Determination of secnidazole in tablets and human serum by cathodic adsorptive stripping voltammetry

2.1 Reagents

All reagents were of analytical-reagent grade or better and high-purity water was used throughout. Secnidazole was kindly provided by the Egyptian International Pharmaceutical Industries Co. (EIPICo), 10th of Ramadan City, Egypt. A stock solution of secnidazole (1 × 10⁻³ mol L⁻¹) in ethanol was prepared and stored in the refrigerator. Working solutions were prepared daily by appropriate dilutions with de-ionized water.

Britton–Robinson buffer solutions (pH 2.0–11.8), used as supporting electrolytes, were prepared by adjusting the pH of a mixture of 0.04 mol L⁻¹ acetic acid, 0.04 mol L⁻¹ boric acid and 0.04 mol L⁻¹ orthophosphoric acid, with appropriate amounts of 0.2 mol L⁻¹ sodium hydroxide. Acetate buffer (pH 3.5–5.5; 0.04 mol L⁻¹) was prepared with acetic acid and sodium acetate.

Phosphate buffers (pH 6.0–8.0; 0.04 mol L⁻¹) were prepared using di-sodium hydrogen phosphate and mono-sodium hydrogen phosphate. Other supporting electrolytes used for stripping quantitative analysis: KCl (0.1 mol L⁻¹), HCl (0.1 mol L⁻¹), HClO₄ (0.1 mol L⁻¹) and H₂SO₄ (0.1 mol L⁻¹) were prepared in water as a stock (1 mol L⁻¹) and added to the working solution.

2.2 Apparatus

dc-Polarographic measurements were performed using a Sargent–Welch apparatus model 3001 (USA). A cell with two electrodes: dropping mercury electrode (DME) as working electrode (m = 0.37 mg s⁻¹, t = 3.6 s and h = 60 cm) and a saturated calomel electrode (SCE) as reference, was used.

Voltammetric and stripping measurements were carried out using a potentiostat model 273 A (Advanced Analytics, USA) with a software model HQ 2030. The three-electrode system used consisted of a glassy carbon working electrode (IJ Cambria Scientific Ltd. of a 3 mm diameter), Ag/AgCl (3.5 mol L⁻¹ KCl) reference electrode and a Pt auxiliary electrode.

Before each measurement, the glassy carbon electrode was polished with an aqueous slurry of 0.03 μm alumina powder on a piece of cloth until a mirror-like finish was obtained; then it was rinsed with distilled water and dried with a non-abrasive tissue paper. Working solutions were purged with pure nitrogen for 5 min at room temperature, before voltammetric measurements.

The pH values were measured using a pH-meter model HI 8014, Hanna Instruments (Italy).

2.3 Procedure

The general procedure used for dc-polarography and voltammetry measurements was as follows: an aliquot (9.0 mL) of BR buffer of the desired pH value was placed in the working cell and deoxygenated for 5 min, then a negatively directed dc scan was initiated between 0.0 and −2.0 V. After background current was obtained, the run was repeated in the presence of the analyte.

The operational parameters of the linear scan stripping voltammetry were: scan rate 500 mV s⁻¹ and step height 5 mV. The analyzed solutions were purged with pure nitrogen gas for 5 min and the solutions were stirred at the same stirring rate during the accumulation step followed by a rest period of 10 s before applying the potential.

3.1 Polarographic behavior of secnidazole

The dc polarographic behavior of 1 × 10⁻⁴ mol L⁻¹ secnidazole was examined in BR buffer (pH 2.0–11.8). Two reduction waves were produced in acidic solutions, the height of the second wave equals to the half height of the first one. In alkaline solutions, the height of the second wave decreased with increasing the pH till it vanished at pH > 9, Fig. 1.

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Figure 1

dc-Polarograms of 1 × 10⁻⁴ mol L⁻¹ secnidazole in BR buffer solutions of different pH values: (a) 2.4; (b) 3.6; (c) 4.5; (d) 5.4; (e) 6.4; (f) 7.3; (g) 8.5; (h) 9.6; (i) 11.8.

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The half-wave potentials (E_1/2) of both waves are dependant on pH and shifted towards more negative potential on increasing the pH of the medium. This behavior indicates that protonation takes place before first electron uptake (Meites, 1965). The height of the first wave was found to be pH-independent, whereas the height of the second wave decreased with increasing the pH in the entire pH range. The heights of the waves were found to be limited by diffusion as evidenced by the linearity of plots of i_d vs √h and i_d vs depolarizer concentration.

The logarithmic analysis of the polarograms confirmed the irreversible nature of the electrode process (Zuman, 1969). The plots of E_de vs log(i/i_d − i) for the reduction wave at different pH values gave straight lines of slope values S₁ mV (S₁ = 59/αn_a). The estimated values of α (transfer coefficient) and n_a (number of electrons participating in the rate-determining step) are listed in Table 1. The plots of E_1/2 vs pH are linear with slopes (S₂) 60.5 and 140 mV pH⁻¹, for the first and second waves, respectively.

The number of hydrogen ions ( $Z_{\text{H}}^{+}$ ) participated in the rate-determining step was calculated using the relation: $$Z_{\text{H}}^{+}=S_2/S_1$$

The values of the transfer coefficient α estimated either from S₁ (α = 59/S₁n_a) or S₂ (α = (59/S₂)( $Z_{\text{H}}^{+}$ /n_a)) are listed in Table 1. It is clear from the data obtained that one proton participated in the rate-determining step for the two waves, whereas two electrons or one electron participated in the rate-determining step for the first and the second waves, respectively.

3.2 Coulometric analysis

From coulometric analysis, the number of electrons involved in an acidic media is six while four electrons involved in the reduction process in an alkaline media. A mechanism can be proposed for the reduction of this compound which is analogous for the reduction of aromatic nitro derivatives (Rubinstein, 1985; Tallec et al., 2000; Radi and El Ries, 1999; Fotouhi and Faramarzi, 2004), Scheme 2.

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Scheme 2

Electrode reduction mechanism of secnidazole at different pH values.

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3.3 Cyclic voltammetry

The nature of the electrochemical process was studied by cyclic voltammetry. A typical cyclic voltammogram of 1 × 10⁻⁴ mol L⁻¹ secnidazole at glassy carbon electrode in BR buffer at pH 7.34 without accumulation is shown in Fig. 2. One cathodic peak was observed over the entire pH range used (2.0–11.8), its height decreased with increasing pH. On the reverse scan, no anodic peak was observed, indicating that the reduction of secnidazole at GCE is an irreversible process.

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Figure 2

Cyclic voltammogram of 1 × 10⁻⁴ mol L⁻¹ secnidazole in BR buffer at pH 7.34 at scan rate of 500 mV s⁻¹.

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The effect of sweep rate (υ) on the cyclic voltammograms was examined from 25 to 500 mV s⁻¹ at a fixed concentration of 1 × 10⁻⁴ mol L⁻¹ secnidazole. The peak potential (E_p) shifted cathodically, as expected for irreversible reduction process. The peak current (i_p) increased steadily with increasing (υ) and a straight line was observed when (i_p) was plotted against (υ^1/2), revealing that the cathodic reduction is mainly controlled by diffusion.

A straight line was obtained from E_p–log υ relationship; described by the equation: E_p= 0.1443 log υ + 0.5016 (n = 5, r = 0.994) at pH 3.10. From the slope of the straight line, αn_a value was calculated by applying the equation, ΔE/Δlog υ = 30/αn_a (Sawyer et al., 1995). The value of α was found to be 0.32, confirming the irreversible nature of the reduction reaction.

A plot of log i_p vs log υ gave a straight line with slope of 0.63, indicating that the reaction is controlled mainly by diffusion with slight adsorption contribution (Laviron et al., 1980).

3.4 Cathodic adsorptive stripping voltammetry

3.4.2

3.4.2 Effect of accumulation potential

The effect of the accumulation potential (E_acc) on the adsorptive stripping current was evaluated over the range 0.0 to −0.4 V for 5 × 10⁻⁵ mol L⁻¹ secnidazole in BR buffer of pH 5.3, for an accumulation period of 360 s, Fig. 3. The results obtained shown that a maximum i_p value was obtained at accumulation potential −0.4 V.

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Figure 3

Effect of deposition potential on 5 × 10⁻⁵ mol L⁻¹ secnidazole in BR buffer solution of pH 5.3 at t_acc= 60 s, υ = 500 mV s⁻¹ and step height = 5 mV.

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3.4.3

3.4.3 Effect of accumulation time

The dependence of peak current on accumulation time (t_acc) was studied at concentration 5 × 10⁻⁵ mol L⁻¹ secnidazole. The peak current increased with increasing accumulation time from 0.0 to 60 s, then it decreased with increasing time (Fig. 4). Hence, an accumulation time of 60 s was chosen to evaluate the best work conditions to the proposed method.

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Figure 4

Effect of deposition time on 5 × 10⁻⁵ mol L⁻¹ secnidazole in BR buffer solution of pH 5.3 at E_acc= −0.4 V, υ = 500 mV s⁻¹ and step height = 5 mV.

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3.5 Instrumental parameters

Fig. 5 displays the resulting peak current vs scan rate (υ) for 5 × 10⁻⁵ mol L⁻¹ secnidazole. Maximum response was obtained at a scan rate of 500 mV s⁻¹, so this value of scan rate was chosen for analytical purposes. The dependence of peak current on the step height was examined in the range 5–30 mV. The peak current increased slightly with increasing step height, but a well-defined peak was observed at a step height of 5 mV, so this value was the best value for quantitative applications.

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Figure 5

Effect of scan rate on 5 × 10⁻⁵ mol L⁻¹ secnidazole in BR buffer solution of pH 5.3 at E_acc= −0.4 V, t_acc= 30 s and step height = 5 mV.

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3.6 Calibration plots

Using the optimized experimental conditions for the determination of secnidazole, the linearity of the i_p response to different analyte concentrations was evaluated. Linear sweep adsorptive voltammograms were recorded at scan rate 500 mV s⁻¹ using an accumulation potential of −0.4 V for an accumulation time of 60 s, for solutions containing increasing concentrations of secnidazole. The results showed that the peak current increased linearly with the analyte concentration in the range 4.0 × 10⁻⁶–1.2 × 10⁻⁴ mol L⁻¹ (0.66–20.4 μg mL⁻¹). The peak current is related to the concentration of secnidazole (C) by the following equation: $$i_{\text{p}}(\mathrm{\mu }\text{A})=3.35+9.025\times {10}^{7}C(\text{mol}{\text{L}}^{-1})$$

The linearity of this relationship was evaluated from the correlation coefficient (r = 0.9996) at n = 10. The detection limit of the proposed method is 1.2 × 10⁻⁶ mol L⁻¹, which was calculated as the concentration that gives a signal to background noise ratio equal to 3 (Househam et al., 1987). The validity of the method was supported by the constancy of i_p/C. Fig. 6 shows the dependence of the cathodic adsorptive peak current on secnidazole concentration under optimal conditions.

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Figure 6

CAdS voltammograms for different concentrations of secnidazole at optimal conditions: (a) background; (b) 4 × 10⁻⁶; (c) 6 × 10⁻⁶; (d) 8 × 10⁻⁶; (e) 1 × 10⁻⁵; (f) 2 × 10⁻⁵; (g) 4 × 10⁻⁵; (h) 6 × 10⁻⁵; (i) 8 × 10⁻⁵; (j) 1 × 10⁻⁴; (k) 1.2 × 10⁻⁴ mol L⁻¹.

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3.7 Precision and accuracy

In order to assess the precision, as percentage relative standard deviation (RSD%) and the accuracy, as percentage relative error (E_r%) of the proposed method, solutions containing five different concentrations of secnidazole were prepared and analyzed in five replicates. The data obtained are summarized in Table 2.

3.8 Analysis of pharmaceutical formulations

The validity of the proposed voltammetric method was investigated by assaying Secnidazole® tablets (each is labeled to contain 500 mg secnidazole per tablet). The recoveries were calculated with reference to the calibration graph. The statistical calculations for the assay results show a good precision of the proposed method, Table 3.

3.9 Analysis of serum

The proposed method was applied to the determination of secnidazole in spiked human serum using the standard addition method. The direct determination of secnidazole in human serum was found to be possible after dilution of the sample with the supporting electrolyte. The percentage recovery of the drug in serum, based on the average of four replicate measurements, is listed in Table 4. The values obtained for recovery are acceptable for biological fluids.