Diclofenac on Boron-Doped Diamond Electrode: From

Article pubs.acs.org/Langmuir

Diclofenac on Boron-Doped Diamond Electrode: From Electroanalytical Determination to Prediction of the Electrooxidation Mechanism with HPLC-ESI/HRMS and Computational Simulations Francisco Willian de S. Lucas,†,‡ Lucia H. Mascaro,‡ Taicia P. Fill,‡ Edson Rodrigues-Filho,‡ Edison Franco-Junior,§ Paula Homem-de-Mello,§ Pedro de Lima-Neto,† and Adriana N. Correia*,† †

Departamento de Química Analítica e Físico-Química, Centro de Ciências, Universidade Federal do Ceará, Bloco 940 Campus do Pici, 60440-900, Fortaleza - CE Brazil ‡ Departamento de Química, Universidade Federal de São Carlos, Caixa Postal 676, 13565-905, São Carlos - SP Brazil § Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Av. dos Estados, 5001, Bloco B, sala 1017, 09210-580 Santo André, SP Brazil S Supporting Information *

ABSTRACT: Using square-wave voltammetry coupled to the boron-doped diamond electrode (BDDE), it was possible to develop an analytical methodology for identification and quantification of diclofenac (DCL) in tablets and synthetic urine. The electroanalytical procedure was validated, with results being statistically equal to those obtained by chromatographic standard method, showing linear range of 4.94 × 10−7 to 4.43 × 10−6 mol L−1, detection limit of 1.15 × 10−7 mol L−1, quantification limit of 3.85 × 10−7 mol L−1, repeatability of 3.05% (n = 10), and reproducibility of 1.27% (n = 5). The association of electrochemical techniques with UV-vis spectroscopy, computational simulations and HPLC-ESI/HRMS led us to conclude that the electrooxidation of DCL on the BDDE involved two electrons and two protons, where the products are colorful and easily hydrolyzable dimers. Density functional theory calculations allowed to evaluate the stability of dimers A, B, and C, suggesting dimer C was more stable than the other two proposed structures, ca. 4 kcal mol−1. The comparison of the dimers stabilities with the stabilities of the molecular ions observed in the MS, the compounds that showed retention time (RT) of 15.53, 21.44, and 22.39 min were identified as the dimers B, C, and A, respectively. Corroborating the observed chromatographic profile, dimer B had a dipole moment almost twice higher than that of dimers A and C. As expected, dimer B has really shorter RT than dimers A and C. The majority dimer was the A (71%) and the C (19.8%) should be the minority dimer. However, the minority was the dimer B, which was formed in the proportion of 9.2%. This inversion between the formation proportion of dimer B and dimer C can be explained by preferential conformation of the intermediaries (cation-radicals) on the surface.

1. INTRODUCTION The use of pharmaceutical products is growing and becoming a new environmental problem. In some places, such as Canada and the United States, this fact is becoming even worse due existence of an aging population that consumes four times the healthcare resources comparing with the general population.1 Therefore, it is possible that high concentrations of such pharmaceutical products can reach and contaminate natural water through human excretions. In addition, the majority of pharmaceutical compounds are not totally eliminated in wastewater treatments. Among the top selling pharmaceutical products, the most widely used are the anti-inflammatory drugs, such as the diclofenac that is often found as a persistent toxic waste and one of the most widely available drugs in the world.2 Diclofenac, 2-[(2,6-dichlorophenyl)amino]phenylacetic acid (Figure 1), and its monosodium or monopotassium salts © 2014 American Chemical Society

(DCL) belong to a class of the nonsteroidal anti-inflammatory drugs (NSAIDs). It exhibits a half-life of 1.9 h, being eliminated specially by hepatic metabolism as hydroxylated diclofenac products, with different level of hydroxylation at DCL aromatic rings. However, between 15% and 30% of the dose administered is excreted as unchanged diclofenac in the urine during 24 h after administration.3 DCL has shown antiinflammatory, analgesic, and antipyretic activity, being used in the treatment of osteoarthritis, rheumatoid arthritis, and ankylosing spondylitis. DCL may cause, in rare instances, hepatic injury in patients, but it is well tolerated and seldom produces gastrointestinal ulceration or other serious side Received: November 15, 2013 Revised: March 11, 2014 Published: April 29, 2014 5645

dx.doi.org/10.1021/la4044123 | Langmuir 2014, 30, 5645−5654

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for application to an industrial analysis routine. These characteristics encouraged us in choosing the BDDE as electrode. To the best of our knowledge, there is no report regarding the determination of the DCL using square-wave voltammetry (SWV) coupling with BDDE. The electrooxidation mechanism of the DCL has been investigated by some authors (Scheme S1 in the Supporting Information).26,30 This mechanism was suggested based on the identification of the product obtained after exhaustive electrolysis. However, the experimental conditions reported there led us to believe that the identified product is in fact a result of the hydrolysis of the major electrooxidation product. It is known41 that the electrooxidation of aniline derivatives can lead to the formation of dimers, easily hydrolyzed. Here, we also prove dimers formation by the electrooxidation of the DCL. The aim of this paper is to describe the development of simple, inexpensive, and reliable electrochemical methodology for determination of the DCL using BDDE coupled with square-wave voltammetry. Moreover, as far as the authors know, information about the oxidation process employing computational simulations and liquid chromatography coupled to electrospray ionization high-resolution mass spectrometry (HPLC-ESI/HRMS) is inexistent. In order to obtain practical applications, studies dealing with pharmaceuticals (tablets) and biological fluids (synthetic urine) were also evaluated.

Figure 1. Chemical structure of the diclofenac. Carbon (dark gray balls), hydrogen (light gray balls), oxygen (red balls), nitrogen (blue ball), chlorine (green balls).

effects. Thus, in the treatment of acute and chronic painful and inflammatory conditions, this drug can be regarded as one of the few NSAIDs of “first choice”.4 Many papers describe the degradation and toxicity of diclofenac1,5−7 due its difficulty to be removed during wastewater treatment.8 Its frequent occurrence in the environment, allied to DCL risks to affect wildlife, has made it to be recently included in the substance priority list within the Water Framework Directive.9 This compound may negatively affect living organisms in the aquatic environment and it is reported to cause histological changes in several organs of fish at concentrations of approximately 1 μg L−1.10 The paper published in Nature in 2004 shows the reduction of the vulture population on the Indian subcontinent as a consequence of exposure to diclofenac via the ingestion of contaminated carcasses.11 Taking this many points into consideration, the development of determination methods and the knowledge of oxidation mechanisms of diclofenac in water are important issues in many areas of science. It has been developed numerous analytical methods for the quantitative determination of this drug in pharmaceutical formulations and in biological samples, such as electrophoresis,12 spectrophotometry,13,14 fluorometry,15,16 and different chromatographic methods.17−22 However, most of these techniques require expensive instrumentation, highly skilled technicians, complicated and time-consuming procedures; for these reasons, they are not suitable for routine analysis. In contrast, some electrochemical methods for quantification of the DCL have been developed,23−32 specially because they are rapid, convenient, low-cost, and environmentally friendly.26,33 Among the electrode materials used in electroanalytical methodologies, the boron-doped diamond electrode (BDDE) has received much attention in recent years,34−37 due to its quasi-metallic conductivity, corrosion and electrochemical stability, very low and stable background current, high response sensitivity, very wide working potential window (larger than 3.5 V), inertness considering the adsorptions of chemical species, and easiness of surface cleanup, when compared to other electrode surfaces.38−40 These characteristics make it one of the more suitable electrode for applications in electroanalysis. Some authors24,29,30 reported that the electrooxidation of the DCL generates a high-adsorbed product on some surfaces. This poisoning of the electrode surface interferes with the electroanalytical determination of the DCL. Moreover, most of the used electrodes were modified in the composite form and, thus, the reproducibility in the preparation is not suitable

2. EXPERIMENTAL SECTION 2.1. Chemicals. All the reagents were of analytical grade, and they were used directly without further purification. The aqueous solutions were prepared with water purified via a Milli-Q system from Millipore Corporation (resistivity greater than 18 MΩ cm). The supporting electrolyte used in most of the electrochemical experiments was 0.04 mol L−1 Britton-Robinson (BR) buffer, prepared as described in a previous paper.42 When it was necessary to adjust pH solution, a pH meter (Micronal B474) was used and appropriate amounts of standard 1.0 mol L−1 NaOH solution were added. Standard 1.0 × 10−3 mol L−1 DCL (Sigma-Aldrich Brazil, 99.8%, CAS: 15307-79-6) solution was daily prepared in water (for electrochemical experiments) or methanol (HPLC solvent J.T. Baker, for chromatography experiments) and stored in a dark flask. 2.2. Apparatus and Software. The voltammetric experiments were performed using a potentiostat/galvanostat (Autolab PGSTAT30, Metrohm - Ecochemie) controlled by GPES 4.9 software. A threeelectrode electrochemical cell with a volume of 50 mL was used. A plate of boron-doped diamond (8,000 ppm) was used as working electrode, Pt-wire as auxiliary electrode, and Ag/AgCl/Cl− (saturated KCl) as reference electrode. The working electrode material was obtained from the Centre Suisse de Electronique et de Microtechnique S.A., Neuchatêl, Switzerland, and the electrode was prepared as described elsewhere.43 Periodically, the BDDE (0.25 cm2 geometric area) was anodically (3.0 V) and cathodically (−2.0 V) pretreated in 0.50 mol L−1 H2SO4 solution during 120 s. The analytical chromatographic experiments were accomplished using high performance liquid chromatography (HPLC), using an instrument from Shimadzu (model LC-20A), with a ultraviolet-visible (UV-vis) photodiode detector (SPD M20A/Shimadzu) and column LC-18 (250 mm × 4.6 mm, 5.0 μm) from KNAUER Company. The volume injection was 20 μL. Instrument operation and data processing were carried out with Labsolution software (LCsolution, release 5.3). For the mechanistic studies, it was performed UV-vis spectroscopy analysis with a ultraviolet-visible-near-infrared spectrophotometer (Cary 5G, Varian), and the HPLC-ESI/HRMS analyses were carried out on a LTQ-Orbitrap Thermo Fisher Scientific mass spectrometry system (Bremen, Germany), with the resolution set at 60 K. HPLC was fitted with a 5 μ phenyl-hexyl column (4.6 × 250 mm, Phenomenex), and samples were eluted using a linear gradient elution 5646


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with acetonitrile and water from 50% to 90% over 30 min at a flow rate of 0.7 mL min−1. 2.3. Electrochemical Experiments. Electrochemical experiments were performed using 10 mL of the supporting electrolyte, and, between each experiment, N2(g) was bubbled (ultrapure, from White Martins Praxair Inc., Brazil) on the electrode surface for 2 min, thus ensuring the reproducibility of the all experiments. Cyclic voltammetry (CV) technique was used in order to evaluate the determining step of the DCL electrooxidation and the adsorptive character of the DCL and of its electrooxidation products on BDDE. Using SWV technique for detection and quantification, the optimization of the analytical procedure was carried out following a systematic study of the experimental parameters that affect the responses, such as pH, supporting electrolyte, pulse potential frequency (f), amplitude of the pulse (a), and height of the potential step (ΔEs). All parameters were properly optimized in relation to the maximum value of peak current and the maximum selectivity (halfpeak width, ΔEp/2), since their values exert high influence on the sensitivity of voltammetric analysis. After optimization of the voltammetric parameters, analytical curves were obtained in supporting electrolyte from the correlation between peak current (Ip) and DCL concentration ([DCL]) by the standard addition method. The standard deviation of the mean current measured at the oxidation potential of the DCL compound for 10 voltammograms of the blank solutions (Sb) in supporting electrolyte was used in the determination of the detection and quantification limits (DL and QL, respectively) together with the slope of the straight line(s) of the analytical curves. The procedures used for determination of the LD and LQ were established by IUPAC.44 Recovery experiments were carried out by adding a certain volume from the solution of the pharmaceutical formulations, with a predetermined concentration of the drug, to the supporting electrolyte, followed by standard additions of the DCL stock solution. All measurements were taken in triplicate at a 95% level of confidence, and the value [DCL]found refers to the concentration obtained by extrapolation of the analytical curves of the corresponding artificially spiked samples. The precision of the proposed procedure was evaluated based on reproducibility experiments conducted with different standard solutions of DCL on different days (intraday). The accuracy was evaluated from experiments of the repeatability obtained in ten replicated determinations with the same DCL solution (interday). 2.4. Analytical Application in Pharmaceutical Preparation and Synthetic Urine. The validation of the proposed method was performed from of a comparative statistical analysis with the standard chromatograph method in the determination of the DCL in pharmaceutical preparation (tablets). For this analysis, the average mass of 10 tablets was determined. These tablets were finely powdered and homogenized in a mortar. An appropriate accurately weighed amount of the homogenized powder was transferred into a flask containing 10 mL of methanol. The contents of the flask were sonicated for 30 min, and the undissolved excipients were removed by filtration, using a 0.45 μm membrane (Millipore Schleicher & Schuell). Then, this solution was transferred into a 50 mL calibrated flask and it was completed with the same supporting electrolyte used in the electrochemical experiments or with methanol for the chromatographic measurements. The tablet samples obtained from a local pharmacy were analyzed by the standard addition method.45 After validation, the proposed method was applied to determine of DCL in synthetic urine. Synthetic urine had been used in some previous studies.46−49 The characteristics of the synthetic urine used in this study was described by Xu et al.49 Synthetic urine was fortified with 4.34 μmol L−1 of DCL and the content of DCL in the sample was analyzed by the standard addition method.45 2.5. Computational Simulations. For the computational calculations, we have employed the Adsorption Locator module of Materials Studio package50,51 to obtain adsorption configurations. Besides that, all structural and electronic properties for DCL were obtained with density functional theory (DFT) calculations, using the B3LYP functional and the 6-311G(d) basis set as implemented in

Gaussian 09 package.52 Atomic charges derived from electrostatic surface potential, in the ChelpG scheme, were obtained in aqueous solution simulated by the continuum model IEFPCM, using Gaussian 03 defaults. 2.6. Mechanistic Study. A possible pathway for DCL electrooxidation was proposed with the aid of computational simulations, UV-vis experiments, and HPLC-ESI/HRMS characterization of the products to prove the reliability of the mechanism. For the identification of the oxidation products, 1.0 × 10−3 mol L−1 DCL was exhaustively electrolyzed by applying a constant potential equal to the oxidation peak potential determined in the electrochemical experiments. The exhaustively electrolyzed solution was removed from the electrochemical cell and immediately taken for chromatographic analysis by HPLC-ESI/HRMS operating in scan mode. To prevent hydrolysis of the products, the sample was immediately cooled and, protected from light, taken for chromatographic analysis. All chromatographic peaks were characterized by high-resolution mass spectrometer.

3. RESULTS AND DISCUSSION 3.1. Investigation of the Electrochemical Behavior. 3.1.1. Preliminary Studies. CV and SWV techniques were used to study the electrochemical behavior of DCL on BDDE and all the studies were performed in triplicate. The DCL electroactivity on the BDDE surface was evaluated by CV experiments at 50 mV s−1 in the potential range between −0.90 and 1.60 V. During the forward scan, for more positive potentials, it was only observed an oxidative electrochemical process around 0.95 V, assigned to irreversible oxidation of the DCL. In the backward scan, an electrochemical process was observed at 0.62 V (with cathodic current), with redox couple at 0.65 V (with anodic current). These last redox processes were attributed to electroactive products, since they were formed after electrooxidation of the DCL and the peak currents increase with the number of potential scans. Similar electrochemical behavior of the DCL was observed by other authors.24,26,30,31 Aiming to observe the adsorptive process of the molecule or of its products, potential scans were carried out for 10 consecutive cycles, with 30 s delay between each cycle for maintenance of diffusion layer, in the potential range from 0.55 to 1.55 V. The decrease in the Ip at 0.95 V with the number of cycles can be associated with adsorption of the products of reaction on the electrode surface, what significantly reduce the active area. Aiming to evaluate if the diffusion or the adsorption was the rate-determining step on the electrooxidation mechanism of the DCL, the influence of the scan rate (ν) was studied. The relationship between the logarithm of the Ip and the logarithm of the ν showed a linear response with linear correlation coefficient (r2) of 0.9974 and slope of 0.78. This slope is in between the theoretical values of 0.5 and 1.0 for diffusion- and adsorption-controlled electrode process, respectively,53 clearly indicating that the process had more than one step. From this, it can be concluded that the electrochemical reaction of the DCL had adsorptive and diffusive determining steps. This behavior was also observed for others electrochemical systems.54−56 Also, it was observed that the Ep values shifted to more positive potential values with increase of the logarithm of the ν, showing r2 of 0.9983 and slope of 0.056. From this behavior, based on the diagnostic criteria of the CV,53 it can be estimated the number of electrons (n) and the value of the charge transfer coefficient (α) involved in the determining step of the reaction. Applying these criteria, it was calculated the value of αn = 1.05, which can be associated with a charge transfer coefficient of 5647


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electrolyte and SWV parameters, the analytical curves were obtained in triplicate for concentrations of DCL ranging from 4.94 × 10−7 to 5.41 × 10−6 mol L−1. The voltammetric profiles and the linear correlation between peak currents and the [DCL] are shown in Figure 2.

about 0.5 and two electrons (n = 2) involved in the DCL electrooxidation. This value is in accordance with that observed by several authors.24,26,30 3.1.2. Study of the pH and of the Supporting Electrolyte. The effect of the pH in the electrochemical response of the DCL was evaluated by SWV using BR buffer (f = 100 s−1, a = 50 mV, and ΔEs = 2 mV) from pH 2 to pH 8. The dependence between potential peak with the pH is shown in Figure S1 (Supporting Information). It was observed a linear relationship according to Ep (V) = 1.00 ( ±0.01) − 0.042 ( ±0.002) pH r 2 = 0.9985

(1)

showing that the molecule protonation was the determining rate step of the electrooxidation mechanism of the DCL and, by the slope value measured, it can be concluded that this step involved equal number of protons and electrons, since that the relationship between the peak potential and the pH is proportional to 0.059 V/pH unit,53 at 298 K, multiplied by ration between the number of protons and electrons involved in the electrochemical reaction. This slope value was a little lower than 0.059 V/pH unit, probably due the variation of the activity of the DCL with the pH. Values lower than 0.059 V/pH unit also were obtained by other authors, like Yang et al.29 and Arvand et al.,26 that obtained a slope of 0.045 V/pH unit. Thus, considering the number of electrons estimated by CV, it was concluded that the electrochemical oxidation of the DCL involved also the participation of two protons. Higher Ip values were observed at pH 2, and this pH was chosen as and optimized condition in the subsequent experiments. The effect of the supporting electrolyte in the electrochemical profile of the DCL was evaluated using 0.04 mol L−1 buffers in pH 2 (BR, biftalate and McIlvaine57) and SWV (f = 100 s−1, ΔEs = 2 mV, and a = 50 mV). In BR buffer, the electrochemical process exhibited higher intensity of the Ip and lower ΔEp/2, resulting in higher sensitivity and selectivity, respectively, when compared to other buffers. 3.1.3. Study of the SWV Parameters. It was evaluated the dependence between Ip and Ep in relation to f. Ip values increased with f values from 5 to 140 s−1. Then 140 s−1 was chosen as optimized condition. It was also observed that, with increasing frequency, Ep values shifted to more positive potentials. There was a linear behavior of the Ep with log f, according to

Figure 2. Square-wave voltammograms (f = 140 s−1, a = 20 mV, ΔEs = 4 mV) for DCL in BR buffer pH 2 on BDDE and concentrations of DCL in the interval from 4.94 × 10−7 to 5.41 × 10−6 mol L−1. Inset corresponds to the analytical curve obtained from voltammograms.

The corresponding analytical equation is Ip/A = 3.15 × 10−7 (± 2.69 × 10−8) + 1.28 (± 0.01)[DCL/mol L−1] r 2 = 0.9998

The evaluation of possible random errors was conducted by a test of significance,45 whose results suggested that the value of intercept was statistically zero. Detection and quantification limits (DL and QL) for DCL determination were obtained by using the procedure recommended by International Union of Pure and Applied Chemistry (IUPAC), as described in the subsection 2.3 of the Experimental Section.44 Interday and intraday experiments were performed, with relative standard deviation (RSD) values indicating good repeatability and reproducibility. Also, the recovery percentages in supporting electrolyte showed that the species present do not interfere in the determination of the DCL, since excellent values were obtained. All the figures of merit can be seen in Table 1. The DL values were also in the same magnitude order and even better than those obtained by others electrochemical methods (Table S1). However, most of the electrodes used in the referred methodologies were modified in the form of composite and, thus, it was difficult to obtain reproducibility in routine analysis, reinforcing that BDDE was a more suitable electrode material to detect DCL. DL values between square brackets shown in Table S1 were calculated from the standard deviation of only three analytical blanks, which is not in accordance with the procedure valid for any statistical analytical calculation of DL. 3.3. DCL Determination in Pharmaceutical Preparation and Synthetic Urine. DCL contents in the analyzed tablets were of 49.32 ± 0.07 mg of DCL/tablet, according the standard chromatographic method, and 49.32 ± 2.12 mg of

Ep (V) = 0.81 ( ±0.005) + 0.06 ( ± 0.002) log(f /s−1) r 2 = 0.9974

(3)

(2)

Applying the SWV diagnostic criteria53,58 for irreversible systems, αn = 0.98, from slope, it was seen that the process can be associated with a charge transfer coefficient of about 0.5 and two electrons (n = 2) involved for the electrooxidation of the DCL, which is in accordance with the relationship previous obtained by CV and mentioned in previous works.24,26,30 Evaluating the behavior for Ip and ΔEp/2 with a and ΔEs, it was possible to choose the optimum values of these parameters, resulting in higher analytical sensitivity and resolution.58 The values chosen for amplitude and increment potential were 20 and 4 mV, respectively. Thus, the optimized parameters of the SWV were f = 140 s−1, a = 20 mV, and ΔEs = 4 mV. 3.2. Analytical Curve and Methodology Validation. Using the optimized experimental conditions of pH, supporting 5648


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Table 1. Figures of Merit for DCL Determination Using SWV on BDDE in BR Buffer pH 2 and HPLC-UVa parameter

SWV

linearity range (mol L−1) intercept slope Sb r2 DL (mol L−1) QL (mol L−1) %RSD (repeatability) %RSD (reproducibility) %recovery in SE

4.94 × 10−7 to 4.43 × 10−6 3.15 × 10−7 ± 2.69 × 10−8 (A) 1.28 ± 0.01 (A/mol L−1) 4.93 × 10−8 (A) 0.9998 1.15 × 10−7 3.85 × 10−7 3.05 (n = 10) 1.27 (n = 5) 97.25 ± 0.70

HPLC-UV −371.73 ± 77.58 (a.u.) 1.14 × 108 ± 1.98 × 106 (a.u./mol L−1) 27.6 (a.u.) 0.9995 7.27 × 10−7 2.42 × 10−6

a All the statistical treatment was expressed at a 95% level of confidence. r2, linear correlation coefficient; Sb, standard deviation from the arithmetic mean of 10 blank solutions; DL, detection limit; QL, quantification limit; RSD, relative standard deviation; recovery in SE = recovery in supporting electrolyte; n, number of replicates.

Figure 3. Adsorptions conformations of DCL on boron-doped diamond cluster. Dark gray balls are carbon atoms, green balls are chlorine atoms, light gray balls are hydrogen atoms, red balls are oxygen atoms, blue ball is nitrogen atom, and pink ball is a boron atom.

associated with the formation of dimers, since dimers present a more extend conjugated system and are more stable than Ncation-radical. So, we propose that radicals formed by electrooxidation process can combine, forming the dimers (true electrooxidation product of the DCL), whose suffered hydrolysis to 5-OH DCL, compound identified by Goyal coworkers.24,30 To propose the structures of radicals and dimers, computational simulations were performed. First, the geometry of DCL with B3LYP/6-311G(d) calculations (frequency calculations were performed to ensure it is a minimum in the energy surface potential) was obtained and its adsorption on BDDE was evaluated. The concentration of boron on BDDE was 8000 ppm (one boron atom for 104 carbon atoms). Since we were interested in verify the preferential adsorption structure, we have modeled the electrode with two minima clusters, both with 110 atoms, one without boron atoms and other with one boron atoms. In this approach, these models furnished an evaluation of the dependence of the adsorption with boron concentration, at low computational costs. In our experiments, electrode surface received a final cathodic treatment, so its surface should be Hterminated. As shown in Supporting Information (Figures S5− S7), this type of termination has no important effects on adsorption structures, since we are using classical simulations and the surface is hydrophobic in both models. Twenty adsorption structures were obtained that can be grouped in three types, as displayed in Figure 3. Independent of the presence of boron atoms or H-termination, adsorption onto both clusters was very similar; higher adsorption energy (in absolute values) was obtained for the same geometry of

DCL/tablet, from the proposed electroanalytical method, at a 95% level of confidence. These results are organized in Table S2 and the recovery curves can be seen in Figure S2 in the Supporting Information. Using the statistical Student’s t test procedure45 it can be concluded that the obtained results by electroanalytical method were statistically equal to the obtained by standard method. Thus, the proposed electrochemical method can be efficiently used for determination of DCL with accuracy and precision. These contents of DCL per tablet were in agreement with the DCL nominal written on the tablet label (50 mg DCL/tablet), since the Brazilian Pharmacopoeia allows a range of ±10% of nominal value.59 The determination of DCL in synthetic urine showed recovery of 103.0 ± 5.8%, at a 95% level of confidence. The results of the individual samples are organized in Table S2, and the recovery curves can be seen in Figure S3 in the Supporting Information. The recovery experiments were carried out to evaluate matrix effects. The added nominal content of DCL was 4.34 μmol L−1. The good average recovery indicates that there were no important matrix interferences and the novel proposed method may be effectively and advantageously used for DCL determination in pharmaceutical preparation and synthetic urine, since it was very simple, inexpensive, and rapid. 3.4. Mechanistic Studies. The electrooxidation reaction was followed using UV-vis absorption spectroscopy. It was detected a band at 450 nm (Figure S4) which disappears after 30 min due to the hydrolysis of the generated product. Goyal et al.24 attributed the yellow coloration to the N-cation-radical formed at the first step of DCL electrooxidation (structure of this compound can be seen in the Scheme S1, Supporting Information). However, we believe that this coloration is 5649


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adsorption (structure “a” of Figure 3), with the chlorinated ring and the carboxylic group laid on the surface. We have also evaluated the DCL cation-radical (charge = +1), the first intermediary of the electrooxidation. Spin density (Figure 4) indicates that the 2C, 25C, and 18N have radical

structures, and if so three combinations are possible to form dimers, as indicated in Scheme 2. Spin density and ESP atomic charges indicated that the C2-structure is more reliable than C1, and thus, based only on this point, dimer A should be the main product, followed by dimer B. Electrochemical studies indicated that the oxidation of DCL involved two electrons and two protons. First, DCL lose one electron, forming the cation-radical C2 (or C1) and enabling the dimerization. In the next step, protons bonded to bridge carbons (carbons whose bonding forms the dimer) leave the molecule as indicated in Scheme 2. Thus, we performed DFT calculations to evaluate the stability of dimers A, B, and C and also time-dependent DFT calculations to simulate each electronic absorption spectrum. As can be seen from Table 2, dimer C (formed by the junction of two C1 radicals) was more stable than the other two proposed structures, ca. 4 kcal mol−1. Figure S8 shows electronic spectra obtained for the three dimers as well as for DCL molecule and the N-cation-radical and 5-OH DCL proposed by Goyal et al. (the structures of these compounds can be seen in the Scheme S1).30 Even if absorption spectra calculated with TD-DFT/B3LYP presented errors about 20% in relation to experimental spectra,60 this is an important tool to compare different molecules. Dimers A, B, and C absorb in higher wavelength than DCL and products proposed in the literature. The lines around 350 nm (Figure S8) were in good agreement with the band observed in experimental spectrum (Figure S4), indicating that the formation of dimers is the pathway more likely of the DCL electrooxidation reaction. To verify the results obtained by UV-vis spectroscopy and computational simulations, the DCL electrooxidation products were separated and identified by HPLC-ESI/HRMS. It was possible to identify the formation of only three products, which showed retention times of 15.53 (9.2%), 21.44 (19.8%), and 22.39 min (71%), as can be seen in Figure 5. The mass spectra (MS2 and MS3) and high-resolution MS1 spectra of molecular ion for each product are shown in Figure 6 and Figure S10, respectively. In mass spectrometry, the presence of isotopes can be easily distinguished and gives rise to a series of characteristic patterns with different intensities peaks. This can be predicted based on the abundance of each isotope in nature and the relative peak heights can also be used to assist in the deduction of the empirical formula of the molecule being analyzed. In the negative ionization mode, the molecular ion is presented with one proton less ([M − H]−). Thus, three major products of the electrooxidation of the DCL were identified from its highresolution mass spectrum (Figure S10). For all products, it was observed the same HRMS profile for the [M − H]−, showing m/z values of 587.01093, 588.01520, 589.00719, and 590.01053.

Figure 4. Spin density (purple surface) calculated for DCL cationradical (isosurface = 0.005 electrons Å−3). Dark gray balls are carbon atoms, green balls are chlorine atoms, light gray balls are hydrogen atoms, red balls are oxygen atoms, and blue ball is nitrogen atom.

character, but the radical character is predominately in the 2C. So, we have three possibilities for cation-radical, as proposed in Scheme 1, but the mainly specie is the C2-structure. Goyal et al.24 proposed that the nitrogen atom had cation-radical character (N-cation-radical). Energy surface potential (ESP) atomic charges can be used to assist the mechanistic studies. Charges obtained in aqueous solution are presented for the heaviest atoms in Table S3. From our calculations, it was possible to verify that the positive charge is distributed along the whole molecule. Comparing the ESP atomic charges of the atoms of the DCL and of the DCL cation-radical, nitrogen atom has its negative charge diminished in 0.152, but 2C was more affected, and its negative charge diminished 0.175. Then, by the analysis of atomic charges, the radical predominantly formed in the first oxidation step should be represented as C2-structure, in Scheme 1. Other important electronic property that supports the mechanistic studies was the frontier molecular orbitals. The highest occupied molecular orbital (HOMO) for DCL and DCL cation-radical, the lowest unoccupied molecular orbital (LUMO) for DCL, and the semioccupied molecular orbital (SOMO) for DCL cation-radical were calculated (Figure S9). SOMO and LUMO have important contributions from the same atoms, but HOMO orbitals are very different. The radical form has HOMO concentrated on chlorinated ring, indicating that the radical formed in the first oxidation step should be also represented as C1-structure, in Scheme 1. So, based on all electronic properties obtained, both structures C1 and C2 in Scheme 1 could be representative Scheme 1. Proposed DCL Cation-Radical Forms

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Scheme 2. Dimerization Reaction of the Radical DCL and Deprotonating of the Dimerization Products

Table 2. Total Electronic (Eele) and Free Energies of Formation (ΔGform) and Differences (ΔEestab and ΔGestab) for Dimers A, B, and Ca dimer Eele (a.u.) ΔEstab (kcal mol−1) ΔGform (a.u.) ΔGstab (kcal mol−1)

A

B

C

−3330.6881 3.83 −3330.3381 3.75

−3330.6868 4.66 −3330.3380 3.81

−3330.6942 0.00 −3330.3441 0.00

a ΔEestab and ΔGestab are electronic and free energy differences, respectively, in relation to the more stable structure.

Figure 5. Chromatogram obtained with HPLC-ESI/HRMS for identifying the electrooxidation products of the DCL.

Comparing the characteristic isotopic pattern seen in this experimentally detected peak cluster with the pattern simulated using MassLynx 4.1 software, the identity of the molecular formulas was confirmed with match of 100%. These m/z values were related to the molecular formulas C 28H 19N2 Cl4O4 (1.36914 ppm error), C27(13C)H19N2Cl4O4 (1.39343 ppm error), C28H19N2Cl3(37Cl)O4 (1.35613 ppm error), and C27(13C)H19N2Cl3(37Cl)O4 (1.32885 ppm error), respectively, which have the same elemental composition of the dimers shown in Scheme 2. MS2 and MS3 experiments were also performed for the DCL dimers in order to confirm their

Figure 6. Mass spectra (MS2) of the compounds with retention times of (A) 15.53 min, (B) 21.44 min, and (C) 22.39 min. Inset: the respective MS3.

chemical structure. The mass spectra (MS2 and MS3) for each product are shown in Figure 6. 5651


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4.94 × 10−7 to 4.43 × 10−6 mol L−1, a detection limit of 1.15 × 10−7 mol L−1, a quantification limit of 3.85 × 10−7 mol L−1, repeatability of 3.05% (n = 10), and reproducibility of 1.27% (n = 5). The recovery studies indicated that the ionic species present in the supporting electrolyte (BR buffer pH 2.0) did not interfere in the accuracy and precision of the assay, showing a recovery value of 97.25 ± 0.70% at a 95% level of confidence, and a BIAS value of 2.74%. The applications of the proposed methodology for determining DCL in tablets and synthetic urine were efficient. The content of DCL in the analyzed tablets was statistically equal to that obtained by chromatographic standard method and in agreement with the nominal content written on the label. The average recovery value for DCL determination in synthetic urine was 103.0 ± 5.8% at a 95% level of confidence and BIAS of 2.99%. UV-vis spectroscopy study and computational simulations indicated that the electrochemical oxidation of the DCL leads to the formation of dimers. With the aid of the HPLC-ESI/HRMS and computational simulations, it was possible to identify the three formed dimers A, B, and C (dimer C was more stable than the other two proposed structures, ca. 4 kcal mol−1). By comparison of the stabilities of the molecular ions with dimers stabilities, the compounds that showed RT of 15.53, 21.44, and 22.39 min were identified as dimers B, C, and A, respectively. Corroborating the observed chromatographic profile, dimer B had a dipole moment almost twice higher than that of dimers A and C. As expected, dimer B has really shorter RT than that of dimers A and C. The majority dimer was A (71%), and C (19.8%) should be the minority dimer, since it was formed by two less stable intermediaries. However, the minority was dimer B, which was formed in the proportion of 9.2%. This inversion between the formation proportion of dimer B and dimer C can be explained by preferential conformation of the cation-radicals on the surface, which increases the effectiveness of the collision between two less stable intermediaries.

The CID fragmentation of m/z 587 produced by the dimers with retention time (RT) of 15.53 and 21.44 min showed losses of two CO2 molecules (44 Da each) as neutral fragments to form the peaks at m/z 543 and m/z 499, respectively. In contrast, the dimer with RT of 22.39 min showed only one loss of 44 units, but it was possible to detect a fragment ion at m/z 339 (C20H15NCl2) in MS3 experiments. This fragmentation can be explained by an N−Ar homolytic cleavage after the CO2 losses, which somehow works very well for dimer A, but it is not a productive path for dimer B (see Scheme S2). This same path applied to dimer C structure would result in an ion with m/z 392, which is not detected in the MS2 or in MS3 spectra. Then, the 22.39 min dimer possibly is not dimer C. A really interesting behavior to be observed is the relative stability of the molecular ions (m/z 587) detected for the dimers. As can be seen in Figure 6, the dimers with RT of 21.44 and 15.53 min showed the most and the less stable [M − H]− under CID fragmentation, respectively. The stability of the [M − H]− is directly related with the molecular stability. Thus, observing the stability of the dimers obtained from DFT calculations (Table 2), we can infer that the dimer with RT of 21.44 min was the dimer C and with RT of 15.53 min was dimer B, and consequently the dimer A had RT of 22.39 min. To check these hypotheses, we have evaluated the dipolemoment obtained with DFT calculations to predict the chromatographic elution order, since the HPLC experiments were performed with a reversed-phase (nonpolar) column and also using a hydrophilic polar eluent. The calculations showed that the dimers A, B, and C had a dipole moment of 2.82, 4.10, and 2.58 D, respectively. Based on these values, we expect that dimer B has really shorter RT than dimers A and C, which should have similar RT. The sequence predicted by dipole moment describes well the behavior observed in the chromatogram of Figure 5. Thus, as inferred from comparison of the stabilities of the molecular ions, the compounds that showed RT of 15.53, 21.44, and 22.39 min were identified as the dimers B, C, and A, respectively. As predicted by the stability of cation-radical, the majority dimer was A (71%), and C (formed in the proportion of 19.8%) should be the minority dimer. However, the minority was dimer B, which was formed in the proportion of 9.2%. This inversion between the formation proportion of dimer B and dimer C can be explained by preferential conformation of the cation-radicals on the surface. As can be seen in Figure 3, the more energetically favorable monomer conformation is where the chlorinated ring is parallel to the surface, increasing the effectiveness of the collision between two monomer with C1-structure (in Scheme 1, monomer with radical portion on chlorined aromatic ring) and promoting the inversion between the formation proportion of dimer B and dimer C. Thus, we can conclude that both stability and the preferential conformation of the cation-radicals on the surface directed the proportion between the formed dimers.

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ASSOCIATED CONTENT

S Supporting Information *

Comparison of the DL values for different electrochemical methods of DCL determination, data of the recovery studies for DCL determination in tablets and synthetic urine, atomic charges for selected atoms of DCL and DCL cation-radical, incomplete DCL electrooxidation mechanism proposed in the literature, the CID fragmentation pathway, DCL electrochemical behavior as pH function, recovery curves for DCL determination in tablet and synthetic urine, monitoring of the DCL electrooxidation by UV-vis absorption spectroscopy, adsorption studies of the dimer on pure and H-terminated diamond, simulated electronic absorption spectra, frontiers molecular orbitals diagrams for DCL and DCL cation-radical, and EIS/HR mass spectrum profile of the dimers. This material is available free of charge via the Internet at http://pubs.acs.org.

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4. CONCLUSIONS The electrooxidation of the DCL on the BDD is an irreversible reaction that shows an adsorption/diffusion mixed control, involving two electrons and two protons, where the products are colorful and easily hydrolyzable. The electroanalytical procedure for determination and quantification of this nonsteroidal anti-inflammatory drug using SWV and BDDE was validated, with results statistically equal to those obtained by chromatographic standard method, showing a linear range of

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: +55 85 3366 9050. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from the Brazilian funding agencies FINEP, CNPq and CAPES. 5652


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F.W.S.L., L.H.M., E.F.-J., and P.H.d.M. wish to thank the São Paulo Research Foundation (FAPESP), Grants 2012/10947-2 and 2012/20653-6.

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Diclofenac on Boron-Doped Diamond Electrode: From

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