Application of Carbon Nanotube-Graphite Mixture for the Determination
of Diclofenac Sodium in Pharmaceutical and Biological Samples
Abdolmajid Bayandori Moghaddam1, Ali Mohammadi2,3* and Mozhgan Fathabadi2,4
1Department of Engineering Science, College of Engineering, University of Tehran, P.O. Box 11155-4563, Tehran, Iran
2Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
3Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, P.O. Box 14155-6451, Iran
4Pharmaceutical Quality Assurance Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
*Corresponding author:
Ali Mohammadi
Department of Drug and Food Control
Faculty of Pharmacy
Tehran University of Medical Sciences
Tehran, Iran E-mail:
alimohammadi@tums.ac.ir
Received July 02, 2012; Accepted July 14, 2012; Published July 18, 2012
Citation: Moghaddam AB, Mohammadi A, Fathabadi M (2012) Application of
Carbon Nanotube-Graphite Mixture for the Determination of Diclofenac Sodium
in Pharmaceutical and Biological Samples. Pharmaceut Anal Acta 3:161.
doi:10.4172/2153-2435.1000161
In this study, the main purpose is to fabricate a sensitive and selective electrochemical sensor through the multiwalled
carbon nanotube-graphite/Ag electrode (MWCNTs-G/Ag). The application of this sensor was developed for
the determination of diclofenac sodium in pharmaceutical dosage form, urine and human plasma. MWCNTs-graphite
mixture improved the electroactive surface area due to its porous structure and a remarkable increase in the peak
currents was observed. It demonstrated a catalytic effect and speeded up the rate of redox process. Application of
MWCNTs-G/Ag resulted in a sensitivity augmentation. It is found that a maximum current response for the sensor in
the Britton-Robinson buffer solution can be obtained in pH 3. The prepared sensor showed good standard calibration
curves during 3 consecutive days over the concentration range of 45-2000 ng/mL and RSD values ranging from
1.95–7.11%. The limit of quantitation and detection limit were 45 and 15 ng/mL, respectively.
Carbon based electrode has been commonly used in electrochemical
systems. In many aspects, it is an ideal material as electrode in the
electrochemical experiments due to its attractive properties, including
the good corrosion resistance, high electrical conductivity, low cost and
a broad anodic potential window in aqueous media [1,2]. According to
the degree of graphitization, it is morphologically diverse, existing in
a various forms from carbon black to glassy carbon, carbon fibers and
pyrolytic graphite [3]. Another interesting form of carbon, are carbon
nanotubes (CNTs). They are allotropes of carbon with a cylindrical
nanostructure. Allotropes of carbon, with their unique and fascinating
one-dimensional nanostructure, are presently under examination as
new tools for various applications [4-7]. The length-to-diameter ratio of
CNTs is considerably larger than other materials. CNTs are assigned as
two typical classes: single-walled (SWCNTs), and multi-walled carbon
nanotubes (MWCNTs) [8]. SWCNTs consist of a single graphitic
sheet rolled up into a cylindrical form, while MWCNTs are composed
of concentric graphite tubules. They show extraordinary strength
and unique electrical properties and can be considered as attractive
candidates in diverse nanotechnological applications. Furthermore,
CNTs illustrate wide potential window, chemical inertness, low cost
and suitability for various sensing and detection. The subtle electronic
properties suggest that CNTs have the ability to promote electrontransfer
reactions, when used as an electrode in chemical reactions
[9,10]. Carbon nanotubes have been widely used in electrochemical
studies, where carbon nanotube modified electrodes employed for
sensing applications. Recent studies showed that modified electrodes
by carbon nanotubes and other nanostructures could impart
electrocatalytic activity to the electrochemical studies [11-13].
Diclofenac, 2-(2,6-dichloranilino) phenylacetic acid, is a nonsteroidal
anti-inflammatory drug. It is used mainly as a sodium
salt for the relief or pain and inflammation in various conditions:
musculoskeletal and joint disorders such as rheumatoid arthritis,
osteoarthritis, spondylarthritis, ankylosing spondylitis [14].
An additional indication is the treatment of acute migraines.
Diclofenac is used commonly to treat mild to moderate post-operative
or post-traumatic pain, particularly when inflammation is also present,
and is effective against menstrual pain and endometriosis [15]. It
has been determined by a variety of analytical techniques, such as
spectrophotometry [16], reflectometry [17], thin-layer chromatography
[18,19], gas chromatography [20,21], high-performance liquid
chromatography-mass spectrometry [22], high performance liquid
chromatography [23], capillary zone electrophoresis [24], polarographic
[25], and potentiometric analysis [26]. Recently, some sensors reported
for the diclofenac analysis [27,28]. In addition, Blanco-Lopez et al was
developed voltammetric sensors for the determination of diclofenac,
based on the molecular recognition of the analyte by molecularly
imprinted polymers [29,30]. Most of these methods require either
sophisticated instruments or expensive reagents or involve several
manipulation and derivatization steps. To the best of our knowledge,
there is very little voltammetric methods reported in the literature for
determination of diclofenac sodium [31]. The voltammetric methods
are very interesting and show to have several advantages for the
sensitive determination of a range of drug compounds [32-35]. In
electrochemical techniques, sample preparation usually consists in
dissolving the active compound from the pharmaceutical dosage forms
in a suitable solvent and performing a direct analysis on an aliquot
of this solution. The specificity and selectivity of the voltammetric techniques are usually excellent because the analyte can be readily
identified by its voltammetric peak potential.
In this work, MWNTs-graphite/Ag electrode was prepared to achieve
a high electroactive surface area for development of an electrochemical
sensor for the determination of diclofenac sodium. This electrode
has the advantages of easy preparation, rapid and simple operation,
reproducibility and very low interference and high accuracy in tablets
and biological samples. Some experimental parameters including, pH,
scan rate, and electrochemical technique were scrutinized to find the
best function of the sensor. The proposed method was successfully
applied to determine diclofenac sodium in pharmaceutical formulation
and biological fluids.
Experimental Section
Chemicals and materials
All chemicals used in this work were of analytical-reagent-grade
chemicals from Merck Co. (www.merck.com). The multi-walled
carbon nanotubes (MWCNTs) were purchased from Neutrino Co.
(www.neunano.com, Iran). The MWCNTs had an outer wall diameter
distribution of <10 nm, a length of between 5-15μm and amorphous
carbon <3%. Distilled water was used to prepare all solution and in all
experiments. The 0.04 M Britton-Robinson (B-R) buffer solutions of
pH 3-8 ranges were freshly prepared. Buffer solutions (pH 3-7) were
prepared by mixing corresponding amounts of 0.04 mol/L H3BO3, 0.04
mol/L CH3COOH, 0.04 mol/L H3PO4 and 0.2 mol/L NaOH. Diclofenac
sodium was obtained from Iranian Quality Control Lab Ministry of
Health and Medical Education (www.behdasht.gov.ir). Fresh frozen
plasma was obtained from Iranian Blood Research and Fractionation
Holding Company (http://ibrf.ir/EN/Concern.asp). Drug free human
urine was obtained from healthy volunteers (25-30 years).
Apparatus
All the electrochemical measurements were performed by the
μ-AUTOLAB TYP III (www.metrohm-autolab.com). A three-electrode
cell was also used, the working electrodes were glassy-carbon (GC)
and MWCNTs-G/Ag electrode. A Pt wire and an Ag/AgCl/KCl (sat.)
(both from Azar Electrode Co., Iran) were used as the counter and
reference electrodes, respectively. (KCl-saturated, 0.197 V versus a
normal hydrogen electrode (NHE)). All experiments were performed
at 25 ± 1°C. The sonication was performed by using an ultrasonic bath
system TECNO-GAZ, Tecna 6 (50–60 Hz, 230 ± 10% V, 0.138 KW).
Furthermore a Philips model X-30 scanning electron microscope was
used to capture images.
Fabrication of carbon nanotube-graphite mixture
Before modification, the surface of Ag electrode was prepared
by polishing on a polishing cloth with aqueous alumina slurries.
The remaining particles on the surface were removed by ultrasonic
treatment in ethanol for a few minutes. Finally it was rinsed with doubly
distilled water. This work was carried out for every renewal paste on
Ag electrode. For making paste the MWCNT:graphite-based electrode
was prepared from a mixture of MWCNTs and graphite powder with
different weight ratio. After weighing carbon and Graphite, they mixed
together truly for 10 min and then the mixture was added to paraffin oil
and again the mixture was mixed for 15 min. A thin layer of the paste
was packed into the end of an Ag electrode. Table 1 shows the amount
of each component for making different pastes.
Table 1:The components of electrodes.
Preparation of standard solutions
A suitable amount of working standard powder of diclofenac
sodium was dissolved in 25mL distilled water to form a stock solution
at a concentration of 5mM. Additional dilute solutions with different
concentrations in different pHs were prepared daily by accurate
dilution by B-R buffer. The stock solution was protected from light
using aluminium foil and stored at 4˚C for three days. Plasma and urine
standard solutions (200 ng/mL, 1000 ng/mL and 2000 ng/mL) were
prepared by spiking of aqueous stock solution of diclofenac to drug free
plasma and urine samples.
Extraction procedure
For the determination of diclofenac sodium in biological fluid
0.5mL of 0.5 M HCl was added to 0.5 mL plasma/urine standard
solution and vortexed for 2 min. This mixture was then blended with 5
mL ethyl acetate, vortexed for 3 min. and centrifuged at 4500 rpm for
5 min. After removal of the organic phase the extraction was repeated
on the residual aqueous phase. The ethyl acetate phase were pooled and
dried at 60°C under a gentle stream of nitrogen. After drying, samples
were diluted with 20 mL of B-R buffer in pH 3 and transferred to
electrochemical cell for analysis.
Tablet assay procedure
For the analysis of diclofenac tablets, 20 tablets were weighed and
powdered well in a pestle. An appropriate, accurately weighted amount
of the powder equivalent to the weight of one tablet was dissolved in
doubly distilled water. Finally, aliquots of this solution were diluted
with B-R buffer at PH 3 to obtain a concentration of 1000 ng/mL.
Validation of the method
Validation of this procedure for the quantitative assay of the drug
was examined via evaluation of the linearity, limit of detection (LOD),
limit of quantification (LOQ), precision, accuracy and selectivity.
Square wave voltammograms (SWVs) of diclofenac solutions with
concentrations range (45-2000 ng/mL) at pH 3 at MWCNTs-graphite/
Ag electrode (1:1) in B-R buffer were recorded.
Results and Discussion
Characterization of MWCNTs and MWCNTs: graphite
mixture
The microscopic structure of MWCNTs and MVCNTs-graphite
mixture were characterized using SEM images. (Figure 1a) shows SEM
image of MWCNTs while (Figure 1b) shows SEM image of MWCNTsgraphite
mixture. The presence of paraffin oil in MWCNTs - graphite
mixture could bridge the isolated carbon materials and MWCNTs
could be clearly observed in (Figure 1b).
Figure 1:SEM images of a MWCNTs and b MWCNTs-G mixture (1:1), scale
bars are 500 nm.
Voltammetric studies of the drug on different electrodes
In this study, voltammetric methods such as cyclic voltammetry
(CV) and differential pulse voltammetry (DPV) were carried out at
pH range 3-7 in diclofenac solutions 15000 ng/mL at both GC and
MWCNTs-graphite/Ag electrodes with different ratios of MWCNTs-G/
Ag electrode in B-R buffer. The results show that the best response is
obtained on MWCNTs-G/Ag electrode with ratio of (1:1). So this paste was chosen for analyzing of diclofenac in this study. By overlaying CVs
and DPVs in different pH at different electrodes (the results are not
showed) there was found that the best pH was 3.
The peak potential (Ep) shifts to more negative values with the
pH increase. The peak current had higher values in acid solutions,
and decreased with increasing pH. Taking into account that from an
analytical point of view high peak currents together with low oxidation
potentials are desirable, this should be a good pH region for analytical
determinations. For investing drug’s stability in acidic media, t-test [36]
was done in 3 days continuously in concentrations of 500 ng/mL and
2000 ng/mL in pH 3. The values of tcalculated were then compared to a
ttabulated with 4 degrees of freedom at the 95% confidence level (t =2.776).
The calculated t-values (2.66 and 0.93 for concentration of 500 ng/mL
and 2000 ng/mL, respectively) were smaller than the theoretical ones.
These results indicate that drug is stable in pH 3.
The enhanced voltammetric behaviour of diclofenac sodium
at MWCNTs-G/Ag electrode
The electrochemical oxidation of 15000 ng/mL diclofenac sodium
at the MWCNTs-G/Ag electrode in B-R buffer solution (pH 3.0) was
examined by cyclic voltammetry (CV). The cyclic voltammograms are
illustrated in (Figure 2). A well-defined oxidation peak is observed
at about 0.8 V. In order to illuminate the enhancement properties of
MWCNTs-G/Ag electrode for the oxidation of diclofenac sodium, CVs
of 15000 ng/mL diclofenac sodium in B-R buffer pH 3 are tested at
different working electrodes including Graphite (curve a), bare GCE
(curve b), MWCNTs-G/Ag electrode (curve c) in (Figure 2a-c). The
peak current of diclofenac sodium greatly increases at MWCNTs-G/
Ag electrode. It is believed that MWCNTs show highly effective
enhancement to diclofenac sodium oxidation because of its large
specific surface area and subtle electrical properties, which provides
enough effective reaction sites to increase the electron exchange rate.
Figure 2:Comparative CVs of diclofenac sodium (15000 ng/mL) in B-R buffer
(pH 3) at different electrodes; a glassy carbon, b graphite, c MWCNTs-G/Ag
electrode (1:1).
Effect of pH on the peak potentials and peak currents
The peak potential and the peak current are closely depending on the
pH of solution. The cyclic voltammograms of 15000 ng/mL diclofenac
in buffer solution in the range of pH from 3 to 7 at MWCNTs-G/Ag
electrode are obtained (Figure 3a). The obtained voltammograms
demonstrate that in pH 3 we have the sharpest response rather than
other pH values. The (Figure 3b) shows a linear relationship between
pH values and anodic peak potentials (Epa), a linear shift of the peak potential towards more negative values was observed as the pH
increased from 3 to 7. Epa decreased by about 63.54 mV per pH, with
equation of Epa = -63.54 pH + 1064.6 (R² = 0.99). The slope of 63.54
mV per pH was close to the theoretic value 59 mV per pH, suggesting
one electron and one proton transferred in the oxidation of diclofenac
sodium. The oxidative reaction of diclofenac sodium is a one electron,
one-proton transfer process resulting in the radical species [37,38].
Figure 3:a CVs at MWCNTs-G/Ag electrode (1:1) in B-R buffer at various pH (right to left); 3, 4, 5, 6 and 7, scan rate: 80 mV/s. b Relationship between pH and
anodic peak potential.
Calibration curves
A typical calibration curve for diclofenac was demonstrated
in (Figure 4a-c). The relationship between diclofenac sodium
concentration and the oxidation peak current can be described with
the following linear regression equations in the range of concentration
from 45 ng/mL to 2000 ng/mL at 3 consecutive days:
Figure 4:Plot of peak current vs. concentration of diclofenac sodium in B-R buffer (pH 3) for various concentrations; 45, 75, 100, 200, 500, 1000 and 2000ng/mL.
Ipa = 0.0008C + 0.0724 (R2 = 0.999)
Ipa = 0.0009C + 0.0756 (R2 = 0.998)
Ipa = 0.0009C + 0.0559 (R2 = 0.998)
According to the obtained results, the best regression equation for
the calibration curve was found to be Ipa = 0.0008C + 0.0724 (R2 =
0.999). The relative standard deviation (%RSD) values ranging from
1.95–7.11% across the concentration range studied were obtained. Under the optimized experiment conditions described above, LOQ and
LOD of diclofenac sodium were obtained to be 45 ng/mL and 15 ng/
mL, respectively.
Repeatability, stability and reproducibility of the modified
electrode
Precision of the method was investigated with respect to both
repeatability and reproducibility. The repeatability of the modified
electrode was investigated by repetitive recording at a fixed diclofenac
concentration of 15000 ng/mL. A decrease for the peak currents in
CVs based on 7 replicates was 1.05% indicating good repeatability
of the response of modified electrode with the %RSD value less than
2.54. The reproducibility was evaluated by measuring the oxidation
current values for fresh solutions of each of the 45, 1000 and 2000 ng/
mL standards over a period of 3 days. The mean concentrations were
found to be 48.32, 978.02 and 2027.12 ng/mL with associated %R.S.D.
values of 7.11, 1.95 and 3.2, respectively. Accuracy of the assay was
determined by interpolation of replicate (n=3) peak areas of three
accuracy standards (45 ng/mL, 1000 ng/mL and 2000 ng/mL) from a
calibration curve prepared as previously described. In each case, the
percent relevant error was calculated. The resultant concentrations
were 47.28 ± 2.95 ng/mL (mean ± S.D.), 1044.07 ± 38.57 ng/mL and 1972.99 ± 28.65 ng/mL with percent relevant errors of 5.1, 4.41 and
-1.35%, respectively.
Response characteristic
Some voltammetric procedures have been reported for the
determination of diclofenac in pharmaceutical tablets and biological
fluids. But in accordance to our knowledge, there is no report regarding
the use of MWCNTs-graphite/Ag electrode for the determination
of diclofenac. By this electrode, we could determine diclofenac
with the LOD value of 15 ng/mL which is the lowest of LOD for the
determination of this drug at different electrodes until now. In (Table
2), the response of the proposed method is compared with those
obtained by reported methods. The results showed that Ag electrode
modified with carbon nanotubes is suitable for sensitive and selective
determination of diclofenac.
Table 2:Different reported electrodes for determination of diclofenac.
Determination of diclofenac in pharmaceutical samples
The applicability of the proposed voltammetric method for
pharmaceutical dosage forms was examined by the analyzing the
tablets. The result of the assay of diclofenac tablets yielded a recovery
of 101.03% (R.S.D. =2.35%) of label claim for the tablets. In order
to evaluate the accuracy of this method and to know whether the
excipients in pharmaceutical dosage forms show any interference
with the analysis, the proposed voltammetric method was checked
by recovery experiments using the standard addition method. After
addition of known quantities of analyte to the drug product, the
mixture was analyzed by the proposed method. The recovery of
diclofenac was calculated using the corresponding regression equations
of previously plotted calibration curves. The results of recovery
experiments are presented in (Table 3). The results indicate the absence
of interference from commonly pharmaceutical excipients used in
the selected formulations. Therefore, the method can be applied to
the determination of diclofenac in pharmaceutical forms without any
interference from inactive ingredients.
Table 3:Determination and recovery of diclofenac sodium in commercial tablets.
Determination of diclofenac in human plasma
The prepared modified electrode was also applied to the analysis of
the human plasma samples using SWV method. The drug-free plasma
samples were spiked with different amounts of standard diclofenac and
their SWV were recorded using the modified electrode. In our tests,
no interference peak was detected in healthy plasma. In (Figure 5I) the
buffer and blank plasma are shown. Three concentrations of diclofenac
spiked plasma were tested (200, 1000 and 2000 ng/mL). The results of
recovery evaluations have been shown in Table 4)
Figure 5:(I) SWVs of the a buffer, b extracted blank plasma c extracted
plasma spiked sample (2000ng/mL). (II) SWVs in a buffer b urine blank
previous extraction, c urine blank after extraction and d extracted urine spiked
sample (2000 ng/mL). Frequency = 50 Hz, pulse amplitude = 30 mV and step
potential = 5 mV.
Table 4:Recovery percent of diclofenac sodium electrochemical determination in
plasma (n = 3) by MWCNTs-G/Ag electrode.
Determination of diclofenac in human urine
The practical analytical application of the proposed method was
further established by estimation of diclofenac in human urine samples.
The voltammograms of human urine sample without and with the
standard solution of diclofenac sodium are shown in (Figure 5II). To
determine the extraction efficiency, diclofenac sodium was spiked in
urine at concentrations of 200, 1000 and 2000 ng/mL and extracted
using the stated extraction procedure. The results obtained are listed
in Table 5.
Table 5:Recovery percent of diclofenac sodium electrochemical determination in
human urine (n = 3) by MWCNTs-G/Ag electrode.
Conclusion
MWCNTs have large specific surface area and strong adsorptive
properties providing more reaction sites. MWCNTs-G paste on
the electrode surface is porous. Because of these characteristics the
adsorption of diclofenac sodium to the electrode surface becomes
easy. Correspondingly, the concentration of diclofenac sodium at
the electrode surface was enlarged. So the oxidation peak current of
diclofenac sodium was greatly enhanced at MWCNTs-G paste modified
electrode. In this study, several ratio of carbon nanotube - graphite
paste electrode were made and voltammetric studies of them were
compared to GCE. The best paste was carbon nanotube - graphite with
ratio of (1:1) that other studies focused on it. We had the lowest LOD
(15 ng/mL) for diclofenac determination compare to other electrodes
which have been reported until now. High sensitivity and improved
detection limit of the MWCNTs-G/Ag electrode are promising for
the determination of diclofenac content in biological fluids as well as
pharmaceutical preparations.
Acknowledgements
The authors would like to acknowledge financial assistance from Tehran
University of Medical Sciences, Tehran, Iran. Also, the authors thank the University
of Tehran for the supply of some instruments.
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