molecules

Article
Preparation and Characterization of Carbon Paste
Electrode Bulk-Modified with Multiwalled Carbon
Nanotubes and Its Application in a Sensitive Assay of
Antihyperlipidemic Simvastatin in
Biological Samples
Amir M. Ashrafi and Lukáš Richtera *
Department of Microelectronics, Faculty of Electrical Engineering and Communication, Brno University of
Technology, Technická 3058/10, CZ-616 00 Brno, Czech Republic; amirmansoor.ashrafi@mendelu.cz
* Correspondence: richtera@mendelu.cz; Tel.: +420-545-133-311

Academic Editor: Ondrej Zitka




Received: 20 May 2019; Accepted: 12 June 2019; Published: 13 June 2019

Abstract: Determination of an antihyperlipidemic drug simvastatin (SIM) was carried out using
a carbon paste electrode bulk-modified with multiwalled carbon nanotubes (MWCNT-CPE).
Scanning electrochemical microscopy (SECM), scanning electron microscopy (SEM), and atomic
force microscopy (AFM) were used for the characterization of the prepared electrodes. Different
electrodes were prepared varying in mass percentage of MWCNTs to find out the optimum amount
of MWCNTs in the paste. The MWCNT-CPE in which the mass percentage of MWCNTs was 25%
(w/w) was found as the optimum. Then, the prepared electrode was used in a mechanistic study and
sensitive assay of SIM in pharmaceutical dosage form and a spiked human plasma sample using
differential pulse voltammetry (DPV). The prepared electrode shows better sensitivity compared
to the bare carbon paste and glassy carbon electrode (GCE). The detection limit and the limit of
quantification were calculated to be 2.4 × 10−7 and 8 × 10−7 , respectively. The reproducibility of the
electrode was confirmed by the low value of the relative standard deviation (RSD% = 4.8%) when eight
measurements of the same sample were carried out. Determination of SIM in pharmaceutical dosage
form was successfully performed with a bias of 0.3% and relative recovery rate of 99.7%. Furthermore,
the human plasma as a more complicated matrix was spiked with a known concentration of SIM and
the spiking recovery rate was determined with the developed method to be 99.5%.

Keywords: multiwalled carbon nanotube; carbon paste electrode; scanning electrochemical

microscopy; simvastatin determination; mechanism study; biological sample

1. Introduction
Simvastatin (SIM) is a long-established hydroxy-methylglutaryl coenzyme which was first
introduced in 1998 [1]. It belongs to a group of drugs called statins. The oral administration of SIM is
followed by its hydrolysis, generating the corresponding β-hydroxyacid form [2]. Then, the content
of the low-density lipoprotein (LDL) cholesterol in plasma is reduced effectively by inhibition of
3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase [3]. This enzyme acts by catalyzing the
conversion of HMG-CoA to mevalonate, which is an early and rate-limiting step in the biosynthesis of
cholesterol [4]. SIM is synthesized from a fermentation product of Aspergillus terreus [5]. In addition to
the well-defined function of statins, some clinical trials showed that this group of drugs can protect
against cardiovascular disease (CVD) through another mechanism [6]. Statins are among the most
widely prescribed drugs in the world [7].

Molecules 2019, 24, 2215; doi:10.3390/molecules24122215 www.mdpi.com/journal/molecules

Molecules 2019, 24, 2215 2 of 14

Due to the in vivo hydrolysis, the concentration of SIM in human plasma is very low (1–15 nM) [8–11].
Moreover, for the determination of SIM in plasma, a separation step is required. Thus, it is highly
demanded to develop a sensitive analytical method for the determination of SIM in pharmaceutical
dosage form and human plasma. The developed analytical methods for the determination of SIM consist
of methods which provide the separation process, including high-performance liquid chromatography
(HPLC) [2,12–15], gas chromatography (GC) [16], and capillary electrophoresis [17] in combination
with detection methods including ultraviolet (UV) spectroscopy [12], fluorescence detection [14], mass
spectrometry [2,13,15,16], and colorimetry [18].
However, the inherent features of electroanalytical methods, such as good selectivity and sensitivity,
even in a complicated matrix, as well as being inexpensive, quick, and simple to execute, make them
advantageous compared to the other analytical methods.
Several electroanalytical methods were applied for the determination of SIM, including adsorption
of the SIM–Cd(II) complex at the mercury drop electrode and determination of SIM from the reduction
peak of cadmium [19], cathodic square-wave stripping voltammetry using the adsorption of SIM onto
the mercury electrode surface [20], direct voltammetry using the bare glassy carbon electrode (GCE) [21],
GCE immobilized with sodium dodecyl sulfate [22], and GCE modified with a multiwalled carbon
nanotube (MWCNT)–dihexadecyl hydrogen phosphate composite [23]. Although, these electroanalytical
techniques showed good sensitivity, there are still some disadvantages, such as toxicity of the mercury
and the short life of the surface-modified electrodes.
Blending the graphite powder with a pasting liquid forms a heterogeneous mixture which is
known as carbon paste. Carbon paste is an attractive electrode material due to its electrochemical
characteristics, including a very low background current, low ohmic resistance, cost-effectiveness,
and simple electrode surface renewal [24]. Furthermore, the modification of the carbon paste can be
simply carried out by adding the modifier to the paste [25–27]. To decrease the electrical resistance of
the paste, room-temperature ionic liquids were used as a pasting liquid [28].
As a modifier, carbon nanotubes (CNTs) enhance the electrode performance by increasing the
velocity of the electron transfer [29]. Moreover, reduced over-potential and improved selectivity are
caused by modifying the electrode with CNTs [30]. In addition, CNTs possess a significant adsorption
capability, which provides pre-concentration of the analyte at the electrode surface and consequently
increases the sensitivity of the electrode. The sensitivity of the electrode is also improved due to the
high surface area of CNTs [31].
This work aimed to prepare a carbon paste electrode (CPE) bulk-modified with MWCNTs for
sensitive determination of SIM. Differential pulse voltammetry was used for the determination.
The MWCNTs can increase the sensitivity of the concentration determination compared to the bare GCE
and CPE. The preparation and surface renewing of the MWCNTs/CPE are rapid and easy. Moreover,
compared to the previous studies, any toxic compounds such as mercury or other heavy metals are
not used. Then, the developed electrode was used to determine SIM in human plasma and in the
pharmaceutical dosage form.

2. Results and Discussion

2.1. Scanning Electrochemical Microscopy (SECM) Analysis

SECM images of three MWCNT-CPEs which differ in mass percentage of MWCNTs are shown in
Figure 1. In SECM, a constant height and a homogeneous surface represent a slight current gradient, while
the surfaces with lower homogeneity show a significant current gradient. The MWCNT-CPE with 25%
MWCNTs shows a relatively homogeneous current which can be attributed to a higher degree of surface
homogeneity. The current gradient shows some degrees of inhomogeneity in the cases of 15% and 35%
MWCNTs, where some peaks or continuous changes in current profile can be observed. Recalling that the
paste is a heterogeneous mixture of three components, it can be concluded that the MWCNT-CPE with
25% MWCNTs is optimal to achieve the highest homogeneity of the mixture. As a matter of experience,
Molecules 2019, 24, x 3 of 14
Molecules 2019, 24, 2215 3 of 14
the highest homogeneity of the mixture. As a matter of experience, MWCNTs adsorb more oil
compared to graphite. Hence, the 15% MWCNT-CPE is more lubricated. On the other hand, the 35%
MWCNTs adsorb more oil compared to graphite. Hence, the 15% MWCNT-CPE is more lubricated.
MWCNT-CPE paste is drier. Thus, the binding of the component is not efficient. The effect of the
On the other hand, the 35% MWCNT-CPE paste is drier. Thus, the binding of the component is not
binder/graphite powder ratio on the carbon paste properties was investigated using different
efficient. The effect of the binder/graphite powder ratio on the carbon paste properties was investigated
characterization methods. It was shown that, with a low amount of organic oil, the binding of the
using different characterization methods. It was shown that, with a low amount of organic oil, the binding
graphite powder is not well established, while a high amount of oil causes increased resistivity of the
of the graphite powder is not well established, while a high amount of oil causes increased resistivity
paste [32]. It is worth noting that each SECM analysis was repeated at least three times for each
of the paste [32]. It is worth noting that each SECM analysis was repeated at least three times for each
prepared electrode, and similar results were obtained.
prepared electrode, and similar results were obtained.

Figure1.1.Scanning
Figure Scanningelectrochemical
electrochemicalmicroscopy
microscopy(SECM) (SECM)images
imagesofofthe
themultiwalled
multiwalledcarbon
carbonnanotube
nanotube
carbon
carbonpaste
pasteelectrodes
electrodes(MWCNT-CPEs):
(MWCNT-CPEs): (A) (A) 15%
15% MWCNT-CPE;
MWCNT-CPE; (B) (B) 25%
25% MWCNT-CPE;
MWCNT-CPE; (C) (C) 35%
35%
MWCNT-CPE.
MWCNT-CPE.Measurement
Measurementconditions:
conditions:Pt Ptultra-microelectrode
ultra-microelectrode(UME)
(UME)withwith10 µmdiameter
10µm diameterand
andRGRG
(the ratio of the insulator radius to the electrode radius) ≥ 10, FcOH 0.2 mM, and KCl 0.1
(the ratio of the insulator radius to the electrode radius) ≥ 10, FcOH 0.2 mM, and KCl 0.1 M; 0.6 V was M; 0.6 V was
applied −1
appliedat atthe
thetip;
tip;the
thescan
scanraterateofofthe
thetip
tiptotothe
thesubstrate
substratewas
was0.5 µm∙s−1. .The
0.5µm·s Thetip
tipapproached
approachedto tothe
the
substrate until i = 1.25
substrate until T𝑖 = 1.25T,∞i .
𝑖 .
,

2.2. SEM Analysis

2.2. SEM Analysis
The SEM images of the three kinds of prepared electrodes are presented in Figure 2. The inhomogeneity
The SEM images of the three kinds of prepared electrodes are presented in Figure 2. The
of the 15% and 35% MWCNT electrodes can be attributed to more defects, which are visible on the electrode
inhomogeneity of the 15% and 35% MWCNT electrodes can be attributed to more defects, which are
surface. The defects are formed due to the ineffective binding of the paste components because of the
visible on the electrode surface. The defects are formed due to the ineffective binding of the paste
 Molecules 2019, 24, x 4 of 14

Molecules 2019, 24, 2215 4 of 14

components because of the unjustified mass ratio of the components. The surface of the 25% MWCNT
electrode shows less defects, which indicates higher homogeneity of the electrode surface.
Based
unjustified masson theofobtained
ratio results, the
the components. The25% MWCNT-CPE
surface was selected
of the 25% MWCNT as the
electrode optimum
shows electrode
less defects,
and
which used for
indicates the further
higher experiments.
homogeneity It was used
of the electrode for the determination of SIM to get insight into its
surface.
electroanalytical properties.

Figure
Figure 2. The2.SEM
The SEM images
images of the of the prepared
prepared MWCNT-CPEs:
MWCNT-CPEs: (A) 15%(A) 15% MWCNT-CPE;
MWCNT-CPE; (B) 25% MWCNT-
(B) 25% MWCNT-CPE;
CPE; (C) 35%
(C) 35% MWCNT-CPE. MWCNT-CPE.

Based
2.3. on the
Atomic obtained
Force results,
Microscopy (AFM)theAnalysis
25% MWCNT-CPE was selected as the optimum electrode
and used for the further experiments. It was used for the determination of SIM to get insight into its
The three-dimensional (3D) AFM images of the developed electrodes are shown in Figure 3. The
electroanalytical properties.
two-dimensional (2D) AFM images are also available in Figure S1. The presence of the MWCNTs on
each prepared
2.3. Atomic electrode
Force Microscopy is apparent.
(AFM) Analysis As seen, the MWCNTs are randomly distributed over the
electrode surface. The bright spots around the MWCNTs are probably attributed to the mineral oil.
The
As three-dimensional
seen in Figure S1, the (3D) AFM images of
35% MWCNT-CPE the developed
seems to be dry aselectrodes
the MWCNTsare shown
are notin Figure 3. by
surrounded
The two-dimensional (2D) AFM images are also available in Figure S1. The presence of
the mineral oil. On the other hand, the MWCNTs in the 15% and 25% MWCNT-CPEs are well mixed the MWCNTs
on each
withprepared electrode
the mineral oil. Theisdryness
apparent. Aselectrode
of the seen, thewith
MWCNTs are randomly
35% MWCNTs stems distributed overcapacity
from the higher the
electrode
of the MWCNTs in the adsorption of the mineral oil. Hence, the higher mass percentageoil.
surface. The bright spots around the MWCNTs are probably attributed to the mineral of the
As seen in Figure S1, the 35% MWCNT-CPE
MWCNTs requires more mineral oil. seems to be dry as the MWCNTs are not surrounded
by the mineral oil. On the other hand, the MWCNTs in the 15% and 25% MWCNT-CPEs are well
Molecules 2019, 24, 2215 5 of 14

mixed with the mineral oil. The dryness of the electrode with 35% MWCNTs stems from the higher
capacity of the MWCNTs in the adsorption of the mineral oil. Hence, the higher mass percentage of
the MWCNTs
Molecules 2019, 24,requires
x more mineral oil. 5 of 14

Figure 3. Atomic
Figure 3. forcemicroscopy
Atomic force microscopy(AFM)
(AFM)images
images
ofof
thethe prepared
prepared MW-CNTCPEs:
MW-CNTCPEs: (A) (A)
15%15% MWCNT-
MWCNT-CPE;
CPE;
(B) (B)MWCNT-CPE;
25% 25% MWCNT-CPE; (C)MWCNT-CPE.
(C) 35% 35% MWCNT-CPE.

2.4. Electrochemical
2.4. Electrochemical Behavior
Behavior of
of SIM
SIM on
on the
the MWCNT-CPEs
MWCNT-CPEs
The CV
The CV of SIM shows
of SIM shows aa distinctive
distinctive irreversible
irreversible oxidation
oxidationtaking
takingplace
placeat
at1.2
1.2VVin
in0.1
0.1M MHH22SO pH
SO44 pH
(Figure 4). The probable oxidation mechanism is also shown in Figure 4. To get more insight
(Figure 4). The probable oxidation mechanism is also shown in Figure 4. To get more insight into the into the
nature of
nature of the
the oxidation
oxidation of
of the
the SIM,
SIM, the
the pH
pH dependence
dependence ofof the
the peak
peak current
current and
and potential
potential was
was studied.
studied.
The changes in the peak potential and peak current are presented in Figure 5. As seen in Figure
5B, the peak potential shifts to less positive potentials upon increasing the pH from 1 to 7. In this pH
range, the pH dependency of the peak potential follows Equation (1). (R = 0.948)
(1)
𝐸 (mV) = −30.693 · pH + 1240.5
Molecules 2019, 24, x 6 of 14
Molecules 2019, 24, 2215 6 of 14

TheofCV of MWCNT-CPEs −4
of 5of× 510× 10
Figure Figure
4. The4.CV MWCNT-CPEs inin0.1
0.1MMHH22SO
SO44 at
atand
andthe CVCV
the M−4simvastatin (SIM) (SIM)
M simvastatin in in
0.1 M H2 SO4 with MWCNTs, at a potential scan rate of 50 mV·s−1−1 . The inset shows the electrochemical
0.1 M H2SO4 with MWCNTs, at a potential scan rate of 50 mV∙s . The inset shows the electrochemical
oxidation mechanism of SIM.
oxidation mechanism of SIM.
The changes in the peak potential and peak current are presented in Figure 5. As seen in Figure 5B,
However, in the pH
the peak potential range
shifts to lessof 7 to 9,potentials
positive the peakuponpotential increases
increasing the pH from with1 to
the7. pH. The
In this pHslope
range, of the
the pH dependency
pH dependency of thepotential
of the peak peak potential
(pH follows Equation
= 1 to 7) is close(1). the= theoretical
to (R 0.948) value corresponding to
the mechanism in which two protons and an electron are involved. However, the redox mechanism
Ep (mV) = −30.693·pH + 1240.5 (1)
reported for the oxidation of SIM is a reaction involving two electrons and two protons. The abnormal
variation ofHowever,
the peakinpotential withofpH,
the pH range 7 towhich was also
9, the peak previously
potential increasesobserved [21,23],
with the pH. mightofarise
The slope the from
pH dependency of the peak potential (pH = 1 to 7) is close to the theoretical
the coexistence of SIM and its hydrolyzed product, since SIM might be hydrolyzed into simvastatin value corresponding to
the mechanism in
acid (β,δ-dihydroxyl which[23,33].
acid). two protons and an electron are involved. However, the redox mechanism
reported for the oxidation of SIM is a reaction involving two electrons and two protons. The abnormal
The dependence of the oxidation peak current on the potential scan rate was also studied. The
variation of the peak potential with pH, which was also previously observed [21,23], might arise from
results are shown in Figure 6A,B. The oxidation peak shifts to a more positive potential upon
the coexistence of SIM and its hydrolyzed product, since SIM might be hydrolyzed into simvastatin
increasing the scan rate, which
acid (β,δ-dihydroxyl confirms the irreversibility of the oxidation of SIM. The dependence
acid). [23,33].
of the peakThe
current on the of
dependence potential scan rate
the oxidation peakincurrent
logarithmic
on the coordinates
potential scanisrate
shown in Figure
was also studied.6C. The
dependency follows
The results Equation
are shown = 0.988).
(2) (𝑅6A,B.
in Figure The oxidation peak shifts to a more positive potential upon
increasing the scan rate, which confirms the irreversibility of the oxidation of SIM. The dependence (2)
of the peak current on the log𝐼(µA)
potential = scan
0.8007rate· log𝜈 (mVs ) −
in logarithmic 0.7908. is shown in Figure 6C.
coordinates
Furthermore,
The dependency thefollows
dependency
Equation of(2)
the(Rpeak current on the square root of the scan rate (Figure 6D)
= 0.988).
is described by Equation (3) (𝑅 = 0.977).  
log I (µA) = 0.8007· log ν mVs−1 − 0.7908. (2) (3)
𝐼(µA) = 1.0837 · 𝜈 . (mVs ) − 3.0866.
Furthermore,
Since the slope ofthe dependency
Equation (2) ofis the peak current
between on the squarevalues
the theoretical root of of
the0.5
scanand
rate 1.0,
(Figure 6D) isare the
which
described
theoretical valuesby of
Equation (3) (R = 0.977).
the diffusion- and adsorption-controlled mechanisms, respectively [34], it can be
concluded that a combination of the two mechanisms  is responsible for the mass transport to the
I (µA) = 1.0837·ν0.5 mVs−1 − 3.0866. (3)
electrode. However, since the slope is close to 1.0, the dominant mechanism is adsorption.
Furthermore, thethe
Since poor linearity
slope of Equation
of Equation (3) confirms
(2) is between that diffusion
the theoretical values of is0.5not
andthe
1.0,main
whichmechanism
are the of
theoretical
the mass transport.values of the diffusion- and adsorption-controlled mechanisms, respectively [34], it can
be concluded that a combination of the two mechanisms is responsible for the mass transport to
 Molecules 2019, 24, 2215 7 of 14

the electrode. However, since the slope is close to 1.0, the dominant mechanism is adsorption.
Furthermore, the poor linearity of Equation (3) confirms that diffusion is not the main mechanism of
the mass transport.
The mechanism of the oxidation of SIM on the developed electrode was also investigated by
performing 10 repetitive CVs of SIM in 0.1 M H2 SO4 (Figure S2). The first cycle shows an oxidation
peak at 1.10 V. Upon increasing the number of cycles, the peak current decreases and the peak potential
shifts to more positive potentials. The shift of the oxidation peak to more positive potentials is due to
the adsorption of SIM onto the electrode surface, which changes the physicochemical properties of the
electrode surface, including its conductivity and catalytic capability.
The developed electrode was used to drive the Tafel plot in a 5 × 10−4 M solution of SIM at
a potential scan rate of 50 mV·s−1 . The Tafel equation (Equation (4)) [35] was used to determine the
transfer coefficient and the exchange current.

(1 − α)nF
log I = logI0 η, (4)
2.30RT
where I0 is the exchange current, I is the current, η is the overvoltage, and n is the number of transferred
electrons.
Molecules 2019, 24, The
x transfer coefficient α was calculated to be 0.07 for this two-electron transfer process.7 of 14
The exchange current was also estimated from the intercept to be 1.8 × 10−9 A.

−4
Figure
Figure (A) Forward
5. Forward
5. (A) scanscan of CVs
of CVs ofof5 5× ×1010
−4 MM SIMinin0.1
SIM 0.1M
MH H22SO
SO44 at
at various
variouspH
pHvalues,
values,with
with a
a potential scan rate 50 mV·s −1 ; (B) pH dependence of the peak potential; (C) pH dependence of the
potential scan rate 50 mV∙s ; (B) pH dependence of the peak potential; (C) pH dependence of the
−1

peak peak current.

current.

The mechanism of the oxidation of SIM on the developed electrode was also investigated by
performing 10 repetitive CVs of SIM in 0.1 M H2SO4 (Figure S2). The first cycle shows an oxidation
peak at 1.10 V. Upon increasing the number of cycles, the peak current decreases and the peak
potential shifts to more positive potentials. The shift of the oxidation peak to more positive potentials
is due to the adsorption of SIM onto the electrode surface, which changes the physicochemical
Molecules 2019, 24, x 8 of 14
Molecules 2019, 24, x 8 of 14
Molecules 2019, 24, 2215 8 of 14

Figure 6. (A) CV of 5 × 10−4 M SIM in 0.1 M H2SO4 at 100 mV∙s−1 ; (B) the forward scan of CVs at various
(A)CV
CV 5× × −4 M SIM in 0.1 M H SO at 100 −1mV·s−1 ; (B) the forward scan of CVs at
Figure
potential
Figure 6. (A)
6. scan ofof5 (10,
rates 1010
20,M50,
−4 SIM 100, 200,
in 0.1 M300,
H2SO and
42at 500
4 mV∙s ); (B)
100 (only
theaforward
potential range
scan related
of CVs to the
at various
various −1 ) (only a potential range related to
peak is potential
potential shown toscan
scan rates see rates
(10,the (10,
50, 20,
20,peaks 50,200,
well);
100, 100,
(C) 200,
scan
300, 300,
and anddependence
rate
500 500 mV·s
mV∙s −1) (onlyofa the oxidation
potential peak
range current
related in
to the
the
peak peak
logarithmic is shown
is shown to see
coordinates;
to see the
the(D) peaks
dependence
peaks well);
well); of(C)
(C) the
scanscanraterate
oxidation dependence
peak current
dependence ofonthe
of the theoxidation
squarepeak
oxidation peak
root of current
the
currentscan
in
in logarithmic
rate.
logarithmic coordinates;
coordinates; (D)(D) dependence
dependence of the
of the oxidation
oxidation peakpeak current
current onsquare
on the the square
root root
of theofscan
the
scan
rate. rate.
2.5. Analytical Parameters and Validation of the Method
2.5. Analytical Parameters and Validation of the Method
2.5. Analytical Parameters and Validation of the Method
Figure 7 shows the differential pulse voltammogram (DPV) of SIM on three kinds of electrodes.
Figure 7 shows the differential pulse voltammogram (DPV) of SIM on three kinds of electrodes.
As seen, the7SIM
Figure showsoxidation peak possesses
the differential the lowest baseline
pulse voltammogram (DPV) current
of SIMinon
thethree
casekinds
of CPE. Compared
of electrodes.
As seen, the SIM oxidation peak possesses the lowest baseline current in the case of CPE. Compared to
to CPE
As seen,and
the GC,
SIM the developed
oxidation peakelectrode
possessesshows a higher
the lowest current
baseline attitude
current response,
in the which
case of CPE. is due to
Compared
CPE and GC, the developed electrode shows a higher current attitude response, which is due to
theCPE
to synergetic
and GC, effect of the MWCNTs
the developed incorporated
electrode in thecurrent
shows a higher carbon attitude
paste. Probably
response,due to the
which higher
is due to
the synergetic effect of the MWCNTs incorporated in the carbon paste. Probably due to the higher
hydrophobicity
the of theofcarbon
synergetic effect paste, theincorporated
the MWCNTs adsorption of
in the
the SIM ontopaste.
carbon its surface takes
Probably place,
due while
to the the
higher
hydrophobicity of the carbon paste, the adsorption of the SIM onto its surface takes place, while
presence of the MWCNTs
hydrophobicity of the carbonincreases
paste, the adsorption
conductivityofofthe
the electrode
SIM onto itsand increases
surface takesthe peak
place, current
while the
the presence of the MWCNTs increases the conductivity of the electrode and increases the peak
attitude. of the MWCNTs increases the conductivity of the electrode and increases the peak current
presence
current attitude.
attitude.

× 10 −5
Figure 7.7.Differential
Figure Differentialpulse voltammogram
pulse voltammogram(DPV) of 2.5of
(DPV) 2.5 ×M10
SIM
−5 Min 0.1
SIM M in
H20.1
SO4Mat various
H2SO4 at electrodes;
various
step potential:
electrodes;
Figure 5 mV; modulation
step potential:
7. Differential pulse5 mV; amplitude: 100 mV;
modulation(DPV)
voltammogram modulation
amplitude:
of 2.5100 time: 0.04
× 10mV;Mmodulation
−5 s; interval
SIM in 0.1 time: time:
M H2SO 0.1
0.044 at s.
s; interval
various
time: 0.1 s. step potential: 5 mV; modulation amplitude: 100 mV; modulation time: 0.04 s; interval
electrodes;
Moreover, the oxidation peak shifts to the less positive potential at the developed electrode due to
time: 0.1 s.
the catalytic effect of the MWCNTs. Thus, the developed electrode was used to study the concentration
Molecules 2019, 24, x 9 of 14
Molecules2019,
Molecules 2019,24,
24,2215
x 99ofof14
14
Moreover, the oxidation peak shifts to the less positive potential at the developed electrode due
to theMoreover,
catalytic the oxidation
effect of the peak shifts toThus,
MWCNTs. the less
thepositive potential
developed at the was
electrode developed
used toelectrode due
study the
dependence of
to the catalytic
concentration the peak current.
effect of the
dependence As
MWCNTs.
of the seen in Figure
Thus,As
peak current. 8A, the
theseen peak
developed current
in Figureelectrodeincreases
8A, the was linearly
peakused with
to study
current the
the
increases
concentration
concentration
linearly ofdependence
with thethe SIM in solution.
concentrationof the The
peak
of the concentration
current.
SIM dependence
As seen
in solution. Figureof8A,
the the
in concentration
The peak current
peak (Figure
current
dependence 8B) is
increases
of the peak
defined
current by Equation
linearly(Figure
with the is(5) (R = 0.998).
8B)concentration
defined of the SIM
by Equation = 0.998).The concentration dependence of the peak
(5)in(𝑅solution.
current (Figure 8B) is defined by Equation (5) (𝑅 = 0.998). (5)
𝐼(µA)
I (µA)==0.1206
0.1206·C· 𝐶((µ𝑀)
µM) −−0.0531.
0.0531. (5)
(5)
𝐼(µA) = 0.1206 · 𝐶 (µ𝑀) − 0.0531.

Figure 8. (A) DPV of SIM recorded at different concentrations (0, 3.75, 7.5, 11.25, 15, 17.5, and 20 µM);
Figure
Figure
(B) 8.8.(A)
(A)DPV of
ofSIM
SIMrecorded
DPVdependence
concentration of theat
recorded different
atSIM
differentconcentrations
concentrations
oxidation (0,
(0,3.75,
peak current. 3.75,7.5,
7.5,11.25,
11.25,15,
15,17.5,
17.5,and
and20
20µM);
µM);
(B) concentration dependence of the SIM oxidation peak current.
(B) concentration dependence of the SIM oxidation peak current.
2.6. Determination
2.6. Determination of of SIM
SIM inin Pharmaceutical Dosage Form
Pharmaceutical Dosage Form andand Its
Its Assay
Assay in in Spiked
Spiked Plasma
Plasma
2.6. Determination of SIM in Pharmaceutical Dosage Form and Its Assay in Spiked Plasma
The possibility
The possibility of of the
the application
application of of the
the developed
developed methodmethod for for the
the direct
direct determination
determination of of SIM
SIM
content Theinpossibility
a commercial of the application
tablet was of the developed
investigated using method
the for the
standard direct method
addition determination
without of any
SIM
content in a commercial tablet was investigated using the standard addition method without any
content in
filtration or a
orothercommercial tablet
otherpre-treatments.
pre-treatments. was investigated
The results using
are shown the standard
in Figure addition
9 and1.Tablemethod without any
filtration The results are shown in Figure 9 and Table Based1.onBased on the
the obtained
filtrationresults,
obtained or other thepre-treatments.
determination The
SIMresults are shown in can
Figure 9 and out
Table 1.theBased on the
results, the determination of SIM in ofdrug in drugform
dosage dosagecanform
be carried beoutcarried
with the with
developed developed
method
obtainedwith
method results,
good theaccuracy
determination of recovery
(relative SIM in drug ratedosage
= 99.7%)form andcanreproducibility
be carried out with the developed
(relative standard
with good accuracy (relative recovery rate = 99.7%) and reproducibility (relative standard deviation
method with
deviation good= 1.8).
accuracy (relative recovery ratein= human
99.7%) and reproducibility (relative standard
(RSD%) = (RSD%)
1.8). The The determination
determination of SIM inofhuman
SIM plasma, plasma,
which iswhicha more is complicated
a more complicated
matrix,
deviation (RSD%) = 1.8). The determination of SIMwithin human plasma, which is ofa more complicated
was also performed. The plasma was spiked with a known concentration of SIM and then thethen
matrix, was also performed. The plasma was spiked a known concentration SIM and spikingthe
matrix, recovery
spiking was also rate performed.
was The plasma
determined wasthe
using spiked withaddition
standard a knownmethod.
concentration
The of SIM
results areand then the
presented
recovery rate was determined using the standard addition method. The results are presented in Table 1.
spiking
in Table recovery
1. The naturerate was determined
satisfactory using theanalysis
standardinaddition method. The resultsfrom
are presented
The satisfactory of thenature of the
SIM analysis SIM
in plasma can beplasma
confirmed can from
be confirmed the high
the high reproducibility
in Table 1. The(RSD% satisfactory nature of accuracy
the SIM analysis in plasma can be confirmed from the high
(RSD% = 2.3) and high accuracy of the measurement (spiking recovery rate = 99.5%) (Table 1). It=must
reproducibility = 2.3) and high of the measurement (spiking recovery rate 99.5%)be
reproducibility
(Table 1). It must (RSD%
be = 2.3)
mentioned and high
that theaccuracy of
concentration the measurement
of the SIM in (spiking
plasma isrecovery
very low rate =
(1–15 99.5%)
nM).
mentioned that the concentration of the SIM in plasma is very low (1–15 nM). However, the developed
(Table 1). the
However, It must be mentioned that
can the concentration of the SIM inofplasma is very low (1–15 nM).
method can bedeveloped
used for the method
determinationbe used for the
of SIM determination
concentration in plasma SIMatconcentration in plasma
levels attributed to the
However, the developed method can be used for the determination of SIM concentration in plasma
at levels attributed
pleiotropic to the
effects (1–50 µM) [8–11]. effects (1–50 µM) [8–11].
pleiotropic
at levels attributed to the pleiotropic effects (1–50 µM) [8–11].

Figure 9. (A) DPVs of solution of 7.5 µMµM SIM prepared in tablet form and three standard additions of
5Figure
µM, 7.5
9. µM,
(A) and
DPVs 10
ofµM; (B)
solution standard
of
µM, 7.5 µM, and 10 µM; (B) standard7.5 µMaddition curve
SIM prepared
addition curve in which
intablet the
the extrapolations
whichform showadditions
and three standard
extrapolations the initialof
concentration of SIM in the measuring
5 µM, 7.5 µM, and 10 µM; measuring
(B) standardsolution.
addition curve in which the extrapolations show the initial
solution.
concentration of SIM in the measuring solution.
Molecules 2019, 24, 2215 10 of 14

Table 1. The analytical parameters obtained for the calibration of standards, drug dosage form and
plasma sample.

Calibration with
Drug Dosage Form Spiked Plasma
Standards
Peak potential (V) 1.10 Potential 1.10 Potential 1.10
Linearity range (µM) 3.75–20 Slope 0.1183 Slope 0.154
Slope (µA·µM−1 ) 0.121 ± 0.005 Intercept 0.8854 Intercept 1.532
Intercept 0.041 Correlation coefficient r 0.996 Correlation coefficient r 0.999
Correlation coefficient (r) 0.998 ± 0.004 Labelled claim (mg) 10 Spiked (µM) 10
LOD 1 (µM) 2.4 × 10−7 Found (mg) 9.97 Found (µM) 9.95
LOQ (µM) 8.0 × 10−7 Recovery rate % 99.7 Bias % 0.5
RSD% (n = 8) 4.8 Bias % 0.3 Spiking Recovery rate% 99.5
Confidence interval 2 ±1.0 × 10−8 RSD% (n = 3) 1.8 RSD% (n = 3) 2.3
1 Obtained with 3σ/m (σ: standard deviation, m: slope of the calibration curve); 2 confidence interval of 95% = √ ts
n
(s: standard deviation, t: critical value for eight repetitions: 2.31, n = 8 repetitions). RSD%—relative standard deviation.

2.7. Interference Study

The oxidation peak of 5 × 10−6 M SIM was recorded in the presence of potential interferents
such as ascorbic acid and citric acid in 100-fold higher concentration. It was found that the peak current
and potential were not affected due to the presence of these chemicals (Figure S3).

3. Materials and Methods

3.1. Chemicals
Simvastatin was kindly provided by Prof. Sibel Ozcan (Faculty of Pharmacy, Ankara University,
Department of Analytical Chemistry), and its pharmaceutical dosage form (Simgal) was provided
by TEVA Czech. Ind. (Opava, Czech Republic). All chemicals for the preparation of buffers and
supporting electrolytes were reagent-grade (Sigma-Aldrich, St. Louis, MO, USA). A stock solution of
SIM (10−2 M) was prepared in ethanol, and the required concentration of SIM was prepared by dilution.
The final percentage of ethanol was kept at 10% in all solutions. Solid MWCNTs (purified to more than
95% carbon with an average diameter of 10 nm and an average length of 1.5 µm) were purchased from
Sigma. Expanded graphite for preparation of the CPE was purchased from Graphite Tyn Ltd. (Tyn nad
Vltavou, Czech Republic). The paraffin oil was also purchased from Sigma. Ferrocene methanol
(FcOH), which was used as the mediator for the SECM imaging, was purchased from ABCR GmbH
chemicals (Karlsruhe, Germany).

3.2. Apparatus
Voltammetric measurements were carried out using a potentiostate/galvanostate AUTOLAB
PGSTAT 302 controlled by Nova software (EcoChemie, Utrecht, the Netherlands). The conventional
three-electrode configuration for the electrochemical experiments was used. The constructed CPEs
and MWCNT-CPEs were used as the working electrodes, while an Ag/AgCl (3 M KCl) electrode and
a platinum wire served as the reference and auxiliary electrodes, respectively. The measurements
were carried out in one-compartment voltammetric cells (10–20 mL) at conditioned room temperature
(23 ± 1 C). The pH measurements were performed using a pH meter Model Sentix 81 (WTW, Weilheim,
Germany) with a combined electrode (glass electrode = Ag/AgCl (3 M KCl) reference electrode) with
an accuracy of pH ±0.05.
A CHI 900 set-up (CH Instrument Inc., Bee Cave, TX, USA) was used for the scanning electrochemical
microscopy (SECM) measurements. A Pt ultra-microelectrode (d = 10 µm) served as the SECM tip
(RG factor = 10), while MWCNT-CPEs prepared with different MWCNT mass ratios were used as SECM
substrates. The counter electrode was a platinum wire, and the reference electrode was an Ag/AgCl
(3 M KCl) electrode. FcOH 1 mM in 0.1 M KCl was used as a redox mediator. All potentials are
referred to the reference electrode. The scanning electron microscope MIRA2 LMU (Tescan, a.s., Brno,
Molecules 2019, 24, 2215 11 of 14

Czech Republic) was also used for imaging the prepared MWCN-CPE electrodes. An accelerating voltage
of 15 kV was applied to produce an electron beam of about 1 nA to visualize the electrode surface at
a working distance of 4 mm. Atomic force microscopy (AFM) for the electrode surface characterization
was carried out with a dimension FastScan Bio from Bruker (Billerica, MA, USA) operating with Gwydion
2.52 for data visualization [36].

3.3. Preparation of the Working Electrode

The MWCNT-modified carbon paste electrodes (MWCNT-CPEs) were prepared by intimately
hand-mixing graphite powder and MWCNTs with highly viscous paraffin oil in three different ratios
(a: 55%/15%/30%, b: 45%/25%/30%, and c: 35%/35%/30% graphite powder, MWCNTs, and paraffin oil,
respectively). All the components were homogenized to obtain a mixture that was subsequently packed
into a piston-driven carbon paste holder (with an internal diameter of 2 mm). Whenever needed,
the surface of the MWCNT-CPEs was mechanically renewed by extruding ca. 0.5 mm of the paste out of
the electrode holder and smoothing with a wet filter paper. To find the optimum combination, various
mass percentages of MWCNTs in the paste were utilized and examined.

3.4. Pharmaceutical Dosage Form Assay Procedure

Ten tablets of Simgal (each tablet contains 10 mg of SIM) were weighed and finely powdered.
An amount of pharmaceutical powder equivalent to 10−2 M SIM (10 mL) was dissolved in ethanol.
The complete dissolution was ensured by 10 min of sonication. The desired concentration of SIM
solution was then prepared by taking the aliquots of the clear supernatant liquor and diluting with the
supporting electrolyte. For pharmaceutical dosage form analysis, the standard addition method was
carried out, in which a known concentration of SIM solution prepared using the tablet was subjected
to three additions of small volumes of stock solution of SIM.

3.5. Recovery Studies in Spiked Human Plasma Samples

The human plasma samples, which were obtained from healthy individuals, were stored frozen
until the assay. To prepare 2 mL of 10−3 M SIM in plasma, after gentle melting at room temperature,
the aliquots of plasma were spiked with the required amount of SIM stock solution and treated with
700 µL of acetonitrile as a plasma protein precipitating agent; then, the same plasma sample was
added to reach 2 mL. After that, the sample was subjected to 5 min of agitation and then 10 min of
centrifugation at 16,500 RCF in order to precipitate the protein residues. The desired concentrations of
the plasma samples were prepared by taking the supernatants and mixing them with the required
amount of the supporting electrolyte. It must be mentioned that the ethanol percentages in all the
analyzed samples were the same.

3.6. Procedure
DPV was used for electroanalytical determination of SIM with the following parameters: step
potential: 5 mV; modulation amplitude: 100 mV; modulation time: 0.04 s; interval time: 0.1 s.
To record the approach curve, a potential of 600 mV was applied to the tip where the oxidation
of FcOH to FcOHox takes place. At the conductive surface, the generated FcOHox is reduced to the
parent FcOH, which is transferred to the tip and increases the tip current. On the contrary, where the
substrate surface insulates the process, the diffusion of the FcOH from the bulk solution to the tip is
limited, which decreases the tip current.
The probe was then approached to the substrate at a tip scan rate of 0.5 µm·s−1 . When the tip is
far from the substrate and the potential is applied, FcOHox is generated and the steady-state current
iT,∞ is obtained, which is controlled by the rate of mass transfer by diffusion of FcOH from the bulk
solution to the electrode surface.
The current iT increases upon approaching the tip to the substrate because of positive feedback.
The approach was interrupted when iT = 1.25 iT,∞ . At this magnitude of current, a distance of 10 µm
Molecules 2019, 24, 2215 12 of 14

between the tip and the substrate is expected based on the theoretical curve of distance (d) dependency
of iT for the utilized ultra-microelectrode (UME) [37]. The constant height mode was used to record
the images at a tip scan rate of 1 µm·s−1 and an area of 100 µm × 100 µm of the samples.

4. Conclusions
The bulk-modified MWCNT–CPEs were prepared and characterized to find the optimum amount
of MWCNTs in the paste. The characterization was performed using SECM, SEM, and AFM. Based on
the obtained results, the 25% MWCNT–CPE was the optimum electrode in terms of the homogeneity of
the prepared paste. Then, the 25% MWCNT–CPE was used for the analysis of SIM in pharmaceutical
dosage form, as well as in spiked human plasma. The results confirmed the satisfactory nature
of the developed method considering the calculated parameters including bias, relative standard
deviation (RSD%), and the recovery rate, as reported in Table 1. The low limit of detection and limit of
quantification, in addition to the slope of the calibration curve, proved the sensitivity of the prepared
electrode. The advantages of the developed method compared to other electroanalytical methods
include simple preparation of the electrode and renewal of the electrode surface, whereas any toxic
compounds such as mercury or other heavy metals were not used, and its sensitivity is higher compared
to the bare GC and CPE. The developed method offers a simple screening approach for preliminary
monitoring. Furthermore, the developed method can be used for rapid but accurate determination of
SIM in pharmaceutical dosage form and in plasma samples when its concentration corresponds to the
pleiotropic effects.

Supplementary Materials: The following are available online: Figures S1–S3.

Author Contributions: A.M.A. conceived and designed the experiments, analyzed the data, and wrote the paper;
L.R. was the lab manager and supervised the writing of the manuscript.
Funding: This research was funded by the Czech Ministry of Education in the framework of the National
Sustainability Program, grant number LO1401 INWITE. For the research, the infrastructure of the SIX Center
was used.
Acknowledgments: We thank Tereza Sojková for her technical assistance. We also acknowledge Sibel Ozcan for
kindly providing the SIM and MU L. S. for providing Simgal. Furthermore, Jan Pribyl is acknowledged because of
providing AFM images.
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Not available.

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