Analysis of sulfamethoxazole by square wave voltammetry using new carbon paste electrode

In this work a new model of carbon paste electrode was employed to determine sulfamethoxazole (SMX), an antibiotic used to treat infections in human and veterinary medicine, by the square wave voltammetric modality (SWV). More specifically, the electrochemical behavior of SMX was investigated by cyclic voltammetry (CV), and the quantitative analysis of SMX was provided by SWV. The analytical curve was obtained with a linear correlation coefficient (r) of 0.985 and standard deviation (SD) of 0.005 μA. Limits of detection and quantification were found as 2.3×10-6 and 7.7×10-6 mol L-1, respectively. According to the obtained results, the new carbon paste prototype electrode can successfully be employed in this kind of electroanalytical applications.


Introduction
Concerning a drug analysis, electroanalytical methods offer several advantages: they are versatile, fast, sensitive, inexpensive, and environmentally friendly due to the limited use of chemicals [1].Recently developed electrochemical devices efficiently monitor pollutants through direct or indirect reactions between the contaminant and the electrode surface, what makes them potentially applicable in situ [2][3][4].The large-scale use of this technology relies on the scientific knowledge and nowadays, scientists have investigated many electrodes for this purpose [5].Gold electrode, glassy carbon electrode, platinum disc electrode, graphite electrode, chemically modified electrodes, carbon nanotube and carbon paste electrodes (CPEs) are some of the examples already reported in the literature [1,5].

Carbon paste electrode construction
The carbon paste electrode was prepared by mixing 2.25 g of graphite powder with 0.75 g of mineral oil.This mixture was homogenized by magnetic stirring in a 25 mL beaker containing 10 mL of chloroform (Merck).The paste was obtained after evaporation of the solvent.The carbon paste was packed into a versatile electrode body fabricated in our laboratory.It consisted of a glass cylindrical tube in the form of a syringe (o.d.7 mm, i.d. 3 mm) and contained a platinum rod to establish the electric contact (Figure 2).

Instrumentation
The voltammetric experiments were conducted using a potentiostat model μAUTOLAB III (Eco Chemie) connected to a personal computer.The experiments were carried out in a three-electrode system consisting of a carbon paste working electrode, platinum spiral wire auxiliary electrode and Ag/AgCl, 3.0 mol L -1 KCl, reference electrode, all arranged in a 5-mL electrochemical cell (Figure 3).The solutions were deoxygenated with nitrogen for 15 min prior measurements.

Voltammetric analysis
The voltammetric behavior of the carbon paste electrode was initially investigated by CV measurements performed in different solutions, such as 0.1 mol L -1 KCl, 0.1 mol L -1 NaNO3 containing 0.01 mol L -1 K3Fe(CN)6, 0.04 mol L -1 BRbuffer (pH 2.18) and 0.1 mol L -1 NaClO4.CV analysis was carried out at potentials ranging from 0.1 V up to 1.8 V and scan rates from 10 to 100 mV s -1 .Subsequently, the quantitative analysis of SMX was conducted by SWV with previously optimized experimental parameters (frequency and pulse amplitude) and a constant step potential of 50 mV.Potential window ranged from 0.1 to 1.8 V vs. Ag/AgCl and 0.04 mol L -1 BRbuffer (pH 2.18) was used as the supporting electrolyte.The voltammetric parameters investigated for the SMX assay were: frequencies of 8, 12, 18, 20, and 24 Hz and pulse amplitude ranging from 10 to 100 mV.SMX was analyzed at different concentrations by the standard addition method, i.e. the analytical curve was constructed by adding aliquots of the SMX stock solution to the electrochemical cell.A linear curve was achieved for SMX concentrations ranging from 7.9 to 24.0 µmol L -1 .

Electrochemical properties of CPE in different electrolyte solutions
The CPE was initially tested in various aqueous systems, in order to provide information about its basic response and stability in different electrolytes.Figure 4(A-D) illustrates the cyclic voltammograms of CPE measured at different scan rates in 0.1 mol L -1 KCl, 0.1 mol L -1 NaNO3 containing 0.01 mol L -1 K3Fe(CN)6, 0.01 mol L -1 NaClO4, and 0.04 mol L -1 BR-buffer (pH 2.18).Among almost all presented CVs suggesting stability of CPE with not specific response in the specific electrolyte and given potential region, Figure 4B presents a typical cyclic voltammogram for the K3Fe(CN)6 4-/3-redox couple.This particular reaction usually served as the test for the redox activity of an electrode.Peaks at 0.36 V (Epa) and 0.19 V (Epc) suggest reversible behavior of redox couple at the CPE in the potential range from 0.1 V to 0.7 V vs. Ag/AgCl, with scan rate between 10 to 100 mV s -1 [12].Literature reveals that the supporting electrolyte plays an essential role in the voltammetric signal of SMX [29,[40][41][42].Besides, the sulfa drugs have two dissociation constants (pKa), which in the case of the SMX correspond to the amino functional group with pKa value of 1.8 and amide functional group with a pKa value of 5.6 [29,33,37,40,44].Therefore the BRbuffer solution (0.04 mol L -1 in acetic, phosphoric and boric acids) (pH 2.18), with CV shown in Figure 4C was chosen as the experimental medium in the voltammetric studies of SMX.
Figure 5(A-B) displays representative CVs of 0.005 mol L -1 SMX together with the corresponding background currents recorded for the proposed CPE and a commercial glassy carbon electrode, respectively.Figure 5A shows CV profiles of CPE in a blank solution of 0.04 mol L -1 BRbuffer (pH 2.18) and in the same solution containing 0.005 mol L -1 SMX, whereas Figure 5B shows CV profiles of glassy carbon in a blank solution of 0.05 mol L -1 s acid/methanol 70:3 (v/v) (pH 1.38) and in the same solution containing 0.005 mol L -1 SMX.An irreversible two-electron oxidation voltammetric peak appeared in both cases when 0.005 mol L -1 SMX was present in the solution [29,40].Also, much lower background current that was obtained for the CPE as compared with the solid glassy carbon, suggests that the proposed CPE electrode could be more sensitive for SMX oxidation [6][7][8][9][10][11][12][13][14][15][16].The higher background current observed for the glassy carbon electrode in Figure 5B stems from the oxygen evolution [43].

CPE electrochemical response toward SMX
The optimized experimental parameters that pointed out the best results for SMX determination using SWV technique were obtained by variations of pulse amplitude and frequency.The pulse amplitude was varied in the range of 10-100 mV, at the constant frequency of 12 Hz.In this case, the optimized result was defined as the parameter value that produced increase of the peak current without shifting the peak potential or making any significant increase in the peak width.Hence, 100 mV was chosen as the square-wave pulse amplitude value.Afterwards, the effect of frequency was evaluated in the range 8-24 Hz, keeping constant the pulse amplitude of 100 mV.The best result was achieved at f = 12 Hz.
According to previous literature investigations, sulfonamide oxidation results in a formation of the corresponding iminobenzoquinone intermediate, shown as peak (1) in Figure 6.The SWV responses presented in Figure 6 showed that the oxidation current peaks increased with increase of the frequency.Hence the frequency of 12 Hz was chosen for further analysis because of the best resolution of the voltammetric peak (Figure 6, peak (2)).
E / V vs. Ag/AgCl According to the literature, the SMX electrochemical oxidation occurs at the primary amino groups (-NH2) [29,40].Figure 7 illustrates the mechanism of SMX oxidation that as a two-electron and pH dependent reaction, possibly takes place in an acid medium.

Figure 7. SMX oxidation at carbon paste electrode in acid medium
The electrochemical behavior of SMX in different concentrations was assessed by successive additions of this drug in concentrations ranging from 7.9 to 24.0 µmol L -1 to the electrochemical cell.As seen in Figure 8(A), the anodic peak current at 1.07 V vs. Ag/AgCl (irreversible oxidation peak) increased upon rising of the SMX concentration.The analytical curve drawn in Figure 8(B) shows a linear correlation coefficient r = 0.985 with a standard deviation SD = 0.005μA.The corresponding linear equation was adjusted as ipa = 0.24 μA + 6.5×10 3 μA /mol L -1 [SMX].The limit of detection calculated according to the criterion 3SD/m ratio, where m is the slope of the analytical curve, gave 2.3×10 -6 mol L -1 , while the limit of quantification based on the criterion of 10SD/m ratio, was adjusted as 7.7×10 -6 mol L -1 .

Conclusions
The novel and efficient support for the carbon paste substrate is developed allowing determination of sulfamethoxazole at the mol L -1 level.The developed CPE showed an excellent stability in different electrolyte media and excellent voltammetric response for the K3Fe(CN)6 4-/3- redox couple probe.Oxidation of SMX at the CPE occurring at about 1.07 V vs. Ag/AgCl was found to be an irreversible 2-electron and pH dependent process.An electrocatalytic effect was observed in comparison with glassy carbon electrode.Another peak occurring at about 0.49 V vs. Ag/AgCl was observed when frequency values higher than 12 Hz were applied and ascribed to the formation of the corresponding iminobenzoquinone intermediate.The developed CPE is an inexpensive and versatile electrode, having high potential for application as a transducer in a device serving for determination of sulfamethoxazole.

Figure 2 .
Figure 2. Carbon paste electrode developed in our research group.