Electrochemical behavior of anticancer drug 5-fluorouracil at carbon paste electrode and its analytical application
© Bukkitgar and Shetti. 2016
Received: 19 May 2015
Accepted: 20 December 2015
Published: 11 January 2016
A set of pyrimidine nucleobase present in all living systems as a component of nucleic acid constitutes uracil together with thymine and cytosine. A diverse physiological activity is exhibited by many N-substituted uracil derivatives. In oncology, 5-FU is widely used as an important anticancer drug.
Electrochemical behavior was studied using cyclic voltammetric method, and the analytical application was studied using differential pulse voltammetric method. Solution pH has been measured by pH meter.
The process on the surface of electrode was found to be irreversible and diffusion controlled. The charge transfer coefficient, heterogeneous rate constant, and the number of electron transferred were calculated. Possible reaction mechanism taking place on the surface of electrode was proposed. Calibration plot constructed using differential pulse voltammetric technique was used for quantitative analysis in pharmaceutical and human urine sample. Limit of detection (LOD) and limit of quantification (LOQ) were calculated to be 12.25 and 40.8 nM, respectively.
In the present work, we described the electrochemical behavior of anticancer drug and its determination in human urine and pharmaceutical samples. The method shows the development of a sensor for selective and sensitive determination of 5-FU.
Ribonucleic acid consists of a pyrimidine base called uracil which forms base pair with adenine. For biosynthesis of nucleic acid in tumors, uracil is preferentially used (Rutman R.J. et al. 1954). A drastic change in the biological properties of uracil resulted from the substitution of hydrogen atom at fifth position by halogen atom (Voet D. and Voet J.G. 1995). Amongst the variety of uracil derivatives reported as antitumor and antiviral agent, 5-FU has acquired a position of particular importance. For the treatment of solid tumor of the breast and rectum, 5-FU has been used extensively as an antineoplastic agent (Heidlberg C. and Ansfield F.J. 1963). One of the major mechanisms responsible for antitumor activity of 5-FU is by inhibition of thymidylate synthesis (Hartmann K.U. and Heidelberger C. 1961). Detailed studies have pointed to 5-FU interference with DNA and protein synthesis, because of conversion to the corresponding ribose nucleoside and substitution into RNA, as an equally important mechanism of toxicity (Myers C.E. 1981). The studies on oxidation—reduction behavior of compounds of biological significance—is of considerable value, as they provide deep insight into the biological relevant redox reactions of these compounds. Although the actual biological redox reactions may be of more complexity due to enzymatic interactions, much more information can be derived from the study of these compounds in aqueous solution of known pH.
Electrochemical methods have proved to be sensitive for the determination of organic molecules, including drugs and related molecules in pharmaceutical dosage forms and biological fluids and their oxidizable property (Padmini V. 2010; Hegde R.N et al. 2008). Carbon electrodes, especially paste electrodes, are widely used in the electrochemical investigations because of their low background current, wide potential windows, chemical inertness, low cost, and suitability for detection of various organic and biological compounds (Genxi L. and Peng M. 2013).
Many advantages such as very low background current, low cost, large potential window, simple surface renewal process, and easiness of miniaturization of carbon paste electrode (CPE) are widely applicable in both electrochemical studies and electroanalysis. In addition, easy fabrication of the electrode can be achieved by incorporating different substances during paste preparation which results in the so-called modified electrode with desired composition and predetermined properties (Khoobi A. et al. 2013; Mokhtari A. et al. 2012; Díaz C. et al. 2013; Gholivand M.B. and Mohammadi-Behzad L. 2014; Mazloum-Ardakani M. et al. 2010; Raoof J.B et al. 2007; Dönmez S. et al. 2014).
Apparatus and chemicals
Electrochemical analyzer (CHI Company, D630, USA) was used to study the electrochemical deeds of the drug under investigation at an ambient temperature of 25 ± 0.1 °C. A three-electrode system consisting of carbon paste electrode as working electrode, platinum wire as counter electrode, and Ag/AgCl (3 M KCl) as reference electrode were used in a 10-ml single compartment. In all the measurements, background subtraction was made. The phosphate buffer solutions ranging 3.0–11.2 pH (I = 0.2) were prepared according to literature (Christian G.D. and Purdy W.C. 1962), and pH of the solutions was measured by pH meter (Elico Ltd., LI120, India). 5-FU (Sigma-Aldrich, USA) was used to prepare 1.0 mM stock solution in double distilled water (6.5 × 106 Ω). Double distilled water and analytical grade chemicals and reagents without further purification were used throughout the experiments.
Preparation of electrode
The CPE was prepared by mixing 1.0 g of graphite powder and 0.5 ml of paraffin oil in a small agate mortar, and this mixture was then homogenized. A portion of the resulting paste was packed firmly into a cavity of polytetrafluoroethylene tube (PTFE). The surface of the electrode was smoothed against weighing paper and rinsed with water. The paste was carefully removed prior to pressing a new portion in to the electrode after every measurement. The resulting electrode was noted as CPE. Prior to use, the CPE was activated in phosphate buffer solution of pH 7 by cyclic voltammetric sweeps between 0.4 and 1.4 V with a scan rate 50 mVS−1(Malode S.J. et al. 2013).
In Eq. (1), for 1.0 mM K3Fe (CN)6 and 0.1 M KCl as supporting electrolyte, Ip refers to the anodic peak current, n is the number of electron transferred during the electrode reaction equal to 1. A 0 is the surface area of the electrode, D R is the diffusion coefficient equal to 7.6 × 10−6 cm2 s−1, υ is the scan rate, and C 0 is the concentration of K3Fe (CN)6. From the slope of the plot of Ip vs. υ 1/2, the area of the electrode surface was calculated to be 0.036 ± 0.0014 cm2.
To carry out the pharmaceutical analysis, 5-FU tablets were grounded using a mortar and a fraction corresponding to stock solution of 1 mM was weighed and completed to the volume with double distilled water in a 100-ml calibrated flask. After sonication for 10 min, to affect complete dissolution suitable aliquots of the clear supernatant, liquid was taken and diluted with buffer solution of pH 7. The oxidation peak current of 5-FU was measured using differential pulse voltammetric technique. Standard addition method was to study the accuracy of the projected method and the interference from excipients used in dose forms.
Results and discussion
Electrochemical behavior of 5-FU
Influence of accumulation time
Effect of supporting electrolyte
From the plot of Ip vs. pH (Fig. 3(B)), it is clear that the best result with respect to sensitivity accompanied with sharper response was obtained with pH = 7.0, hence, it was selected for further work (Hegde R. N. et al. 2009). The peak current depends on the deprotonation and protonation form of the electro-active species in electrochemical cell. At pH 7, protonated and deprotonated form of 5-FU dominates. And a gradual change of speciation of 5-FU occurs in the pH range 7 to 9. Further, the magnitude of current is directly proportional to the rate of the electrochemical reaction. Hence, it is apparent to conclude that the oxidation of 5-FU is very high at pH 7 (Ioana P. et al. 2005).
Influence of scan rate
Where E p/2 is the potential where the current is at half the peak value. From the above equation, value of α was to be 0.56. The number of electrons transferred in electrode oxidation was calculated to be 2.3 ≈ 2. Hence, 5-FU may be assumed to undergo two protons and two electron transfer in the electrode reaction. If the value of E 0 is known, the value of k 0 can be determined from the intercept of the above plot. From the intercept of Ep versus υ curve by extrapolating to the vertical axis, at υ = 0, the value of E 0 can be calculated from Eq. 2 (Shetti N.P. et al. 2012). From the intercept of Ep versus log υ which was found to be 1.199, E 0 and k 0 were calculated to be 1.13 and 1.7 × 103 s−1, respectively.
Characteristics of 5-fluorouracil calibration plot using differential pulse voltammetry at carbon paste electrode
Linearity range (M)
1 × 10−7 to 4 × 10−5 M
Slope of the calibration plot (μA M−1)
Correlation coefficient (r)
RSD of slope (%)
RSD of intercept (%)
Number of data points
Repeatability (RSD %)
Reproducibility (RSD %)
Comparison of detection limits of 5-fluorouracil by different reported methods
Glassy carbon electrode modified with bromothymol blue and multi-walled carbon nano-tube
Hua X. et al 2013
Glassy carbon electrode mediated by surfactant cetyltrimethyl ammonium bromide
Sataraddi S. R. and Nandibewoor S. T 2011
Badea I. et al. 2002
Fars K.A. et al. 2009
Ionic liquid modified carbon paste electrode
Tianrong Z. et al. 2011
Carbon paste electrode
Tablet analysis and recovery test
Analysis of 5-fluorouracil in tablets by differential pulse voltammetry and recovery studies
Amount found (mg)a
t test of significant
F test of significant
Effect of interferents
Influence of potential interferents on the voltammetric response of 1.0 × 10−4 M 5-fluorouracil
Signal change (%)
Urine analysis and recovery test
Application of differential pulse for the determination of 5-fluorouracil in spiked human urine
Urine spiked (10−5 M)
Detecteda (10−5 M)
Urine sample 1
Urine sample 2
Urine sample 3
Urine sample 4
Urine sample 5
In the present work, oxidation of 5-FU in phosphate buffer solution (pH = 7) was successfully carried out. The electrode process of 5-FU is diffusion-controlled and irreversible. Suitable electrode reaction mechanism was proposed. A differential pulse voltammetric technique was developed for the determination of 5-FU in pharmaceutical dose and human urine samples. As compared to other methods, the proposed method offered an improvement in simplicity and accuracy.
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