution
in the model. Analysis of results by response surface methodology
(RSM) for plotting ER (%) versus significant variables was investi-
gated and results are shown in Fig. 6. Generally, prefer format of
analytes for quantitative and selective sorption is molecular
neutral form. Therefore, preliminary change in polarity and/or
charge of analytes (ionic or molecular forms) depends on their
sample solution pH [36]. The effect of pH in the range of 4.0–8.0
on the sorption efficiency of CEL reveals that maximum CEL

Fig. 6. Response surfaces for the 25 central composite designs: (a) sonication time (min)-pH, (b) eluent volume (mL)-pH, (c) washing volume (mL)–pH, (d) sorbent amount
(mg)-pH, (e) eluent volume (mL)-sonication time (min), (f) sonication time (min)-washing volume (mL), (g) sorbent amount (mg)-sonication time (min), (h) washing volume
(mL)-eluent volume (mL), (i) sorbent amount (mg)-eluent volume (mL), (j) sorbent amount (mg)-washing volume (mL).
 M. Arabi et al. / Ultrasonics Sonochemistry 33 (2016) 67–76
Fig. 6 (continued)
extraction efficiency was obtained at pH of 7.0. The hydrogen
bonds among CEL and MAA is main mechanism for binding and
accumulation of CEL on sorbent. At low pHs, the acidic moiety of
the polymer and CEL nitrogen atoms simultaneously protonated
while opposite trend was occur at higher pH. Therefore at low
pH due to strong electrostatic repulsive force a strong reduction
in sorption efficiency was found. At both pH limits, generation of
charge centers on polymer lead to significant decrease in ability
and capacity of sorbent for complexation and binding of analyte.
Closeness of optimum pH to plasma pH (7.0–7.70) [37] permit
development of novel compatible strategy without requirement
to preliminary pH adjustment stage as time consuming stage. Mass
transfer of CEL from sample solution to solid phase is one the
important factor on extraction recovery. The effect of ultrasonic
time in the range of 5–10 min (Fig. 6a, e, f, g) reveal that the best
recovery was obtained following 7.5 min sonication. It may be seen
from the figure (Fig. 6e) that the decrease in elution volume dose is
associated with significant increase in sonication time. The surface
plots (Fig. 6i) proof that low eluent volume and mid amount of sor-
bent is associated with more active sorption sites and enhance in
the ER% (95). At low volume of washing solvent and mid eluent
volume CEL quantitatively was eluted and possible achievement
of high sensitivity. As can be seen from Fig. 6a-j, the highest ER%
was obtained at mid amount of sorbent (250 mg), low volume of
73
washing solvent and mid eluent (1.5 and 2 mL respectively) and
mid sonication time (7.5 min) at neutral pH 7. The results suggest
extremely lower consumption of toxic organic solvent. The final
optimized conditions of the variables to obtain maximum response
(ER%) was calculated and their significant interaction was
investigated by RSM. Based on the data analysis semi-empirical
expression for evaluation of ER% was obtained as follow:
ER% ¼ þ93:32
1:52X1 þ 1:62X2
1:34X3
þ 1:59X4
0:23X5 þ 1:04X1 X2
2:94X1 X3
þ 0:85X1 X4 þ 0:36X1 X5
1:68X 2 X3

0:35X2 X4
0:26X 2 X5 þ 0:46X3 X4

3:68X3 X5 þ 0:57X4 X5 þ 0:046X21

0:87X22
0:24X23
0:27X24
3:02X25 ð2Þ
3.4. Validation of the MISPE procedure
3.4.1. Calibration curve linearity
The minimum concentration of CEL that can be determined by
HPLC in standard solutions was 20 lg L
1. The standard curve
was obtained from HPLC–UV analysis of standard solutions
prepared in aqueous solution without pre-concentration process
that was linear in the range of 20–2000 lg L
1 with R2 = 0.99.
74 M. Arabi et al. / Ultrasonics Sonochemistry 33 (2016) 67–76

Due to the importance of determination of analyte at low concen- 120

tration in samples, separation and pre-concentration before analy-
sis are very important. The calibration curve was plotted for the 100
ultrasonic assisted-solid-phase extraction method using MINPs as

Recovery (%)
80
adsorbent. The detection limits and low linear range are calculated
after pre-concentration by the method that is described in Sec-
60
tion 2.4 with linear correlation in the range of 0.2–2000 lg L
1.
The regression equation was y = 538.6x + 3.357 with R2 = 0.980. 40

3.4.2. Reusability 20
The method robustness is an estimation of its capacity to remain
0
unaffected when small variations are deliberately introduced in the
0 2 4 6 8 10 12 14 16
analytical parameters. An indication of the method reliability and
Number of the repeated use
the influence of each analytical parameter during routine operation
can be achieved through this way [36]. In order to show the reusabil- Fig. 7. Reusability of MISPE.
ity of the CEL-MINPs, sorption–elution cycle is repeated 14 times
using the same MINPs. As it is shown in Fig. 7, the result clearly
shows that CEL-MINPs could be used repeatedly without signifi-
cantly losing its adsorption amount until 12 times repeated use. Table 6
Accuracy and precision of the proposed ultrasonic assisted-MISPE-HPLC–UV method
(n = 5).
3.4.3. Precision and accuracy
Added (lg mL
1) Found (lg L
1) Recovery (%) Precision (RSD%)
The ultrasonic assisted-MINPs-SPE-HPLC–UV method applica-
bility in the plasma samples were checked by spiking four concen- Intra-day Inter-day

trations within the calibration range (0.5, 10, 500 and 1000 lg L
1, 1.0 1.025 102.5 8.14 6.42
n = 5) and subsequently recommended procedure were under 10 10.16 101.6 6.35 7.34
100 93.4 93.4 6.65 4.43
taken. The accuracy and precision was examined by standard addi-
500 503.75 100.7 3.37 4.14
tion method in linear form. The inter- and intra-assay precisions 1000 986.5 98.6 2.78 3.14
(Table 6) show that the CEL recoveries for the ultrasonic

Fig. 8. The chromatograms of the plasma samples (a) the blank plasma (b) spiked sample (30 lg mL
1 of celecoxib).
 M. Arabi et al. / Ultrasonics Sonochemistry 33 (2016) 67–76
Table 7
Comparison of different methods for pre-concentration of celecoxib with proposed
method.
Method Selectivity Linear range Recovery% Ref
lg L
1
SPE-HPLC–UV Lack of selectivity 25–2000 85–96 [38]
LLE-HPLC–UV Lack of selectivity 10–2000 92–99 [39]
LLE-HPLC–UV Lack of selectivity 20–2000 80–90 [40]
Ultrasonic Selective 0.2–2000 93.4–102.5 Present
assisted-SPE- work
HPLC–UV
assisted-MISPE-HPLC–UV method are higher than 90% at the
concentrations tested. These acceptable recoveries are sufficient
for quantitative determination of CEL in human plasma. The
intra-assay precision (five repeated injections) investigation show
that each concentration has RSDs less than 8%, while their
inter-day RSDs were in the range of 3.14–7.34%. The so results
suggest high efficiency of ultrasonic assisted-MINPs-SPE to
represent acceptable recovery and precision and candidate it as
clean up and pre-concentration procedure for quantification of
CEL in plasma samples. As it can be seen in Fig. 8, no peaks
detected in blank plasma that interferes with CEL.
3.4.4. Limits of detection and quantification
The limits of detection (LOD) and quantification (LOQ) were
determined based on the signal to noise (S/N) ratio of 3–10.
The LOD and LOQ for CEL standard were 0.08 and 0.18 lg L
1,
respectively. Therefore, the obtained LOD and LOQ were satisfac-
tory and allowed the determination of CEL.
4. Conclusion
In this work, MINPs were synthesized via a non-covalent molec-
ular imprinting for cleaning-up CEL from human plasma samples
prior to quantitative analysis by HPLC–UV. The functional mono-
mer had strong interaction with the template molecule with highly
selective cavities. The effect of factors during extraction process
were evaluated and optimized by central composite design. In this
method ultrasound assisted for decreasing organic solvent for
desorption analyte. The MINPs had high adsorption capacity and
an acceptable linearly range was obtained. The present method
benefits from advantages like cost effective, complete template
removal, low consumption of toxic organic solvents, easy to
operate and excellent LOD. Moreover, MINPs sorbents can be also
easily recycled by rinsing with methanol/DIW. Table 7 collects
some recent methods found for the determinations of the CEL in
plasma samples [38–40]. As it can be seen, compared with other
method the operation of ultrasonic assisted-MINPs-SPE is simpler
and highly selective. This study provides a new tool and method
for the monitoring of CEL.
Acknowledgement
The authors express their appreciation to the Graduate School
and Research Council of the University of Yasouj for financial sup-
port of this work.
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