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Development and characterization of an electrochemical sensor for

furosemide detection based on electropolymerized molecularly imprinted
polymer

Article in Talanta · January 2016

DOI: 10.1016/j.talanta.2015.08.042

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 Talanta 146 (2016) 181–187

Contents lists available at ScienceDirect

Talanta
journal homepage: www.elsevier.com/locate/talanta

Development and characterization of an electrochemical sensor

for furosemide detection based on electropolymerized molecularly
imprinted polymer
Kamalodin Kor, Kobra Zarei n
School of Chemistry, Damghan University, Damghan, Iran

art ic l e i nf o a b s t r a c t

Article history: A novel electrochemical sensor based on a molecularly imprinted polymer, poly(o-phenylenediamine)
Received 6 June 2015 (PoPD), has been developed for selective and sensitive detection of furosemide. The sensor was prepared
Received in revised form by incorporating of furosemide as template molecules during the electropolymerization of o-phenyle-
19 August 2015
nediamine on a gold electrode. To develop the molecularly imprinted polymer (MIP), the template
Accepted 20 August 2015
Available online 21 August 2015
molecules were removed from the modified electrode's surface by washing it with 0.25 mol L
1 NaOH
solution. The imprinted layer was characterized by cyclic voltammetry (CV), electrochemical impedance
Keywords: spectroscopy (EIS) and atomic force microscopy (AFM). The sensor′s preparation conditions including
Furosemide furosemide concentration, the number of CV cycles in the electropolymerization process, extraction
Molecular imprinted polymer
solution of the template from the imprinted film, the incubation time and the pH level were optimized.
Poly(o-phenylenediamine)
The incubation of the MIP-modified electrode, with respect to furosemide concentration, resulted in a
Electropolymerization
Gold electrode suppression of the K4[Fe(CN)6] oxidation process. Under the optimal experimental conditions, the re-
sponse of the imprinted sensor was linear in the range of 1.0  10
7–7.0  10
6 mol L
1 of furosemide.
The detection limit was obtained as 7.0  10
8 mol L
1 for furosemide by using this sensor. The sensor
was successfully used to determine the furosemide amount in the tablet and in human urine samples
with satisfactory results.
& 2015 Elsevier B.V. All rights reserved.

1. Introduction electrochemical techniques there is no affinity or specificity

against analyte. During the past few years, a considerable devel-
Furosemide (Scheme 1) (4-chloro-2-(furan-2-ylmethylamino)- opment of molecular imprinting technique has been seen for
5-sulfamoylbenzoic acid) is a sulfonamide. However this com- electrochemical determination of different analytes due to their
pound was originally used as an antibacterial agent. More recently high affinity to the template molecule.
its diuretic properties made furosemide a valuable drug for the Molecularly imprinted polymers (MIPs) are synthetic polymeric
treatment of hypertension [1,2], fluid retention (edema) in pa- materials with tailored high affinity and selectivity towards a pre-
tients with congestive heart failure, liver disease and kidney dis- defined target molecule. These make them very attractive candi-
order such as nephrotic syndrome [3]. Furosemide is also a com- date for the development of novel sensors [16]. Development of
mon doping agent in sports [4]. MIPs polymers was inspired by the lock-and-key fit principle. This
Several techniques have been reported for detection of fur- principle is observed in many basic biological processes such as
osemide such as electrochemistry [1,3,5–7], fluorescence [8], sensing of scent, signaling between nerve and muscle cells and
spectrophotometry-partial least squares method [9], micellar li- enzyme/substrate recognition. For decades, scientists have tried to
quid chromatography [10], chemiluminescence flow-injection [11] find the right key–lock pairs; the discovery of MIPs allows building
and high-performance liquid chromatography [12–15]. the locks around a desired key by the creation of polymeric ma-
Low cost, high sensitivity and short time analysis of electro- trices containing cavities which complement in size, shape and
chemical techniques caused a revolution in the analysis field in chemical interactions (e.g. dipole–dipole, ionic interactions and
comparison with other techniques, but in the most of hydrogen bonds) with the target molecule (key). The imprinting
process involves the polymerization of functional monomers in
n
Corresponding author. Fax: þ 98 2335235431. the presence of a template, typically the target molecule or a
E-mail address: zarei@du.ac.ir (K. Zarei). molecule of similar size and chemical functionality. Following

http://dx.doi.org/10.1016/j.talanta.2015.08.042
0039-9140/& 2015 Elsevier B.V. All rights reserved.
182 K. Kor, K. Zarei / Talanta 146 (2016) 181–187

at the room temperature. Then the electrode was subjected to

cyclic potential sweeps, between 0.2 and 1.5 V vs. Ag/AgCl, in
0.5 mol L
1 H2SO4, until a stable cyclic voltammogram was ob-
tained. Subsequently, for preparation of MIP, the electrode was
immersed in acetate buffer solution (0.5 mol L
1, pH ¼5.2) con-
taining 5  10
3 mol L
1 OPD and 1  10
3 mol L
1 furosemide.
The electrosynthesis of PoPD film was performed by cyclic vol-
tammetry (20 scans), between 0 and 1.0 V vs. Ag/AgCl, at a scan
rate of 0.1 V s
1. To remove the furosemide template, the MIP
electrode was dipped in the 0.25 mol L
1 NaOH for 5 min, twice.
As a control, the NIP electrode was also prepared in the same way,
Scheme 1. Structure of the neutral form of furosemide.
but without adding furosemide. The prepared MIP and NIP elec-
trodes were stored in a refrigerator at 4 °C.

polymerization the template is removed from the polymeric ma- 2.4. Experimental procedure
trix creating in this way the recognition sites [17]. Molecularly
imprinted polymers (MIPs) have some specific advantages such as The MIP electrode was incubated in phosphate buffer solution
low cost of preparation, high affinity to the template, robustness (pH 7.0) containing appropriate concentrations of furosemide for
and long-term stability [18]. 10 min with stirring. After washing with water to remove any
In the present study, a novel electrochemical sensor based on material that may have been adsorbed on the surface, the elec-
imprinted poly(o-phenylenediamine) (PoPD) membranes at the trochemical measurements were carried out in the presence of
gold electrode, for the selective and sensitive detection of fur- 1  10
3 mol L
1 K4[Fe(CN)6] solution containing 0.1 mol L
1 KCl.
osemide is proposed. This is the first application of electro- Differential pulse voltammograms (DPVs) of the imprinted mem-
polymerization and MIPs for electrochemical determination of branes were recorded in the potential range of 0.0–0.5 V vs. Ag/
furosemide (as we know). AgCl with a scan rate of 50 mV s
1. A blank solution without
furosemide was used to obtain the blank peak current.

2. Experimental
3. Results and discussion
2.1. Apparatus
3.1. Electropolymerization of OPD on the surface of the gold
All electrochemical experiments were performed on an Autolab
electrode
PGSTAT 30 electrochemical system (EcoChemie, Utrecht, Nether-
lands). The Autolab system was run on a PC using GPES and FRA
Electropolymerization of OPD on the surface of the gold elec-
4.9 software. The three-electrode system consisted of bare Au
trode was performed using cyclic voltammetry (Fig. 1). The Au
(3 mm in diameter), non-imprinted electrode/Au (NIP/Au) or MIP/
electrodes were cleaned and were cycled 20 times between 0 and
Au as the working electrode, Ag/AgCl/KCl (saturated) as the re-
1.0 V vs. Ag/AgCl (sat KCl) at a scan rate of 0.1 V s
1 in acetate
ference electrode, and a platinum wire as the auxiliary electrode.
buffer solution (0.5 mol L
1, pH ¼ 5.2). The solution contained
For impedance measurements, a frequency range of 0.10–100 kHz
5  10
3 mol L
1 OPD and 1  10
3 mol L
1 of furosemide.
was employed. The AC voltage amplitude used was 5 mV, and the
As the CV showed in Fig. 1, in the first scan, an irreversible
equilibrium time was 10 min. Atomic force microscopy (AFM) was
anodic oxidation peak appeared at a potential of þ0.4 V. This was
performed using a DME microscope with 95–50 E probe model.
ascribed to the oxidation of the OPD monomer to its dimer state. A
Measurements of pH were made using a PMT 1003 pH meter and a
significant decrease in the anodic oxidation peak during the cycles
combined glass electrode.
that followed indicated the formation of a non-conductive film
onto the electrode's surface. The formation of the film was also
2.2. Reagents
confirmed by CV in the presence of a ferro/ferricyanide redox
probe (curve not shown here). Following electropolymerization a
O-Phenylenediamine (OPD, Z98%, Sigma-Aldrich, USA), po-
tassium ferricyanide (K3[Fe(CN)6], 99%, E. Merck, Germany), po-
tassium ferrocyanide (K4[Fe(CN)6], 99%, E. Merck, Germany) were
used. Furosemide (99.9%) was kindly provided by Aburaihan
pharmaceutical company (Iran).
The stock 0.01 mol L
1 furosemide solution was prepared by
dissolving the required amount of furosemide in alkaline solution
and maintaining the solution in darkness at 4 °C. All other reagents
were of analytical grade and used as received. All solutions were
prepared with redistilled water, and the solutions were deox-
ygenated with high purity nitrogen for 200 s prior to each ex-
periment, which were performed under a nitrogen atmosphere.

2.3. Preparation of MIP and NIP electrodes

The surface of the gold electrode was polished on a micro cloth

with 1.0 and 0.05 μm aqueous slurry of alumina, and then it was Fig. 1. Cyclic voltammograms for the electropolymerization of 5  10
3 mol L
1
cleaned ultrasonically into the water for 5 min to remove any OPD on a gold electrode in acetate buffer (pH 5.2) containing 1  10
3 mol L
1
particles that might be attached at the surface and allowed to dry furosemide: scan rate, 100 mV s
1; number of scans, 20.
 K. Kor, K. Zarei / Talanta 146 (2016) 181–187 183

total loss of the response associated to the redox probe was re- In Fig. 3A, after the polymerization of OPD, the imprinted
corded. This has been reported to be due to the fact that the polymeric film formed an insulating layer on the electrode surface
presence of the non-conductive film hindered the redox probe's and obstructed the electron-conductor of the electrolyte, which
access to the surface of the electrode [19]. resulted in a high interfacial Rct. Fig. 3B shows significant changes
No significant difference was observed between the cyclic in the impedance spectra after treatment for the removal of the
voltammograms obtained in the presence of furosemide and in its template. In the Fig. 3B, after treatment, both the MIP and NIP
absence, which can be explained by the fact that furosemide does electrodes' spectra showed the appearance of a high frequency
not have any electroactivity on the gold electrode in the potential semicircle which represents the ferro/ferricyanide electrode reac-
window chosen for the polymerization [20]. tion. The treatment dramatically increased the rate of electron
transfer between the solution and the electrode due to the re-
3.2. Surface topographical characterization of imprinted sensor using moval of the unstable monomers on the polymerized OPD film
AFM [19]. The radius of the semicircle is smaller in the case of MIP, due
to the successful extraction of furosemide from the imprinted
Atomic force microscope (AFM) was used to investigate the polymer. The Rct values for MIP and NIP were obtained at 41 kΩ
surface topography of the imprinted layer. The AFM images of and 150 kΩ respectively. Treated MIP-modified electrodes showed
imprinted (furosemide-PoPD/Au) and non-imprinted electrodes an electrode process that was approximately 3.6 times faster in
(PoPD/Au) after the template removal are shown in Fig. 2. The AFM comparison with NIP-modified electrodes. In addition to the above
images reveal a marked difference in the roughness of the surfaces
points, Fig. 3B also shows EIS of NIP after a second treatment. It
which can be expressed in terms of the root-mean-square (RMS)
can be seen that Rct values are almost equal for the first and sec-
value (RMS is proportional with roughness). As shown in Fig. 2, the
ond treatment of NIP. Therefore, it appears that after the first
NIP film was relatively flat and compact with a calculated RMS of
treatment, in which unstable monomers were removed, the
value of 8.4 nm, on the other hand MIP showed a rougher surface,
polymer remains stable due to treatment with NaOH.
with a calculated RMS value of 12.6 nm, which may be due to the
The equivalent circuit compatible with the Nyquist diagram is
successful deposition and removal of the furosemide in/from the
shown in Fig. 3(C). In this circuit, Rs, CPEdl, and Rct are the re-
film.
spective following: solution resistance, a constant phase element
corresponding to the double-layer capacitance and the charge-
3.3. Electrochemical characterization of the imprinted sensor
transfer resistance. W is a finite-length Warburg short-circuit term
coupled to Rct, which accounts for the Nernstian diffusion. In this
Cyclic voltammetry (CV) and electrochemical impedance
circuit, the charge transfer resistance of the electrode reaction is
spectroscopy (EIS) were used to characterize the imprinted sensor.
the only circuit element with a simple physical meaning that de-
EIS is an effective method for probing the changes in the surface's
features of the modified electrodes during assembly [21]. It was scribes the rate of charge transfer during electrode reaction with
therefore decided that EIS would be employed to characterize the the electrode potential.
film of the polymer. Double-layer capacitance and interfacial In order to further characterize the prepared sensor, cyclic
electron transfer resistance of the electrode surface can be chan- voltammograms of the stepwise fabrication procedure were re-
ged by modifying the electrode using conducting polymers, na- corded in the presence of [Fe(CN)6]3
/[Fe(CN)6]4
in 0.1 mol L
1
nomaterials and semiconducting materials. KCl as a probe. As shown in Fig. 4, no peak was observed at the MIP
The EIS is comprised of a semicircular part and a linear part. The and NIP electrodes before the template removal (curves A and B).
semicircular part corresponds at higher frequencies (short time Since the formed PoPD film by electropolymerization was very
scales) to a limited electron transfer process and its diameter is compact, and there were almost no channels for the active probe
equivalent to the electron transfer resistance (Rct). The Rct value has to approach the electrode surface. After removal of the furosemide
an inverse relationship with the electrochemical activity of the the MIP electrode showed a couple of redox peaks (curve C). Op-
specimen. The linear part corresponds at lower frequencies (long positely, in the case of NIP, no well-defined redox peaks appeared
time scales) to a diffusion process. Fig. 3 shows the Nyquist dia- as a result of the removal process (curve D). After incubation of the
grams (imaginary impedance Zim vs. real impedance Zre) of MIP and MIP electrode in furosemide solution, the redox peaks disappear,
NIP electrodes before (A) and after (B) the template removing which confirms the specific adsorption of furosemide molecules to
process in 5  10
3 mol L
1 [Fe(CN)6]3
/[Fe(CN)6]4
and the imprinted recognition layer deposited onto the surface of the
0.1 mol L
1 KCl solution at 5 mV (AC) at a frequency of 0.1–100 kHz. electrode (curve E).

Fig. 2. AFM images of (A) non-imprinted electrode (NIP) and (B) imprinted electrode (MIP) after template removal with 0.25 mol L
1 NaOH solution.
184 K. Kor, K. Zarei / Talanta 146 (2016) 181–187

Fig. 3. EIS of MIP and NIP electrode (A) before and (B) after template removing process in 5  10
3 mol L
1 [Fe(CN)6]3
/[Fe(CN)6]4
and 0.1 mol L
1 KCl solution at 5 mV
(AC) at a frequency of 0.1–100 kHz. (C) Equivalent circuit compatible with the Nyquist diagram.

3.4.2. Effect of template concentration

The template concentration in the polymerization mixture is a
significant factor in the response of the sensor. The quantity and
quality of the MIP recognition sites is also a direct function of
template concentration [23]. Fig. 5B shows the effect of different
concentrations of the template on the response of the MIP during
the electropolymerization process. The sensor produced at a lower
template concentration was less sensitive. This is probably because
of the small number of recognition sites formed in the polymer′s
matrix. The sensor prepared in a higher concentration of template
molecule also showed poor sensitivity. This may be due to the
furosemide molecules forming a complex with all of the monomer
molecules, lowering the likelihood of cross-linking and the con-
sequent entrapment of the template molecule in a tri-dimensional
matrix [24].

3.4.3. Template removing treatment

Fig. 4. Cyclic voltammograms obtained with different electrode. (A) MIP and (B)
NIP modified electrodes before template removing. (C) MIP and (D) NIP modified
In order to achieve a good sensitivity and reproducibility
electrodes after template removing and (E) MIP electrode after incubation at template, molecules should be eluted. Extraction with solvent is
5  10
4 mol L
1 furosemide. Conditions: 1  10
3 mol L
1 the most common method. In the presence of solvent, a polymer
[Fe(CN)6]3
/[Fe(CN)6]4
and 0.1 mol L
1 KCl at scan rate of 50 mV s
1. can be swollen so it facilitates the template removal [23]. In this
study, methanol–acetic acid (4:1, v/v), methanol–water (1:1, v/v),
3.4. Optimization of conditions acetonitrile–water (1:1, v/v), ethanol–water (1:1, v/v), acetic acid,
acetonitrile–acetic acid (4:1, v/v), methanol–NaOH 0.5 mol L
1
3.4.1. Number of cycles for polymerization of OPD (1:1 v/v), ethanol–NaOH 0.5 mol L
1 (1:1 v/v) and 0.25 mol L
1
It is known that the thickness of the imprinted PoPD membranes NaOH solution were applied in order to remove the template. The
can influence the amount of furosemide molecules embedded results showed that the 0.25 mol L
1 NaOH solution allowed a
which then influences the sensitivity of the sensor. Changing the faster and more efficient removal of the template. This is because
number of electropolymerization cycles can be done to adjust the furosemide can easily be dissolved in the alkaline solution, while
thickness of the imprinted polymeric membranes. Fig. 5A shows the the other extraction solvents are only able to partially dissolve it. It
effect that the number of cycles has on the response of the MIP. The was found that stirring the imprinting electrode in 5 ml of
volume of imprinted sites increases with the increase of the im- 0.25 mol L
1 NaOH solution for 5 min (twice) could remove the
printed membrane's thickness. However, if the imprinted mem- highest amount of the template in comparison with other solvents
branes are too thick, the imprinted sites situated in the middle and conditions. This extraction procedure was therefore used in
prove difficult to access due to the high mass-transfer resistance. As the continuation of our work.
a result, thick imprinted membranes would give poor site accessi-
bility and low binding capacity [22]. Thus, in this work, it was 3.4.4. Effect of incubation time and pH
decided that 20 cycles was the most appropriate number of cycles The incubation step affects the sensitivity of the MIP-based
to produce a membrane with optimum analytical performances. sensor. After the template removal process, the MIP electrode was
 K. Kor, K. Zarei / Talanta 146 (2016) 181–187 185

Fig. 5. Effect of different parameters on the response of the MIP. (A) Effect of the number of cycles in electropolymerization process. (B) Effect of different concentration of
the furosemide in electropolymerization process. (C) Effect of incubation time. (D) Effect of pH in template binding process. The response was measured through DPV of
1  10
3 mol L
1 K4[Fe(CN)6] solution after immersion of the electrode in a 3  10
6 mol L
1 furosemide solution for 10 min. Scan rate 50 mV s
1.

incubated in 0.2 mol L
1 PBS (pH 7.0) which contained

3  10
6 mol L
1 of furosemide for differing incubation times. The
differential in pulse voltammetric responses in the K4[Fe(CN)6]
solution which contained 0.1 mol L
1 KCl were recorded. Fig. 5C
shows the effect of incubation time on the response of the MIP.
The results show that the peak current decreased rapidly (increase
in ΔI) with the increase in the incubation time. A stable response
was obtained after 10 min, suggesting that the adsorption equili-
brium was reached. Thus, the optimum incubation time was se-
lected to be 10 min for the electrochemical determination of fur-
osemide. Furthermore, different pH levels of 0.2 mol L
1 PBS were
used to estimate the current responses of 3  10
6 mol L
1 fur-
osemide in the K4[Fe(CN)6] solution that contained 0.1 mol L
1 KCl
(Fig. 5D). The results showed that pH ¼7 is the most appropriate
pH of phosphate buffer in the incubation process. This is con-
sidered to be reasonable as furosemide is insoluble in a low pH, so
with the increase of the pH in the incubation step, the amount of
furosemide that can interact with sites on the polymer will in- Fig. 6. Differential pulse voltammograms of MIP-modified electrode after 10 min of
incubation in different concentrations of furosemide from 1.0  10
7 to
crease. Also, in the high pH solutions, it will be difficult for the 7.0  10
6 mol L
1 in buffer (1  10
3 mol L
1 K4[Fe(CN)6] and 0.1 mol L
1 KCl at
furosemide to transfer from alkaline solution to polymer cavities. scan rate of 50 mV s
1).

3.5. Performance of the imprinted sensor furosemide molecules which led to further decrease in the re-
corded current. An analytical curve between the relative oxidation
3.5.1. DPV response and analytical curve peak current and the furosemide concentration is exhibited in
In this study, quantitative analysis of furosemide using the Fig. 6. Considering the absolute currents may vary with the surface
proposed MIP electrode under optimized experimental conditions
areas of the electrodes used and the distance between the working
was performed using DPV, which is relatively sensitive in com-
electrode and auxiliary electrode in each experiment, it was sug-
parison to the conventional CV method.
gested that relative current change (Δi/i0) was used to indicate
After the template removal and background response mea-
current response [20]. Here, Δi¼ i0
ic, i0 and ic are the current
surements, MIP-modified electrodes were dipped into the binding
buffer solutions which contained furosemide at different con- values when the concentration of furosemide is 0 and C mol L
1,
centrations. This was done for 10 min and was followed by the respectively. The linear range was estimated to from 1.0  10
7 to
detection step. When the MIP electrode was immersed in the so- 7.0  10
6 mol L
1 and the detection limit (CLOD ¼3Sb/m, where Sb
lution containing furosemide, the cavities in the film were partially is the standard deviation for 8 replicates' determination of the
occupied by furosemide, which led to the decrease of current blank and m is analytical curve slope) was found to be
signal produced by potassium ferrocyanide. As concentration in- 7.0  10
8 mol L
1. The linear calibration graphs of (Δi/i0) vs.
creased, more and more binding sites in the film were occupied by concentration of furosemide can be described by the following
186 K. Kor, K. Zarei / Talanta 146 (2016) 181–187

equation, Δi/i0 ¼ 0.0955(mmol L
1) þ0.056. The correlation coef- furosemide was 1.7%. Intraday precision study was carried out by
ficient is 0.9989. preparing furosemide solution of same concentration and analyzing
it at three different times in a day. The same procedure was fol-
3.5.2. Binding study lowed for three different days to determine interday precision. The
The obtained DPV data in the previous section was applied to average RSD values for intraday and interday precisions were ob-
study the binding properties of a MIP-modified electrode. The tained as 2.2% and 2.5%, respectively.
binding isotherm of the MIP-modified electrode was fitted using a The inter-electrode reproducibility was estimated by determin-
model for two types of simultaneous binding: on the sites of ing the response of seven different electrodes, which were im-
specific recognition inside polymer film and on the surface of the mersed in 3  10
6 mol L
1 and 4.5  10
6 mol L
1 of furosemide
electrode due to non-specific adsorption [19]: for 10 min respectively. The relative standard deviations (RSD) for
seven successive assays of furosemide were 2.5% and 2.9% respec-
Δi B c
= MAX + Ns c tively, indicating acceptable fabrication for reproducibility.
i0 KD + c (1)
The stability of the electrode was also investigated by mea-
where c is bulk concentration of the analyte, BMAX is the suring the electrode response with 4.5  10
6 mol L
1 of fur-
maximum number of binding sites in the MIP, KD is the equili- osemide every 4 days over a large time frame. Between mea-
brium dissociation constant and NS is the binding constant for surements, the electrode was stored at 4 °C in a refrigerator. The
nonspecific adsorption. The value KD obtained with fitting was current response decreased to 94% after 8 days, while 86% of the
3.3  10
8 mol L
1 (R2 ¼ 0.994). original response was retained after 20 days. The electrode still
retained 80% of its original response after as long as one month.
3.5.3. Selectivity of the imprinted sensor
MIPs have specific selectivity towards the template molecule. 3.5.5. Determination of furosemide in pharmaceutical dosages and
The specific recognition is based on the interaction between the Human urine samples
template and the imprinting sites. There are two important factors The MIP electrode was finally applied successfully for the de-
that play key roles in selectivity. These are the functional groups of tection of furosemide in tablet samples (furosemide 40 mg tablets
the molecules and the size of molecules. Molecules with the same were purchased from Chemidarou Pharmaceutical Co. Iran). The
functional groups and sizes have more interference effect. The tablets were ground into powder, dissolved in alkaline water, fil-
specificity of the MIP-based sensor toward furosemide was eval- tered and then further diluted so the furosemide concentration fell
uated by testing and comparing its responses to furosemide and into the range of the calibration plot. This study was carried out
some possible interfering substances. Such substances included using a standard addition method. The data given in Table 1 shows
chloramphenicol, gabapentin, spironolactone, dopamine, pheny- the satisfactory results for assay of furosemide in drug samples.
lephrine and folic acid. The change of current response of Moreover, because of the importance of the determination of
K4[Fe(CN)6] on the MIP electrode in the same concentration furosemide in biological samples, the MIP electrode was also ap-
plied to the determination of furosemide in the human urine
(3.0  10
6 mol L
1) of each substance was determined using the
sample. The urine sample was diluted twice with the PBS (pH ¼7)
DPV method. As shown in Fig. 7, almost no interference was ob-
before analysis with no further pretreatments. Different amounts
served with spironolactone, chloramphenicol and folic acid. These
of furosemide were then spiked into the urine sample and the
are larger in size than furosemide and cannot enter the imprinting
standard addition method was used to obtain the results. The re-
sites present on the MIP surface. A slight interference was ob-
sults are summarized in Table 1. Statistical t-test showed that
tained with Dopamine, Phenylephrine and Gabapentin. This may
there is an agreement between spiked and obtained results, which
be explained by the fact that these compounds are smaller than
revealed that the proposed MIP electrode can be applied in order
furosemide in molecular size and have some chance of ap-
to determine the furosemide concentration in both pharmaceu-
proaching the imprinting sites.
ticals and urine with satisfactory accuracy and precision.
3.5.4. Precision, reproducibility and storage stability of the imprinted
sensor
Precision studies were carried out to ascertain the reproduci- 4. Conclusions
bility of the proposed method. Repeatability was evaluated by as-
For the first time, a very simple electrochemical sensor was
saying one modified electrode for seven replicate determinations.
proposed for furosemide determination via the electropolymeriza-
The RSD value for seven measurements of 4.5  10
6 mol L
1
tion of furosemide imprinted poly-o-phenylenediamine with a gold

Table 1
Determination of furosemide in tablet and human urine samples.

Sample no.a Concentration of furosemide Recovery (%)

(C  107 mol L
1)

Added Foundb

Tablet 1 0.0c 5.2 7 0.2 –

2 20.0 277 2 109
3 50.0 547 3 98

Human urine 1 10.0 10.4 7 0.5 104.0

2 50.0 48 7 3 96

a
Each sample was assayed in triplicate (n¼ 3).
b
Mean 7 ts (confidence interval 95%).
N
Fig. 7. Selectivity of MIP modified electrode. c
Real amount is 5.0  10
7 mol L
1 in the diluted tablet.
 K. Kor, K. Zarei / Talanta 146 (2016) 181–187 187

Table 2
The comparison of electrochemical determination of furosemide on various electrodes.

Electrode Linear range (mol L
1) Detection limit (mol L
1) Reference

Multi-walled carbon nanotubes–paraffin oil paste electrode 8.0  10
6–2.0  10
4 2.9  10
7 [25]
Graphite–polyurethane composite electrode 7.5  10
7–6.5  10
6 1.5  10
7 [1]
Amperometric detection at carbon fiber microelectrodes coupled to HPLC and FIA 5.0  10
7–1.0  10
4 1.7  10
7 [26]
Poly(vinyl chloride) membrane electrode 1.59  10
4–1.0  10
2 1.19  10
4 [27]
Molecularly imprinted polymer 1.0  10
7–7.0  10
6 7.0  10
8 Present work

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