Nanomolar and selective determination of epinephrine in the presence of norepinephrine using carbon paste electrode modified with carbon nanotubes and novel 2-(4-oxo-3-phenyl-3,4-dihydroquinazolinyl)-n′-phenyl- hydrazinecarbothioamide

Article abstract of DOI:10.1021/ac801854j

A novel modified carbon nanotube paste electrode of 2-(4-oxo-3-phenyl-3,4- dihydro-quinazolinyl)-N′-phenyl-hydrazinecarbothioamide (2PHC) was fabricated, and the electrooxidation of epinephrine (EP), norepinephrine (NE), and their mixture has been studied using electrochemical methods. The modified electrode displayed strong catalytic function for the oxidation of EP and NE and resolved the overlap voltammetric response of EP and NE into two well-defined voltammetric peaks of about 240 mV with square wave voltammetry (SWV). A linear response in the range of (5 × 10^-8)-(5.5 × 10 ^-4) M with a detection limit (S/N = 3) of 9.4 nM for EP was obtained.

Full text of DOI:10.1021/ac801854j

Anal. Chem. 2008, 80, 9848–9851
Nanomolar and Selective Determination of
Epinephrine in the Presence of Norepinephrine
Using Carbon Paste Electrode Modified with Carbon
Nanotubes and Novel 2-(4-Oxo-3-phenyl-3,4-dihydro-
quinazolinyl)-N′-phenyl-hydrazinecarbothioamide
Hadi Beitollahi,*,† Hassan Karimi-Maleh,‡ and Hojatollah Khabazzadeh§
Department of Chemistry, Faculty of Science, Yazd University, Yazd, Iran, College of Chemistry, Isfahan University of
Technology, Isfahan, Iran, and Department of Chemistry, Shahid Bahonar University of Kerman, Kerman, Iran
A novel modified carbon nanotube paste electrode of 2-(4-
oxo-3-phenyl-3,4-dihydro-quinazolinyl)-N′-phenyl-hydra-
zinecarbothioamide (2PHC) was fabricated, and the electro-
oxidation of epinephrine (EP), norepinephrine (NE), and
their mixture has been studied using electrochemical
methods. The modified electrode displayed strong cata-
lytic function for the oxidation of EP and NE and resolved
the overlap voltammetric response of EP and NE into two
well-defined voltammetric peaks of about 240 mV with
square wave voltammetry (SWV). A linear response in the
range of (5 × 10-8)-(5.5 × 10-4) M with a detection limit
(S/N ) 3) of 9.4 nM for EP was obtained.
Also, NE is one of the derivatives of cathecholamines secreted
in the adrenal medulla and plays important physiological roles in
the central nervous system. It affects muscle and tissue control,
stimulates arteriole contraction, decreases peripheral circulation,
and activates lipolysis in adipose tissue.8 It is also critical in mental
disease, heart failure, DNA breaks in cardiac myoblast cells, and
diabetes. Recent reports have indicated that NE enhances adhe-
sion of human immunodeficiency virus-1 (HIV-1)-infected leuko-
cytes to cardiac microvascular endothelial cells and also acceler-
atesHIVreplicationviaproteinkinase.9Therefore,thedeterminations
of EP and NE have attracted much attention of researchers.
In recent years, many methods have been reported for the
determination of EP and NE, such as high-performance liquid
chromatography (HPLC), fluorometry, chemiluminescence, and
spectrophotometry.10-13 However, most of these methods are
complicated because they need derivatization or combination with
various detection methods. Also, some of them suffer from low
sensitivity and low specificity. Because of the simple procedure
and high sensitivity of electroanalysis and the electroactivity of
EP and NE, studying and measuring of these two compounds with
electrochemical methods were carried out.14-17
Carbon nanotubes are a kind of porous nanostructure material
with properties such as high electrical conductivity and high
mechanical strength.1,2 The subtle electronic properties of carbon
nanotubes suggests that they have the ability to promote electron
transfer reaction when used as the electrode material in electro-
chemical reaction, which provides a new way in the electrode
surface modification for designing new electrochemical sensors.3-5
EP, often called adrenaline, is an important catecholamine
neurotransmitter in the mammalian central nervous system. Many
life phenomena are related to the concentration of EP in blood.6
Medically, EP has been used as a common emergency healthcare
medicine.5 Also, low levels of EP have been found in patients with
Parkinson’s disease.7
However, a major problem is that at bare electrodes, the anodic
peak potentials for EP and NE are almost the same, which results
in overlapped current responses, and makes their discrimination
very difficult. There are some promising strategies for the selective
determination of EP and NE, such as using fast scan cyclic
* To whom correspondence should be addressed. E-mail: h.beitollahi@
yahoo.com. Phone: 00983518211670. Fax: 00983518210644.
† Yazd University.
(8) Voet, D.; Voet, J. G. Biochemistry, 2nd ed.; Wiley: New York, 1995.
(9) Cole, S. W.; Korin, Y. D.; Fahey, J. L.; Zack, J. A. J. Immunol. 1998, 161,
‡ Isfahan University of Technology.
610–616
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§ Shahid Bahonar University of Kerman.
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9848 Analytical Chemistry, Vol. 80, No. 24, December 15, 2008
10.1021/ac801854j CCC: $40.75 2008 American Chemical Society
Published on Web 11/19/2008

Figure 2. Cyclic voltammograms of (a) CPE in 0.1 M PBS (pH 7.0),
(b) CPE in 0.5 mM EP, (c) 2PHCMCNPE in 0.1 M PBS, (d) CNPE in
0.5 mM EP, (e) 2PHCMCPE in 0.5 mM EP, and (f) 2PHCMCNPE in
0.5 mM EP. In all cases the scan rate was 30 mV s-1
.
EXPERIMENTAL SECTION
Apparatus and Chemicals. All electrochemical measure-
ments were performed using an Autolab PGSTAT12 potentiostat/
galvanostat (Eco Chemie, Utrecht, The Netherlands). An Ag/
AgCl/KCl 3 M, a platinum wire, and a 2PHCMCNPE were used
as the reference, auxiliary, and working electrodes, respectively.
A digital pH/mV meter (Metrohm model 710) was applied for
pH measurements. Epinephrine, norepinephrine, graphite fine
powder, paraffin oil, and reagents were analytical grade from
Merck. Multiwalled carbon nanotubes (purity more than 95%) with
o.d. between 10 and 20 nm, i.d. between 5 and 10 nm, and tube
length from 0.5 to 200 nm were prepared from Nanostructured
& Amorphous Materials, Inc. The buffer solutions were prepared
from orthophosphoric acid and its salts in the pH range 2.0-11.0.
Synthesis of 2PHC. Anthranilic acid (10 mmol) was dissolved
in 50 mL of acetic acid, and 10 mmol of phenyl isothiocyanate
was added. The mixture was refluxed for 3 h. After cooling, the
resulted precipitate was filtered and recrystalized from ethanol
to give pure 2-mercapto-3-phenyl-3H-quinazolin-4-one (MPQ).
To a solution of 10 mmol of (MPQ) in 50 mL of n-butanol, 20
mL of hydrazine monohydrate was added. The solution was
refluxed for 18 h. Then the reaction mixture was cooled and
filtered off. The resulting precipitate recrystalized from ethanol
to give pure 2-hydrazino-3-phenyl-3H-quinazolin-4-one (HPQ).
A mixture of 10 mmol of (HPQ) and 10 mmol of phenyl
isothiocyanate in 40 mL of DMF was heated at 60 °C for 1 h.
After completion of the reaction, 30 mL of water was added. The
resulting precipitate was recrystalized from acetic acid after
filtration to give 2-(4-oxo-3-phenyl-3,4-dihydro-quinazolinyl)-N′-
phenyl-hydrazinecarbothioamide (2PHC) (Figure 1A).
Figure 1. (A) Structure of 2PHC. (B) The cyclic voltammograms of
(a) 2PHCMCNPE and (b) bare CPE in 0.1 M PBS (pH 7.0) at a scan
rate of 100 mV s-1. (C) The plot of anodic (a) and cathodic (b) peak
currents of 2PHCMCNPE vs v from the cyclic voltammograms of
2PHCMCNPE in 0.1 M PBS (pH 7.0) at various scan rates (data
points from left to right): 20, 50, 100, 200, 300, 400, 500, 600, 700,
800, and 900 mV s-1
.
voltammetry (FSCV)18,19 carbon fiber microelectrodes,20,21 and
chemically modified electrodes (CMEs).15,22
To our knowledge, most of the published electrochemical
studies have utilized glassy carbon electrodes or other kinds of
electrodes15,18-23 for simultaneous determination of EP and NE,
and no study has reported the simultaneous electrocatalytic deter-
mination of EP and NE by using carbon nanotube paste electrodes.
Also, no paper has reported quinazoline or its derivatives as catalyst
for electrocatalysis and specially for electrocatalysis of EP and NE.
Thus, in this paper, we described initially the preparation and
suitability of a 2-(4-oxo-3-phenyl-3,4-dihydro-quinazolinyl)-N′-phenyl-
hydrazinecarbothioamide modified carbon nanotube paste electrode
(2PHCMCNPE) as a new electrocatalyst in the electrocatalysis and
determination of EP in an aqueous buffer solution, then we evaluated
the analytical performance of the modified electrode in quantification
of EP in the presence of NE.
(18) Ciolkowski, E. L.; Cooper, B. R.; Jankowski, J. A.; Jorgenson, J. W.;
Wightman, R. M. J. Am. Chem. Soc. 1992, 114, 2815–2821
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4537
(20) Herna´ndez, P.; Sa´nchez, I.; Pato´n, F.; Herna´ndez, L. Talanta 1998, 46,
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(21) Yuanxi, S.; Baoxian, Y.; Yu, W.; Xiaorong, T.; Xingyao, Z. Microchem. J.
1998, 58, 182–191
(22) Chen, S. M.; Peng, K. T. J. Electroanal. Chem. 2003, 547, 179–189
(23) Michael, D. J.; Wightman, R. M. J. Pharm. Biomed. Anal. 1999, 19, 33–
46
.
.
Preparation of the Electrode. Modified carbon nanotube
paste electrodes were prepared by dissolve 0.01 g of 2PHC in
CH3Cl and hand mixing with 89 times its weight of graphite
powder and 10 times its weight of carbon nanotube with a mortar
and pestle. The solvent was evaporated by stirring. A 70:30 (w/
w) mixture of 2PHC spiked carbon nanotube powder and paraffin
.
.
.
.
Analytical Chemistry, Vol. 80, No. 24, December 15, 2008 9849

oil was blended by hand mixing for 20 min until a uniformly wetted
paste was obtained. The paste was then packed into the end of a
Teflon rod with a hole (2 mm diameter, 5 mm deep) bored at one
end for the electrode filling. Contact was made with a platinum
wire through the center of the rod which screwed to the rotating
disk electrode (RDE) device.
RESULTS AND DISCUSSION
Electrochemistry of 2PHCMCNPE. According to our knowl-
edge, there is no report on the study of the electrochemical
properties and especially the electrocatalytic activity of 2PHCM-
CNPE in aqueous media. Because this compound is insoluble in
aqueous media, we prepared 2PHCMCNPE and studied the
electrochemical behavior of 2PHCMCNPE in pure buffered
aqueous solution by cyclic voltammetry. Figure 1B shows the
cyclic voltammogram of 2PHCMCNPE in a 0.1 M phosphate
buffered solution (PBS).
As Figure 1B shows, the cyclic voltammogram of 2PHCMC-
NPE exhibits an anodic and cathodic peak at the forward and
reverse scans of the potential related to the oxidation and
reduction of the 2PHC, respectively. A pair of reversible peaks
observed at Epa ) 0.21 V and Epc ) 0.11 V. The half-wave potential
Figure 3. Square wave voltammograms of 2PHCMCNPE in 0.1 M
PBS (pH 7.0) containing different concentrations of EP and NE: (from
inner to outer) mixed solutions of 0.36 + 10, 0.55 + 40, 0.67 + 125,
0.80 + 175, 40 + 275, 175 + 400, 350 + 550, and 525 + 800,
respectively, in which the first value is concentration of EP in
micromolar and the second value is concentration of NE in micro-
molar. (A and B) Plots of the peak currents as a function of EP
concentration and (C) plot of the peak currents as a function of NE
concentration.
(E1/2) and ∆E were 0.16 and 0.10 V, respectively. The peak
p
separation potential, ∆E p () Epa - Epc), is greater than the (59/
n) mV expected for a reversible system, which indicates a quasi
reversible behavior for the mediator in an aqueous medium. In
addition, the effect of the scan rate of the potential on the
electrochemical properties of the 2PHCMCNPE was studied in
PBS by cyclic voltammetry. The plots of the anodic and cathodic
peak currents were linearly dependent on the sweep rate (v)
(Figure 1C). This behavior indicates that the nature of the redox
process is diffusionless controlled.
Electrocatalytic Oxidation of EP. Figure 2 depicts the cyclic
voltammetric responses from the electrochemical oxidation of 0.5
mM EP at 2PHCMCNPE (curve f), 2PHC modified CPE (2PH-
CMCPE) (curve e), CNPE (curve d), and bare CPE (curve b). As
can be seen, the anodic peak potential for the oxidation of EP at
2PHCMCNPE (curve f) and 2PHCMCPE (curve e) is about 210
mV, while at the CNPE (curve d) and bare CPE(curve b), peak
potentials are about 410 and 450 mV, respectively. From these
results it is concluded that the best electrocatalytic effect for EP
oxidation is observed at 2PHCMCNPE (curve f). For example,
the results are shown that the peak potential of EP oxidation at
2PHCMCNPE (curve f) shifted by about 200 and 240 mV toward
the negative values compared with that at a CNPE (curve d) and
bare CPE (curve b), respectively. Similarly, when we compared
the oxidation of EP at the 2PHCMCPE (curve e) and 2PHCMC-
NPE (curve f), there is a dramatic enhancement of the anodic
peak current at 2PHCMCNPE relative to the value obtained at
the 2PHCMCPE (curve e). In the other words, the data obtained
clearly show that the combination of carbon nanotube and mediator
(2PHC) definitely improve the characteristics of EP oxidation. The
2PHCMCNPE in 0.1 M PBS, without EP in solution, exhibits a well-
behaved redox reaction (curve c) upon the addition of 0.5 mM EP;
the anodic peak current of the mediator was greatly increased, while
the corresponding cathodic peak disappeared on the reverse scan
of the potential (curve f). This behavior is typical of that expected
for electrocatalysis at chemically modified electrodes.24
Figure 4. Voltammograms of rotating disk 2PHCMCNPE in 0.1 M
PBS (pH 7.0) containing 0.5 mM EP at the various rotation rates
indicated for each voltammogram. Scan rate: 30 mV s-1. Insets: (A)
Levich plots constructed from the modified RDE voltammograms of
solution with (a) 0.5, (b) 1.0, (c) 1.5, and (d) 2.0 mM EP. (B)
Koutecky-Levich plots obtained from Levich plots shown in part A.
Calibration Plot and Limit of Detection. Square wave
voltammetry was used to determine the concentration of EP. The
responses are linear with the EP concentrations ranging from (5.0
× 10-8)-(9.5 × 10-7) M and (9.5 × 10-7)-(5.5 × 10-4) M with
current sensitivities of 7.7494 and 0.0181 µA/µM. The decrease
of sensitivity (slope) in the second linear range is likely to be due
to kinetic limitations.14,24 The detection limit (S/N ) 3) is 9.6 nM.
(24) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and
Applications, 2nd ed.; Wiley: New York, 2001.
9850 Analytical Chemistry, Vol. 80, No. 24, December 15, 2008

Simultaneous Determination of EP and NE. One of the
main objectives of the present study was the development of a
modified electrode capable of the electrocatalytic oxidation of EP
and separation of the electrochemical responses of EP and NE.
The utilization of the 2PHCMCNPE for the simultaneous deter-
mination of EP and NE was demonstrated by simultaneously
changing the concentrations of EP and NE. The square wave
voltammetric results show that the simultaneous determination
of EP and NE with two well-distinguished anodic peaks at
potentials of about 240 mV is possible at the modified electrode
(Figure 3).
It is very interesting to note that the sensitivities of the
modified electrode toward EP in the absence and presence of NE
are virtually the same, which indicates the fact that the oxidation
processes of EP and NE at the 2PHCMCNPE are independent
and therefore simultaneous, or independent measurements of the
two analytes are possible without any interference. If the EP signal
is affected by the NE, the above-mentioned slopes would be
different.
In this case, the Koutecky-Levich plots (Figure 4, inset B)
can be used to determine the value of the catalytic reaction rate
constant, k, between the oxidized form of 2PHC and EP. The
Koutecky-Levich equation can be formulated as follows:24
1 ⁄ Icat ) 1 ⁄ ILev + 1 ⁄ Ik
(1)
Here ILev is the Levich current and Ik is the kinetic current and
are defined by eqs 2 and 3:
ILev ) 0.620nFAD2⁄3v-1⁄6ω1⁄2C
Ik ) nFACkΓ
(2)
(3)
With the use of these equations, the k value was found to be
5.82 × 103 M-1 s-1 for EP. Also, the diffusion coefficient of EP,
D, may be obtained from the slope of the Koutecky-Levich plots.
The mean value of D was found to be 7.87 × 10-6 cm2 s-1
.
Rotating Disk Electrode Voltammetry. To our knowledge,
no paper has used the rotating disk electrode voltammetry
technique with carbon paste and especially carbon nanotube paste
electrodes for the oxidation of EP and in this paper, we used this
technique for the first time by this kind of electrodes for the
oxidation of EP. Thus, the electrocatalytic activity of 2PHCMCNPE
toward oxidation of EP was also evaluated by the RDE voltam-
metry technique (Figure 4). The plots of catalytic currents (I)
measured at 240 mV versus ω1/2 (Levich plots) are shown in inset
A of Figure 4. As can be seen, the clear lack of linearity
immediately suggests that the reaction is limited by kinetics and
not by diffusion.
CONCLUSION
This work demonstrates the construction of a 2PHCMCNPE
and its application in the simultaneous determination of EP and
NE. The high current sensitivity, low detection limit, and high
selectivity of the 2PHCMCNPE for the detection of EP prove its
potential sensing applications.
Received for review September 3, 2008. Accepted
November 1, 2008.
AC801854J
Analytical Chemistry, Vol. 80, No. 24, December 15, 2008 9851