Abstract
Chlorpromazine was used as a homogeneous electrocatalyst in the oxidation of captopril. The anodic peak current of chlorpromazine was increased substantially in the presence of low concentrations of captopril (pH 4). Cyclic voltammetry and chronoamperometry were used to study the kinetics of the catalytic electron transfer reaction. The values of electron transfer coefficient () and catalytic rate constant () were estimated to be 0.34 and , respectively. Linear sweep voltammetry was used for the determination of captopril in the presence of chlorpromazine. A linear calibration curve was obtained in the concentration range of captopril of 10.0–300.0 μM, with a limit of detection of 3.65 μM. The relative standard deviation (RSD%) for 5 replicate measurements of captopril (100 μM) was 1.96%. The method was applied to the determination of captopril in pharmaceutical formulations and blood serum samples with satisfactory results.
1. Introduction
Captopril (CAP), 1-(3-mercapto-2-D-methyl-1-oxopropyl) proline (Scheme 1(a)), is an oral drug and a member of a class of drugs called angiotensin-converting enzyme (ACE) inhibitors. CAP has been widely used as antihypertensive drug and to moderate heart failure [1]. It normally works by lowering blood levels of angiotensin enzyme, to help relax the blood vessels and lower blood pressure. Relaxing the arteries, and as a consequence lowering of blood pressure, improves the pumping efficiency of a failing heart and improves cardiac output in patients with heart failure [2]. CAP with a thiol functional group may also act as a scavenger of free radicals in living systems [3–5].
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Several methods have been applied to the determination of CAP, including high-performance liquid chromatography [6–9], gas chromatography [10, 11], spectrophotometry [12, 13], fluorimetry [14–16], radioimmunoassay [17], chemiluminescence [18–20], atomic absorption spectrophotometry [21], Raman spectroscopy [22], capillary electrophoresis [23, 24], and electrochemical methods [25–30].
Direct electrochemical determination of pharmaceutical compounds has a number of limitations, such as low sensitivity and reproducibility, slow electron transfer kinetics, and high overpotentials. The chemical modifications with redox active materials (homogenous and heterogeneous catalysts) offer significant advantages in the design and development of electrochemical sensors. During the reaction, the mediator shuttles electrons between the analyte and the electrode with significant reduction in the activation overpotential.
In this study, chlorpromazine (CPZ, Scheme 1(b)), an antipsychotic drug, was used as a suitable homogeneous mediator in the electrooxidation of CAP. The proposed method was fast, selective, sensitive, and successful in the determination of CAP in real samples. Cyclic voltammetry (CV) and chronoamperometry were used to characterize the electrochemical properties of CPZ and to investigate its electrocatalytic effect on the CAP oxidation. Kinetic parameters such as electron transfer coefficient and the rate constant of catalytic reaction were estimated.
2. Experimental
2.1. Reagents
CPZ hydrochloride was purchased from Merck and CAP was from Sigma. All solutions were prepared from analytical reagent grade chemicals and were used as received from the suppliers without further purification. Doubly distilled water was used for the preparation of solutions. All experiments were carried out at ambient temperature (25°C). The CAP tablets (25 mg and 50 mg per tablets) were from Tehran Darou Co. (Tehran, Iran), and Iran Daru Co. (Tehran, Iran), respectively. A freshly prepared 10.0 mM aqueous solution of CAP was used for the preparation of more dilute solutions. The stock solution was kept in refrigerator at 4°C in the dark.
2.2. Apparatus
Electrochemical measurements were performed using a Micro-Autolab (μ3AUT-70751), potentiostat/galvanostat instrument connected to a three-electrode cell. The electrochemical data acquisition was performed using the software NOVA 1.6. A conventional three-electrode cell was used containing a glassy carbon disk electrode (GCE) as the working electrode, and a Pt wire was directly immersed in the solution as the auxiliary electrode and a saturated calomel electrode (SCE) as the reference electrode. The geometrical area of the glassy carbon electrode was 0.0314 cm2. A Metrohm 781 pH/ion meter was used for pH measurements.
2.3. Preparation of Real Samples
Ten tablets of CAP (25 or 50 mg per tablets) were completely ground, and 10 mg of the fine powder was accurately weighed and dissolved in 10 mL of phosphate buffer solution (0.05 M, pH 4) using ultrasonication (30 min). The blood serum sample was centrifuged and the supernatant was diluted 50 times with water without any further pretreatment. The standard addition method was used for the determination of CAP in both kinds of real samples.
2.4. General Procedure
The GCE was polished with an alumina fine powder (0.05 μm) in a water slurry using a polishing cloth followed by rinsing thoroughly with distilled water. In a typical experiment, the electrodes were immersed in a solution containing 1.5 mM CPZ and phosphate buffer (pH 4.0, 0.05 M). The potential was swept from +0.2 to +0.9 V versus SCE with a scan rate of 50 mV s−1. The experiment was repeated in the presence of CAP as the sample solution.
3. Results and Discussion
3.1. Electrochemical Oxidation of CPZ at the GCE
The electrochemical behavior of CPZ was studied by cyclic voltammetry, and the results obtained were similar to previous reports [31]. Figure 1 shows the cyclic voltammograms of a 1.5 mM CPZ solution at the surface of GCE and different scan rates (pH 4). CPZ shows a redox couple at about +0.60 and +0.69 V versus SCE at the experimental conditions. The values of half-wave potential [] and peak potential separation () are about 0.64 and 0.09 V at scan rate of 10 mV s−1, respectively. The value of corresponds to a one-electron quasireversible process which can be represented as follows.
Scheme 1 (oxidation of CPZ). where refers to the CPZ cation radical.
Both anodic and cathodic peak currents increased along with the increase in scan rate (Figure 1(A)). The plot of anodic peak current versus square root of sweep rate (10–100 mV s−1) was linear (Figure 1(B)) which suggests a simple diffusion-controlled mechanism. The results of polarization studies (Figure 1(C)) showed that the average Tafel [32] slope, RT, was 89.5 mV. Assuming the rate determining step involves a one-electron process (), a value of 0.34 is obtained for the charge transfer coefficient () of the redox reaction in Scheme 1.
The study of the effect of pH on the voltammetric response of CPZ revealed that the system shows more reversibility in acidic solutions, and a sodium phosphate buffer with pH 4 was selected as a proper medium in further studies.
3.2. Electrocatalytic Behavior of CPZ towards CAP
Figure 2 shows the electrocatalytic oxidation of CAP in the presence of CPZ at a GCE surface. As is obvious, at the potential range studied (0.2–1.0 V), CAP was not electroactive (Figure 2(b)). On the other hand, the anodic current of CPZ was increased substantially in the presence of low concentrations of CAP (Figures 2(c) and 2(d)). This observation is an evidence for electrocatalytic oxidation of CAP by CPZ. The electrocatalytic activity of CPZ was reported in previous reports [33].
The mechanism of EC′ reaction could be represented as follows [34].
Scheme 2 (electrocatalytic effect of CPZ on the oxidation of CAP).
The influence of CPZ concentration on the electrocatalytic peak current was studied at three different concentrations of CAP (Figures 3(a)–3(c)) at pH 4.0, and in the range of 0.10 to 2.40 mM CPZ. The results showed that by increasing the concentration of CPZ up to 1.55 mM the peak current increased, whereas higher concentrations of CPZ caused a slight decrease on the magnitude of peak current, which may be due to the formation of CPZ aggregates. Therefore, 1.50 mM CPZ concentration was selected for further studies.
The scan rate dependence of cyclic voltammograms of a CAP solution (0.5 mM) in the presence of CPZ (1.50 mM) was studied (Figure 4(A)) at a GCE surface. Figure 4(B) shows that the anodic peak current increases linearly with the square root of the sweep rate as expected for a diffusion-controlled reaction. The regression equation is (μA) = 1.2 (mV/s)0.5 + 3.43 with = 0.9957. A plot of the scan rate-normalized current () versus sweep rate (Figure 4(C)) exhibits the characteristic shape typical of an EC′ process [32].
3.3. Chronoamperometric Studies
In order to obtain an estimation of the rate constant of the catalytic oxidation of CAP (), chronoamperometric method was applied to the system. The chronoamperograms obtained for a series of CAP solutions (with a step potential of +0.850 V versus SCE) are shown in Figure 5(A). Using the Cottrell equation (3) [33] it can be seen that the plot of versus under diffusion-controlled conditions is linear (Figure 5(B)) where is the concentration of CAP in mol/cm3 and is the time elapsed in seconds.
The rate constant for the chemical reaction between CPZ and CAP () is determined according to the method described in the literature [30, 34, 35] by chronoamperometry using (4) where is the catalytic current of CPZ in the presence of CAP and is the limiting current in the absence of CAP.
From the slope of versus 1/2 for five different concentrations of CAP, the average value of was calculated to be 8.48 × 102 M−1 sec−1 (Figure 5(C)). This value of rate constant explains the sharp catalytic peak observed for the oxidation of CAP at the surface of GCE.
3.4. Determination of CAP by Its Electrocatalytic Oxidation in the Presence of CPZ
Linear sweep voltammetry (LSV) was used for construction of a calibration plot for CAP in a concentration range of 10.0–300.0 μM under the optimum conditions and a scan rate of 50 mV s−1 (Figure 6). The regression line (inset) equation was = 8 × 10−6 + 0.01 with = 0.997. The limit of detection (3 , where is the standard deviation of the blank signal and is the slope of the calibration curve) equals 3.65 μM CAP. The relative standard deviation (%R.S.D.) for 5 replicate measurements of 100 μM CAP was 1.96%.
The method was applied to the determination of CAP in pharmaceutical preparations (tablets) and biological fluids (spiked blood serum samples) using standard addition technique (Figure 7). As is obvious from Table 1, the results of the proposed voltammetric method, compared to labelled or spiked amounts of CAP, are quite satisfactory.
4. Conclusions
In the present work, chlorpromazine was used as a redox mediator for the homogeneous electrocatalytic oxidation of captopril in aqueous media (pH 4) at the surface of a glassy carbon electrode. The electrochemical characteristics of chlorpromazine and its catalytic effect on the oxidation of captopril were investigated. The rate constant of the catalytic reaction was estimated using chronoamperometry.
Linear sweep voltammetry was successfully applied to the determination of captopril in the presence of an optimum concentration of chlorpromazine at pH 4. The results of the determination of captopril in its tablets as well as blood serum samples spiked with low concentrations of the drug were satisfactory. The proposed electrochemical method was simple, fast, and selective without the need for electrode modification.