Anodic behavior of sertindole and its voltammetric determination in pharmaceuticals and human serum using glassy carbon and boron-doped diamond electrodes

Electrochimica Acta 54 (2009) 1893–1903

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Anodic behavior of sertindole and its voltammetric determination in pharmaceuticals and human serum using glassy carbon and boron-doped diamond electrodes Yuksel Altun a , Burcu Dogan-Topal b , Bengi Uslu b , Sibel A. Ozkan b,∗ a b

Gazi University, Faculty of Education, Department of Chemistry, 06500 Teknikokullar, Ankara, Turkey Ankara University, Faculty of Pharmacy, Department of Analytical Chemistry, 06100 Tandogan, Ankara, Turkey

a r t i c l e

i n f o

Article history: Received 19 August 2008 Received in revised form 3 October 2008 Accepted 8 October 2008 Available online 18 October 2008 Keywords: Sertindole Oxidation mechanism Glassy carbon Boron-doped diamond Voltammetry

a b s t r a c t The electrochemical oxidation of sertindole was investigated using cyclic, linear sweep voltammetry at a glassy carbon and boron-doped diamond electrodes. The aim of this study was to determine sertindole levels in serum and pharmaceutical formulations, by means of electrochemical methods. In cyclic voltammetry, depending on pH values, sertindole showed one or two irreversible oxidation responses. These two responses were found related to the different electroactive part of the molecule. Using second and sharp oxidation peak, two voltammetric methods were described for the determination of sertindole by differential pulse and square wave voltammetry at the glassy carbon and boron-doped diamond electrodes. Under optimized conditions, the current showed a linear dependence with concentration in the range between 1 × 10−6 and 1 × 10−4 M in acetate buffer at pH 3.5 and between 4 × 10−6 and 1 × 10−4 M in spiked human serum samples for both methods. The repeatability, reproducibility, selectivity, precision and accuracy of all the methods in all media were investigated and calculated. These methods were successfully applied for the analysis of sertindole pharmaceutical dosage forms and human serum samples. No electroactive interferences from the tablet excipients and endogenous substances from biological material were found. © 2008 Elsevier Ltd. All rights reserved.

1. Introduction Sertindole is one of the newer atypical antipsychotic medications available, which is thought to give a lower incidence of extra-pyramidal side effects at clinically effective doses than typical antipsychotic drugs. Sertindole is used to help treat the symptoms of schizophrenia. It is an arylpiperidylindole antipsychotic with affinity for central dopamine (D2 ), serotonin 5-HT2 and ␣1 adrenergic receptors [1–3]. Sertindole is slowly and wellabsorbed when administered orally with good penetration of the blood–brain barrier. The peak concentrations occur about 10 h after oral doses. It is about 99.5% bound to plasma proteins and readily crosses the placenta. Its elimination half-life is 3 days. Sertindole is metabolized in the liver mediated by cytochromes p450 2D6 and P450 3A. The two major metabolites, dehydrosertindole and norsertindole, do not appear to have any therapeutic effect [1–3]. Sertindole has been studied and determined by very few procedures. All the reported methods for the determination of

∗ Corresponding author. Tel.: +90 312 2238243; fax: +90 312 2238243. E-mail address: [email protected] (S.A. Ozkan). 0013-4686/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2008.10.010

sertindole in biological fluids rely on the use of separation techniques such as liquid chromatography-UV detection [4,5], liquid chromatography–MS–MS detection [6–8], liquid chromatographyfluorimetric detection [9], and capillary electrophoresis [10,11] techniques. Most of the reported methods required timeconsuming sample pretreatment and solid-phase extraction steps prior to the drug analysis, expensive reagents and equipment, which are not economically feasible for routine use biological media studies. Also these methods were influenced by interference of endogenous substances and potential loss of drugs in the re-extraction procedure and involve lengthy, tedious and timeconsuming plasma sample preparation and extraction processes and requires an expensive instrumentation. In the last decades modern computer-based voltammetric techniques have been used to realize the determination of organic chemicals in diverse types of samples, especially pharmaceutical field [12–17]. The advance in experimental electrochemical techniques in the field of drug analysis is because of their simplicity, low cost, and relatively short analysis times no need for derivatizations or time-consuming extraction steps when compared with other techniques [12–17]. Cyclic voltammetry is the most widely used technique for acquiring qualitative information about electrochemical reactions. The results of such investigations into the

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redox chemistry of biomolecules and drugs might have profound effects on the understanding of their in vivo redox processes or pharmaceutical activity [12–17]. Boron-doped diamond electrodes are used as useful alternatives to other carbon-based electrodes. Glassy carbon electrodes are the most common carbon-based electrodes in current use. However, the use of diamond electrodes in electroanalytical chemistry is in its infancy, but the results obtained to date indicate a bright future for this advanced material. Diamond electrodes open up new opportunities for work under extreme conditions such as at extremely high anodic potentials, in chemically extremely aggressive media, e.g. strongly acidic media, and are now available commercially in polycrystalline boron-doped form [16,17]. The widespread use of sertindole and the need for clinical and pharmacological study require fast and sensitive analytical techniques to assay the presence of the drug in pharmaceutical dosage forms and biological samples. There have been no studies published related to the detailed electrochemical behavior and voltammetric determination of sertindole at glassy carbon and boron-doped diamond electrodes from pharmaceutical dosage forms and biological samples. Most pharmacologically active molecules contain one or more ionizing groups and it is well-known that knowledge of the ionization state of a drug, indicated by the pKa value, is critical for understanding many properties with respect to pH. This information is applied to dispensing problem and dosage-form development, to decide to, what the dosage form should be adjusted to provide optimum bioavailability, and to predict solubility in a solvent media at a given pH and drug concentration [18,19]. Moreover, the study of acid–base behavior of analytes in binary organic–water solvent systems can be a key in predicting influence of pH on electroanalytical characteristics of drugs. In the literature the dissociation constant of the sertindole in water, pure acetonitrile and methanol media were found [20], however, no data were available in 30% (v/v) acetonitrile–water medium that was investigated in our electroanalytical study, using UV spectrophotometry. The goal of this work is to study detailed voltammetric behavior to obtain possible electroactive centers of sertindole at glassy carbon and boron-doped diamond electrodes using cyclic, linear sweep, DPV and SWV techniques. Also the development of new voltammetric methods for the fully validated, simple, rapid, selective and sensitive procedures determination of sertindole in pharmaceutical dosage forms, raw materials and spiked human serum samples without any time-consuming extraction or evaporation steps prior to drug assay were aimed. To find out the relationship between the oxidative behavior and protonation constant, pKa value of sertindole was determined. The proposed methods might be alternatives to the LC techniques in therapeutic drug monitoring or the experimental data might be used for the development LC–EC method. 2. Experimental 2.1. Apparatus All voltammetric measurements at a glassy carbon and borondoped diamond electrodes were performed using a BAS 100 W (Bioanalytical System, USA) electrochemical analyzer. The three electrode system contained either a boron-doped diamond working electrode (Windsor Scientific Ltd.; Ø: 3 mm, diameter) or a glassy carbon working electrode (BAS; Ø: 3 mm diameter), with a platinum wire counter electrode and Ag/AgCl reference electrode (KCl 3 M, BAS) and a standard one-compartment three-electrode cell of 10 mL capacity. Before each experiment, the diamond or

glassy carbon electrode was polished manually with slurries prepared from 0.01 ␮m aluminum oxide on a smooth polishing pad (BAS velvet polishing pat), then rinsed with double-distilled water thoroughly. All measurements were realized at room temperature. The pH values were measured using a pH meter Model 538 (WTW, Austria) using a combined electrode (glass electrodereference electrode) with an accuracy of ±0.05 pH. For analytical application, the following parameters were employed: SW voltammetry pulse amplitude, 25 mV; frequency, 15 Hz; scan increment, 4 mV. DPV parameters were: pulse amplitude, 50 mV; pulse width 50 ms; scan rate, 20 mV s−1 . The pKa value of the sertindole was determined by means of the data obtained from spectrometric titrations in 30% (v/v) acetonitrile–water mixture at 25.0 ± 0.1 ◦ C and in 0.1 mol L−1 ionic strength (NaCl). For this procedure a PerkinElmer Lambda-25 spectrophotometer, with 10 mm path length cells connected to a personal computer was used for acquisition of UV–visible absorbance data. A peristaltic pump equipped with the spectrometer was used to circulate the solution from the titration vessel to the spectrophotometer cell, and vice versa, through Teflon or Tygon tubes in a closed loop circuit with continuous flow. For potentiometric titrations, a special glass vessel that was used also in our earlier studies [21,22] with a double wall, with entries for the combined glass electrode (Mettler Toledo Inlab 412), nitrogen and base from the burette were used. Temperature was maintained constant inside the cell at (25.0 ± 0.1) ◦ C, by circulating water from an external thermostat (Heto CBN 8-30 and temperature control unit Heto HMT 200). The standard emf values, E◦ , of the potentiometric cell with 30% (v/v) acetonitrile–water mixture were evaluated from titrations of diluted HCl solutions in the desired solvent using NaOH solution in the same solvent as the titrant, and checking the calibration parameters from the Gran plots [23–25]. A solution of sertindole (42.0 mL containing 1 × 10−5 mol L−1 sertindole) in 30% (v/v) acetonitrile–water were titrated with NaOH in potentiometric titration cell in the pH range of 2.0–11.0. After each addition of titrant, and accurate reading of emf, the test solution was pumped to a spectrometric flow-cell by means of a peristaltic pump. UV–vis spectra were recorded with 1 nm resolution at 200–400 nm intervals in order to obtain different spectra around the maximum  for each sertindole. 2.2. Computational methods The data evaluation was performed by using STAR program developed by Dr. Jose Luis Beltrán et al. (Stability Constants by Absorbance Readings) [26], which calculates stability constants and molar absorbance values of the pure species by multilinear regression. The STAR program requires a previous model of the chemical equilibria, based upon the existence of certain chemical species, to be postulated. On the other hand, the SPARC online calculator program (SPARC Performs Automated Reasoning in Chemistry, http://ibmlc2. chem.uga.edu/sparc/) [27–29] has been used to compute pKa value of sertindole in water media. Like all chemical reactivity parameters addressed in SPARC, molecular structures are broken into functional units called the reaction center and the perturber in order to estimate pKa in water. After entering molecular structure and the sign of the atom of the ionizable group (the piperidinyl nitrogen in this study), the program generates SMILE strings (Cl-c(ccc1N2c(ccc3F)cc3)cc1C( C2)C(CCN4CCN(C( O)N5)CC5)CC4 for sertindole) and predicts both macroscopic and microscopic pKa values strictly from molecular structure. The temperature of 25 ◦ C was selected for the calculations.

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2.3. Reagents

2.6. Analysis of spiked human serum samples

Sertindole and its pharmaceutical dosage form Serdolect® tablets were kindly supplied by Lundbeck Pharm. Ind. (Istanbul, Turkey). Model compounds, etodolac, melatonin, fluvastatin, pimozide, droperidol, benperidol and haloperidol were kindly supplied from different pharmaceutical companies. Indol, indol3-acetic acid and indol-3-butiric acid were also used as model the compounds and they were supplied from Sigma or Merck. All chemicals for preparation of buffers and supporting electrolytes were reagent grade from Merck or Sigma. Stock solutions of sertindole (1 × 10−3 M) were prepared in acetonitrile and kept in the dark in a refrigerator. Sertindole working solutions under voltammetric investigations were prepared by dilution of the stock solution and contained 30% acetonitrile. The stock solutions of all other compounds were also prepared in methanol or acetonitrile or bi-distilled water and kept in the dark in a refrigerator. Four different supporting electrolytes, namely sulphuric acid (0.1, 0.2, 0.3 and 0.5 M; pH 0.0–1.0), phosphate buffer (0.2 M; pH 3.0–7.99), acetate buffer (0.2 M; pH 3.5–5.5), Britton–Robinson buffer (0.04 M, pH 2.14–12.03) were prepared in bi-distilled water. The calibration curve for DPV and SWV analysis was constructed by plotting the peak current against the sertindole concentration for both electrodes. The ruggedness and precision were checked at different days, within day (n = 5) and between days (n = 5) for two different concentrations. Relative standard deviations were calculated to check the ruggedness and precision of the method [30–33]. The accuracy and precision of the developed methods are described in a quantitative fashion by the use of relative errors (Bias%). One example of the Bias% is the accuracy, which describes the deviation from the expected results. All solutions were protected from light and were used within the same day after preparation to avoid decomposition. However, current–potential curves of sertindole solutions recorded 3 weeks after preparation but did not show any appreciable change in assay values.

Drug-free human serum samples, obtained from healthy subjects (after obtaining their written consent) were stored frozen until assay. After gentle, thawing, an aliquot volume of sample was spiked with sertindole dissolved in acetonitrile. Then the mixture was treated acetonitrile as serum denaturing and precipitating agent, and then the volume was completed with the same serum sample. The final concentration of sertindole was maintained to 1 × 10−3 M. Acetonitrile removes serum proteins effectively; the appropriate ratio of volumes to eliminate the protein was found 1.5–1. After vortexing for 30 s, the mixture was then centrifuged for 10 min at 5000 × g in order to eliminate serum protein residues, and supernatant was taken carefully. Appropriate volumes of this supernatant were transferred into the volumetric flask and diluted up to the volume with the selected supporting electrolytes. The concentration of sertindole varied in the range between 4 × 10−6 and 2 × 10−4 M by DPV, and between 4 × 10−6 and 1 × 10−4 M by SWV methods in human serum samples. Quantifications were performed by means of the calibration curve method from the related calibration equations.

2.4. Serdolect® tablet assay procedure Ten tablets of Serdolect® (Lundbeck Pharm Ind., Istanbul), containing 20 mg sertindole per tablet, were accurately weighed and crushed to a homogeneous fine powder in a mortar. An adequate amount of this powder, corresponding to a stock solution of concentration 1 × 10−3 M, was weighed, transferred into a 50 mL calibrated flask and completed to the volume with acetonitrile. The contents of the flask were sonicated for 10 min to achieve complete dissolution. Analyzed solutions were prepared by taking aliquots of the clear supernatant and diluting with the selected supporting electrolyte. Voltammograms were recorded according to the DPV and SWV parameters and as in pure sertindole using glassy carbon and boron-doped diamond electrodes.

3. Results and discussion The dissociation constant value was determined for the involved equilibria for the sertindole in 30% (v/v) acetonitrile–water mixture at 25.0 ± 0.1 ◦ C with addition of 0.1 M NaCl (ionic strength) was found as 8.22 (0.02). In Scheme 1, it can be seen that the sertindole comprise several nitrogen atoms, one of them is on the piperidine ring. Comparing to piperidine nitrogen, the other nitrogen atoms of the molecule are less basic, and can thus be protonated at too high pH. Therefore it can be concluded that pKa value is associated with the piperidine nitrogen. In Fig. 1, UV–vis spectra of sertindole at 200–350 nm intervals and different pH values were given in 30% acetonitrile–water mixture. The dissociation constant in water media by means of SPARC program was found 8.01. It is necessary to note that SPARC calculation is not influenced by character (properties) of environment in which experimental determination takes place. This fact has to be taken into account when the results are compared. pKa values of sertindole increased depending on the percentage of acetonitrile from 0% to 30% (v/v). This pKa value in 30% (v/v) acetonitrile–water is lower than the pure acetonitrile pKa = 14.6 [20]. This result was found in agreement with previously obtained values in acetonitrile–water mixtures [34–36]. In dissociation of a monocharged cation acid such as

2.5. Recovery experiments Recovery studies have shown the possible interferences from common excipients. To study the accuracy and reproducibility of the proposed techniques, recovery experiments were carried out using the standard addition method. In order to find out whether the excipients show any interference with the analysis, known amounts of pure sertindole were added to the pre-analyzed tablet formulation and the mixtures were analyzed by the proposed methods. The recovery of sertindole was calculated using the corresponding regression equations of previously plotted calibration plots. The recovery results were determined based on five parallel analyses.

Scheme 1. The dissociation equilibrium of sertindole.

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Fig. 1. The wavelength (nm)–absorbance graphic of sertindole as a function of pH in 30% acetonitrile–water mixtures.

ammonium ion of piperidine ring of sertindole, there is no change in the number of charges involved in the process (HA+ ↔ H+ + A), and thus the electrostatic term makes no contribution on the isoelectric dissociation reaction. This behavior observed in the study of the acid–base properties of sertindole could be explained in terms of structural features and preferential solvation by water in

acetonitrile-water mixtures [34,35]. In acetonitrile–water mixtures there are three regions [37–39]. In water-rich region the structure of water (xacetonitrile < 0.15) remains constant, the solutes are preferentially solvated by water and thus the variations of pKa values are minimal [34,35]. Sertindole is an easily oxidizable molecule. There were no previous electrochemical data available concerning redox behavior of sertindole. The peak currents and peak potentials were determined in supporting electrolytes containing 30% acetonitrile (v/v) to maintain solubility. Therefore, several measurements with different electrochemical techniques were performed using various supporting electrolytes and buffers in order to obtain such information. As first step, sertindole was subjected to a cyclic and linear sweep voltammetric studies with the aim to characterize its electrochemical oxidation behavior in detail on the boron-doped diamond and glassy carbon electrodes. For this reason, the electrochemical behavior of sertindole was studied over a wide pH range (0.0–12.0) at a boron-doped diamond and glassy carbon disc electrodes in buffered aqueous media. Cyclic and linear sweep voltammograms of sertindole exhibited one distinct and well defined anodic peak and/or one ill-defined anodic wave at different potential values depending on pH values, supporting electrolyte composition and electrode material (Fig. 2a–d). After pH 5.0 and 6.0, the sharp anodic peak gave a shoulder and splitted as two peaks on both electrodes (Fig. 2c and d). Cyclic voltammetric measurements showed an irreversible nature of the oxidation process. The scanning was started at 0.0 V in the positive direction at pH 3.5 acetate buffer, the anodic oxidation of sertindole did not occur until about +1.20 V on glassy carbon and +1.15 V on boron-doped diamond electrode. By reversing at +1.80 V no reduction signal corresponding to the anodic response was

Fig. 2. Cyclic voltammograms of 1 × 10−4 M sertindole in 0.1 M H2 SO4 (a); Britton–Robinson buffer at pH 3.02 (b); Britton–Robinson buffer at pH 5.02 (c); phosphate buffer at pH 6.0 (d) with constant amount acetonitrile (30%). Scan rate, 100 mV s−1 . (1) Glassy carbon electrode and (2) boron-doped diamond electrode.

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sertindole undergoes one main irreversible oxidation process and one additional ill defined wave, which shifted towards less positive potentials as the pH was increased for both electrodes. Thus the observed pH dependence indicates that the electroactive group which is corresponding to the sertindole main oxidation peak is in acid–base equilibrium with pKa about 8.0. Above pH 8.0 the peak potential nearly becomes pH independent (Figs. 4a and 5a). This can be associated with the pKa of sertindole that was found as 8.22 (0.02) using spectrophotometric method. This break could be due to a change in protonation–deprotonation process of electroactive molecule. The pH-independent zone above pH 8.0 means that there are no proton transfer steps before the electron transfer ratedetermining step. Since, no dissociation occurs before the electron transfer rate-determining step, the oxidation potential remains pHindependent for both electrodes. At pH < pKa , the conjugate base must be formed by a rapid dissociation of the protonated form. In the linear part of Figs. 4a and 5a, a disharmony was obtained between Britton–Robinson (0.04 M) and acetate buffer (0.2 M) in the pH range 3–6. This may be due to the differences of the ionic strength and the ionic species of the buffers (Figs. 4a and 5a). The highly linear segments between pH 2.0 and 8.0 can be expressed by the following equations in Britton–Robinson buffer for the main peak using DPV technique. For glassy carbon electrode; Ep = 1242 − 55.52 pH; r = 0.974 (between pH 2.0 and 8.0)

Fig. 3. Repetitive cyclic voltammograms of 1 × 10−4 M sertindole in acetate buffer at pH 3.5 with constant amount acetonitrile (30%) with glassy carbon electrode (a) and boron-doped diamond electrode (b). Scan rate, 100 mV s−1 . The numbers indicate the number of scan.

observed on the cathodic branch. The sertindole peak decreased to the second or higher cycles for both electrodes (Fig. 3). This phenomenon may be partly attributed to the consumption of adsorbed sertindole on the electrode surface. Comparison of the response at the boron-doped diamond electrode with glassy carbon electrode reveals potential shifts (to less negative potentials) of 50 mV in pH 3.5 acetate buffer. These shifts clearly showed that the investigated compound was easily oxidized on boron-doped diamond electrode than the glassy carbon electrode. The lower background currents were obtained using boron-doped diamond electrode compared with the glassy carbon electrode in Figs. 2 and 3. Also boron-doped diamond electrode demonstrates a better electrochemical reversibility than the glassy carbon electrode at the less positive potential. Nonetheless, the peak potentials shifted to the more positive potentials about 65 mV for boron-doped diamond electrode and about 102 mV for glassy carbon electrode to the anodic direction when the scan rate was increased. Next, the response of pH on the voltammetric waves at glassy carbon and boron-doped diamond electrodes were explored. The effect of pH on peak potential and peak intensity were studied using CV, DPV and SWV techniques for both electrodes, between pH 0.0 and 12.0. All obtained graphs from CV, DPV and SWV were found similar. For this reason, only DPV results for the main oxidation step was given as Ep –pH and Ip –pH graph in Fig. 4a and b for glassy carbon electrode and Fig. 5a and b for boron-doped diamond electrode, respectively. In all instances and techniques,

Fig. 4. Effect of pH on sertindole peak potential (a) and peak current (b); sertindole concentration 1 × 10−4 M with constant amount acetonitrile (30%) with glassy carbon electrode () H2 SO4 (0.1, 0.2, 0.3 and 0.5 M); () Britton–Robinson (0.04 M); () phosphate (0.2 M); and (♦) acetate buffers (0.2 M).

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for the glassy carbon electrode was observed with the square root of the scan rate as follows: Ip (␮A) = 0.191/2 (mV s−1 ) − 0.074;

r = 0.997.

Equation obtained for the boron-doped diamond electrode was as follows; Ip (␮A) = 0.211/2 (mV s−1 ) + 0.029;

r = 0.996.

A plot of the logarithm of the peak current versus the logarithm of the scan rate for glassy carbon and diamond electrodes gave a straight line with a slope of 0.53 and 0.47, respectively, close to the theoretical value of 0.5, which is expected for an ideal reaction of solution species [40]. Such dependence indicated that the oxidation of sertindole was indeed diffusion controlled. It is expected for an ideal reaction of solution species [40]. The equations obtained were: log Ip (␮A) = 0.53 log  (mV s−1 ) − 0.803 (r = 0.997) for glassy carbon electrode logIp (␮A) = 0.47 log  (mV s−1 ) − 0.607 (r = 0.993) for boron-doped diamond electrode

Fig. 5. Effect of pH on sertindole peak potential (a) and peak current (b); sertindole concentration 1 × 10−4 M with constant amount acetonitrile (30%) with borondoped diamond electrode () H2 SO4 (0.1, 0.2, 0.3 and 0.5 M); () Britton–Robinson (0.04 M); () phosphate (0.2 M) and (♦) acetate buffers (0.2 M).

For boron-doped diamond electrode; Ep = 1225.98 − 41.87 pH; r = 0.946 (between pH 2.0 and 8.0) For both electrodes, potential remains pH-independent at lower pH values than 2.0 and higher pH values than 8.0. The slopes of these equations are found as 55.52 mV/pH for glassy carbon electrode and 41.87 mV/pH for boron-doped diamond electrode. According to the obtained slope values of these equations, 2 electrons and 2 protons are involved in the rate-determining steps. These slopes being close to expected theoretical value of 59 mV/pH indicates that the number of proton and electron involved in the oxidation of sertindole is equal [12,13]. The experimental results showed that shapes of the curves and maximum peak current were better in acetate buffer at pH 3.5 supporting electrolyte with 30% constant amount of acetonitrile. The main peak that appeared with less positive potential was the best developed and became sharper and occurred as without shoulder in this supporting electrolyte. For this reason, acetate buffer solution at pH 3.5 was chosen with respect to have sharp response and better peak shape for the calibration equation. Scan rate studies were carried out to assess whether the processes at both electrodes, under diffusion or adsorption control. Using the concentration of 4.0 × 10−5 M in acetate buffer at pH 3.5, the voltammetric peak currents were observed as the scan rate over the range of 5–1000 mV s−1 for both electrodes. A linear response

Tafel analysis of voltammograms from the oxidation of 4.0 × 10−5 M sertindole (at a scan rate of 5 mV s−1 ) was conducted in acetate buffer at pH 3.5. The ˛n value of the anodic reaction corresponding to the voltammetric oxidation peak was obtained using Tafel plot (log I vs. Ep ). The value of 0.20 and 0.57 were obtained in acetate buffer at pH 3.5 for glassy carbon and boron-doped diamond electrodes, respectively. The intercepts for the log I = f(E) plot were obtained at −8.83 and −15.96 ␮A glassy carbon and boron-doped diamond electrodes, respectively. The exchange current densities are obtained as I0 = 1.47 × 10−9 ␮A/cm2 for glassy carbon and 1.09 × 10−16 ␮A/cm2 for boron-doped diamond electrodes. These values together with the absence of cathodic response in cyclic voltammetry (Figs. 2 and 3) confirmed the irreversibility of the oxidation process on both electrodes. When the logarithm of current at a potential of +1.05 V for DPV and +1.13 V for SWV obtained in acetate buffer at pH 3.5 was plotted against the logarithm of sertindole concentration in the range of 2.0 × 10−5 to 2.0 × 10−4 M using glassy carbon electrode. Also +1.03 V for DPV and +1.07 V for SWV obtained in acetate buffer at pH 3.5 was plotted against the logarithm of sertindole concentration in the range of 2.0 × 10−6 to 2.0 × 10−4 M using boron-doped diamond electrode. The linear relationships were obtained with both methods. Related equations were given as follows: log I (␮A) = 2.53 + 0.45 log C (M)

(r = 0.993; DPV);

log I (␮A) = 2.62 + 0.43 log C (M) (r = 0.996; SWV) using glassy carbon electrode; log I (␮A) = 3.68 + 0.76 log C (M) (r = 0.996; DPV); log I (␮A) = 3.67 + 0.73 log C (M) (r = 0.991; SWV) using boron-doped diamond electrode. The sertindole molecule is extensively metabolized in vivo [1–3]. To identify the grouping responsible for the oxidation of sertindole was compared with some selected model compounds. Cyclic voltammetric curves from the redox properties of active compounds and biomolecules might have profound effects on the understanding of the redox mechanism related to the activity of

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compounds. The electrochemical behavior of piperidine group of sertindole at both electrodes, showed that the main oxidation processes for using determination study are located on the piperidine. Sertindole was oxidized at similar potentials with piperidine contain model compounds on both electrodes. Considering the above comparison and the position of the break Ep versus pH plot for the first process of sertindole which is obtained after pH 5.0 and bearing in mind the oxidative process of nitrogen atom in the indole ring [41–47], it was assumed that the first oxidation step is located on the indole ring, similarly with model compounds main peak, and attributed to the oxidation of the nitrogen atom. In sertindole molecule piperidine oxidation process is sharper than the indole oxidation step, because it has a bonded nitrogen atom. This oxidation process can be attributed to a piperidine ring oxidation, to produce an N-oxide derivative [46–49]. Results showed that the anodic oxidation of piperidine looks irreversible and involves the loss of 2 electrons and 2 protons. For this reason, it looks covered with the piperidine oxidation step in more acidic pH values. Droperidol, benperidol, clonixin and pimozid contain piperidine group in its chemical structure and we suggest that the second (main) anodic reaction could be attributed to the oxidation of the nitrogen atom on the piperidine [46–49]. Both redox responses are shown with some selected model compound responses in Fig. 7. Our results on model compounds confirm that the electroactive center corresponding to the first and second anodic peak was the nitrogen atom on the indole ring and on the piperidine moiety, respectively. 3.1. Analytical applications and methods validation

Fig. 6. Cyclic voltammograms of 1 × 10−4 M sertindole in phosphate buffer at pH 3.0 (a); and in phosphate buffer at pH 7.01 (b) with constant amount acetonitrile (30%). Scan rate, 100 mV s−1 . (1) Glassy carbon electrode and (2) boron-doped diamond electrode.

the sertindole compound. Even though the exact oxidation mechanism was not determined, some conclusions about the potentially electroactive centers under working conditions, could be reached. It is assumed that oxidation occurred firstly on the nitrogen atom of indole ring and secondly on the nitrogen atom of piperidine ring of the molecule. Piperidine response was found sharper and measurable for the determination of sertindole than the indole oxidation step. Because of the bonded nitrogen atom of indole ring, oxidative response occurred with small response and it was assumed that the piperidine oxidation response covered in acidic pH values. After the pH value 5.0, both indole and piperidine responses was clearly obtained. However, in some supporting electrolyte solutions these responses were obtained separately before pH 5.0 (Figs. 2, 3 and 6). It may be possible due to the supporting electrolyte nature, composition and concentration. The anodic oxidative behavior of sertindole is comparable to indole and piperidine oxidations that were reported in our previous studies [41–49]. To support the working hypothesis that piperidine (similar to pimozide, droperidol, benperidol and haloperidol oxidation step) and indole groups (similar oxidation pathway with indol, indol-3-acetic acid, etodolac, fluvastatine and melatonin) of sertindole that undergoes oxidation, behavior of small anodic peak ip1 (related with indole moiety) and main anodic peak ip2 (related with piperidine moiety) of sertindole were compared to some model

In order to develop a voltammetric methodology for determining sertindole, DPV and SWV modes were selected. SWV showed similar results with other techniques. In this study DPV were used as an alternative technique. Various electrolytes such as sulphuric acid, Britton–Robinson, phosphate and acetate buffer were examined as a supporting electrolyte. Based on the above study, the best condition for analytical applications proved to be an acetate buffer of pH 3.5. This supporting electrolyte with a constant amount of acetonitrile (as 30%) was chosen for the subsequent experiments. The effects of methanol and acetonitrile on peak current and potential were also studied. As expected, the peak current decreased with changing ionic strength and viscosity of the medium. For example, peak current in solutions containing 40% (v/v) methanol or acetonitrile decreased by ca. 20% of that obtained in aqueous solution. Besides, a very slight decrement in peak potential was observed. Some solubility problems occurred for high concentrations of sertindole using 30% methanol. For analytical purposes, best response (with regard to peak current sensitivity, morphology, reproducibility and solubility) was obtained by working with 30% constant amount of acetonitrile in selected supporting electrolyte for both electrodes. Good correlations were obtained for concentration between 1 × 10−6 and 1 × 10−4 M for glassy carbon electrode for both techniques and using diamond electrode 1 × 10−6 to 2 × 10−4 M was found linear for DPV and 1 × 10−6 to 1 × 10−4 M for SWV. Above this concentration, a loss of linearity was probably due to the adsorption of sertindole on the electrode surface. The characteristics of the calibration plots are summarized in Table 1. Several approaches are given in the ICH guideline to determine the LOD and LOQ values. The detection limit (LOD) and quantification limit (LOQ) of the procedures (Table 1) were calculated according to the 3s/m and 10s/m criterions, respectively, where s is the standard deviation of the peak currents (five runs) and m is the slope of related calibration graph [30–33]. The low values of SE of slope and intercept and greater than 0.999 correlation coefficient nearly in all media established the precision of the proposed method.

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Fig. 7. Cyclic voltammograms of 1 × 10−4 M sertindole (1); melatonine (2); pimozide (3) in acetate buffer at pH 3.5; in phosphate buffer at pH 5.03 and in phosphate buffer at pH 7.01 with constant amount acetonitrile (30%). Scan rate, 100 mV s−1 . (a, c and e) Glassy carbon electrode and (b, d and f) boron-doped diamond electrode. Table 1 Regression and necessary validation data of the calibration lines of sertindole by DPV and SWV in supporting electrolyte with glassy carbon and boron-doped diamond electrodes. Glassy carbon electrode

Measured potential (V) Linearity range (M) Slope (␮A M−1 ) Intercept (␮A) Correlation coefficient SE of slope SE of intercept LOD (M) LOQ (M) Repeatability of peak current (RSD%) Repeatability of peak potential (RSD%) Reproducibility of peak current (RSD%) Reproducibility of peak potential (RSD%)

Boron-doped diamond electrode

DPV

SWV

DPV

SWV

1.08 1 × 10−6 to 1 × 10−4 3.29 × 104 0.0642 0.999 4.70 × 102 0.0211 1.90 × 10−7 6.33 × 10−7 0.55 0.42 0.89 0.31

1.10 1 × 10−6 to 1 × 10−4 4.43 × 104 0.107 0.999 7.69 × 102 0.0346 2.79 × 10−7 9.31 × 10−7 0.83 0.20 0.96 0.60

1.03 1 × 10−6 to 2 × 10−4 4.05 × 104 0.103 0.999 5.03 × 102 0.0362 2.40 × 10−7 7.99 × 10−7 0.84 0.55 1.53 0.81

1.07 1 × 10−6 to 1 × 10−4 6.11 × 104 0.019 0.999 7.64 × 102 0.0343 2.17 × 10−7 7.24 × 10−7 0.67 0.21 1.57 0.21

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Table 2 Results of the assay from the dosage forms and the recovery analysis of sertindole in tablets with glassy carbon and boron-doped diamond electrodes. Glassy carbon electrode

Labeled (mg) Amount found (mg)a RSD% Bias% tvalue Fvalue Added (mg) Found (mg)a Recovery% Bias% RSD% of recovery a

Boron-doped diamond electrode

DPV

SWV

DPV

SWV

20.00 20.02 0.21 −0.10 ttheoretical : 2.31 Ftheoretical : 2.60 10.00 10.03 100.34 −0.30 0.86

20.00 20.23 0.71 −1.15 ttheoretical : 2.31 Ftheoretical : 2.60 10.00 10.01 100.09 −0.10 0.55

20.00 20.01 0.70 −0.05 tcalc : 0.952 Fcalc : 0.042 10.00 10.05 100.44 −0.50 0.59

20.00 19.99 0.93 0.05 tcalc : 0.055 Fcalc : 0.615 10.00 10.02 100.15 −0.20 0.76

Each value is the mean of five experiments.

The developed methods were validated according to the standard procedures [30–33] and the results obtained are shown in Table 1. Accuracy, precision and reproducibility of the proposed method were assessed by performing replicate analysis of the standard solutions in supporting electrolyte and biological media within calibration curves. The within day and between day precision, accuracy and reproducibility were determined as the RSD% and the results are shown in Table 1. The stability of the reference substance and sample solutions were checked by analyzing prepared standard solution of sertindole in supporting electrolyte aged at +4 ◦ C in the dark against freshly prepared sample. 3.2. Determination of sertindole in tablet dosage forms On the basis of above results, both DPV and SWV methods were applied to the direct determination of sertindole in tablet dosage forms, using the related calibration straight lines without any sample extraction, evaporation or filtration other than an adequate dilution step. The mean results for the determinations of both tech-

niques with both electrodes were found very close to the declared value of 20 mg. These results showed that the proposed methods could be applied with great success to sertindole assay in tablet dosage form without any interference (Table 2). For showing the similarities and differences of both DPV and SWV techniques using both electrodes the results of the tablet analysis were compared. Table 2 shows comparison results. The F- and student t-tests were carried out on the data and statistically examined the validity of the obtained results. At 95% of the confidence level, the values of t- and F-tests (calculated from experiments) were less than that of theoretical t- and F-values showing that there were no significant differences between the performances of the both proposed methods using SWV methods were found more rapid, than DPV methods. There is no official method present in any pharmacopoeias (e.g. USP, BP or EP) related to pharmaceutical dosage forms or bulk drugs of sertindole. For checking the accuracy, precision and selectivity of the proposed methods and in order to know whether the excipients in pharmaceutical dosage forms show any interference

Table 3 Regression and necessary validation data of the calibration lines of sertindole by DPV and SWV in spiked serum samples with glassy carbon and boron-doped diamond electrodes. Glassy carbon electrode

Measured potential (V) Linearity range (M) Slope (␮A M−1 ) Intercept (␮A) Correlation coefficient SE of slope SE of intercept LOD (M) LOQ (M) Repeatability of peak current (RSD%) Repeatability of peak potential (RSD%) Reproducibility of peak current (RSD%) Reproducibility of peak potential (RSD%)

Boron-doped diamond electrode

DPV

SWV

DPV

SWV

1.06 4 × 10−6 to 2 × 10−4 2.98 × 104 0.256 0.998 6.27 × 102 0.0494 4.74 × 10−7 1.58 × 10−6 0.69 0.32 0.72 0.67

1.09 4 × 10−6 to 1 × 10−4 4.45 × 104 0.372 0.997 1.32 × 103 0.0656 2.94 × 10−7 9.78 × 10−7 1.02 0.49 1.43 0.48

1.04 4 × 10−6 to 2 × 10−4 4.28 × 104 0.165 0.998 1.13 × 103 0.0561 2.51 × 10−7 8.36 × 10−7 1.05 0.42 1.52 0.41

1.08 4 × 10−6 to 1 × 10−4 5.56 × 104 0.210 0.997 1.59 × 103 0.0789 2.53 × 10−7 8.44 × 10−7 0.62 0.33 0.86 0.30

Table 4 Application of the DPV and SWV methods to the determination of sertindole in spiked serum samples with glassy carbon and boron-doped diamond electrodes and its recovery results. Glassy carbon electrode

Added (M) N Found (M) Average recovery % RSD% Bias%

Boron-doped diamond electrode

DPV

SWV

DPV

SWV

6.00 × 10−5 5 6.01 × 10−5 100.10 0.64 −0.10

6.00 × 10−5 5 6.03 × 10−5 100.49 0.88 −0.49

6.00 × 10−5 5 6.05 × 10−5 100.90 0.41 −0.90

6.00 × 10−5 5 6.04 × 10−5 100.64 0.62 −0.64

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with the analysis, the proposed methods were evaluated by recovery tests after addition of known amounts of pure drug to various pre-analyzed formulations of sertindole. For the recovery study standard addition method was used. Recovery experiments using the developed assay procedure further indicated the absence of interference from commonly encountered pharmaceutical excipients used in the selected formulations (Table 2). These results showed that both methods had adequate precision and accuracy and consequently can be applied to the determination of sertindole in pharmaceuticals without any interference from inactive ingredients.

ent concentrations of sertindole using both electrodes. The peak current was linearly related to sertindole concentrations over the range 4 × 10−6 to 2 × 10−4 M for DPV and 4 × 10−6 to 1 × 10−4 M for SWV using glassy carbon electrode and 4 × 10−6 to 1 × 10−4 M for both method using boron-doped diamond electrode according to the equations: For DPV ip (␮A) = 2.98 × 104 + 0.256C (M) For SWV

ip (␮A) = 4.45 × 104 + 0.372C (M) using glassy carbon electrode;

3.3. Determination of sertindole in spiked biological samples Analysis of drugs from serum samples usually requires extensive time-consuming sample preparation, use of expensive organic solvents and other chemicals. In order to check the applicability of the proposed techniques to the human serum samples, the calibration equation was obtained in spiked biological samples. Acetonitrile and methanol and also mixture of both were used as a serum precipitating agents. Acetonitrile gave the best results when 1.5 volume of acetonitrile for 1 volume of serum samples was used. The preparation of the samples and measurements of sertindole are described in Section 2. Calibration equation parameters and related validation parameters are shown in Table 3 and the results of the methods for spiked serum samples are abridged in Table 4. Also Fig. 8 illustrates DP and SW voltammograms obtained from serum spiked at differ-

For DPV ip (␮A) = 4.28 × 104 + 0.165C (M) For SWV

ip (␮A) = 5.56 × 104 + 0.210C (M) using boron-doped diamond electrode;

In the potential range where the analytical peak appeared there were no oxidation compounds and no extra noise peak found from biological material (Fig. 8). Stability of serum samples kept in refrigerator (+4 ◦ C) was tested by making five consecutive analyses of the sample over a period of approximately 5 h. There were no significant changes observed in the peak currents and potentials between the first and last measurements.

Fig. 8. DPV (a and c) and SWV (b and d) obtained for the determination in spiked serum samples using glassy carbon (a and b) and boron-doped diamond electrode (c and d). (1) Blank; (2) 2 × 10−5 M; (3) 6 × 10−5 M and (4) 1 × 10−4 M sertindole samples in acetate buffer at pH 3.5 with constant amount acetonitrile (30%).

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The proposed methods gave reproducible results, easy to perform, and sensitive enough for the determination of sertindole in human serum samples. 4. Conclusion The electrochemical behavior of sertindole was examined for the first time with this study. The voltammetric oxidation steps of sertindole in different buffer solutions of pH 0.0–12.0 have been elucidated with glassy carbon and boron-doped diamond electrodes. The electrooxidation of sertindole at both electrodes was investigated as details so that the behavior of sertindole at carbon based electrodes might be used for analytical purposes, particularly as a sensor. The obtained results may possibly clarify and aid in understanding oxidation pathways of sertindole. Fully validated, highly selective and sensitive, simple and precise voltammetric procedures were described for determination of sertindole in bulk form, pharmaceutical dosage form and human serum samples without the necessity of sample pre-treatment or any time-consuming extraction and evaporation steps prior to the analysis. Once the instrument is set, just by changing the analyte, within about 2 min, the amount of sertindole can be determined, indicating its potential in high throughput analysis of large number of samples. The method may also be used in analysis of sertindole in biological fluids. The analytical results obtained from pharmaceutical dosage forms by DPV and SWV using both electrodes are in good agreement with each other. Although all voltammetric methods showed similar simplicity, the principal advantage of the proposed voltammetric methods is that the absence of influence of matrix effects, higher selectivity and sensitivity because of the possibility of higher sample dilution. The proposed voltammetric methods can be applied directly to the analysis of pharmaceutical dosage forms and biological samples without the need for separation or complex sample preparation, since there was no interference from the excipients and endogenous substances, respectively. This study is not intended to find out pharmacodynamic properties of sertindole, since only healthy volunteers were used for the sample collection and results may be of no significance. Consequently, the above-presented techniques are a good analytical alternative for determining sertindole in pharmaceutical dosage forms and spiked serum samples. This study only shows the possibility of monitoring this compound that makes the method useful for pharmacokinetic and pharmacodynamic purposes. However, the proposed methods might be alternatives to the HPLC techniques in therapeutic drug monitoring or the experimental data might be used for the development HPLC-EC method. Acknowledgements This work was realized the BAS 100 W equipment which is supplied from Ankara University Scientific Research Foundation Projects (Grant Nos.: 20030803037 and 20030803043). We are grateful to Dr. Jose Luis Beltran from University of Barcelona for the STAR program. References [1] S.C. Sweetman (Ed.), Martindale—The Complete Drug Reference, 35th ed., Pharmaceutical Press, London, 2007.

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