Diclofenac on Boron-Doped Diamond Electrode: FromElectroanalytical Determination to Prediction of the ElectrooxidationMechanism with HPLC-ESI/HRMS and Computational SimulationsFrancisco 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 Ciencias, Universidade Federal do Ceara, Bloco 940 Campus doPici, 60440-900, Fortaleza - CE Brazil‡Departamento de Química, Universidade Federal de Sao Carlos, Caixa Postal 676, 13565-905, Sao Carlos - SP Brazil§Centro de Ciencias Naturais e Humanas, Universidade Federal do ABC, Av. dos Estados, 5001, Bloco B, sala 1017, 09210-580 SantoAndre, SP Brazil
*S Supporting Information
ABSTRACT: Using square-wave voltammetry coupled to theboron-doped diamond electrode (BDDE), it was possible todevelop an analytical methodology for identification andquantification of diclofenac (DCL) in tablets and syntheticurine. The electroanalytical procedure was validated, withresults being statistically equal to those obtained by chromato-graphic 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 of3.05% (n = 10), and reproducibility of 1.27% (n = 5). Theassociation of electrochemical techniques with UV-vis spec-troscopy, computational simulations and HPLC-ESI/HRMSled us to conclude that the electrooxidation of DCL on theBDDE involved two electrons and two protons, where the products are colorful and easily hydrolyzable dimers. Densityfunctional theory calculations allowed to evaluate the stability of dimers A, B, and C, suggesting dimer C was more stable thanthe other two proposed structures, ca. 4 kcal mol−1. The comparison of the dimers stabilities with the stabilities of the molecularions observed in the MS, the compounds that showed retention time (RT) of 15.53, 21.44, and 22.39 min were identified as thedimers B, C, and A, respectively. Corroborating the observed chromatographic profile, dimer B had a dipole moment almosttwice higher than that of dimers A and C. As expected, dimer B has really shorter RT than dimers A and C. The majority dimerwas the A (71%) and the C (19.8%) should be the minority dimer. However, the minority was the dimer B, which was formed inthe proportion of 9.2%. This inversion between the formation proportion of dimer B and dimer C can be explained bypreferential conformation of the intermediaries (cation-radicals) on the surface.
1. INTRODUCTION
The use of pharmaceutical products is growing and becoming anew environmental problem. In some places, such as Canadaand the United States, this fact is becoming even worse dueexistence of an aging population that consumes four times thehealthcare resources comparing with the general population.1
Therefore, it is possible that high concentrations of suchpharmaceutical products can reach and contaminate naturalwater through human excretions. In addition, the majority ofpharmaceutical compounds are not totally eliminated inwastewater treatments. Among the top selling pharmaceuticalproducts, the most widely used are the anti-inflammatory drugs,such as the diclofenac that is often found as a persistent toxicwaste 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
(DCL) belong to a class of the nonsteroidal anti-inflammatorydrugs (NSAIDs). It exhibits a half-life of 1.9 h, being eliminatedspecially by hepatic metabolism as hydroxylated diclofenacproducts, with different level of hydroxylation at DCL aromaticrings. However, between 15% and 30% of the doseadministered is excreted as unchanged diclofenac in the urineduring 24 h after administration.3 DCL has shown anti-inflammatory, analgesic, and antipyretic activity, being used inthe treatment of osteoarthritis, rheumatoid arthritis, andankylosing spondylitis. DCL may cause, in rare instances,hepatic injury in patients, but it is well tolerated and seldomproduces gastrointestinal ulceration or other serious side
Received: November 15, 2013Revised: March 11, 2014Published: April 29, 2014
effects. Thus, in the treatment of acute and chronic painful andinflammatory conditions, this drug can be regarded as one ofthe few NSAIDs of “first choice”.4
Many papers describe the degradation and toxicity ofdiclofenac1,5−7 due its difficulty to be removed duringwastewater treatment.8 Its frequent occurrence in the environ-ment, allied to DCL risks to affect wildlife, has made it to berecently included in the substance priority list within the WaterFramework Directive.9 This compound may negatively affectliving organisms in the aquatic environment and it is reportedto cause histological changes in several organs of fish atconcentrations of approximately 1 μg L−1.10 The paperpublished in Nature in 2004 shows the reduction of thevulture population on the Indian subcontinent as aconsequence of exposure to diclofenac via the ingestion ofcontaminated carcasses.11 Taking this many points intoconsideration, the development of determination methodsand the knowledge of oxidation mechanisms of diclofenac inwater are important issues in many areas of science.It has been developed numerous analytical methods for the
quantitative determination of this drug in pharmaceuticalformulations and in biological samples, such as electro-phoresis,12 spectrophotometry,13,14 fluorometry,15,16 and differ-ent chromatographic methods.17−22 However, most of thesetechniques require expensive instrumentation, highly skilledtechnicians, complicated and time-consuming procedures; forthese reasons, they are not suitable for routine analysis. Incontrast, some electrochemical methods for quantification ofthe DCL have been developed,23−32 specially because they arerapid, convenient, low-cost, and environmentally friendly.26,33
Among the electrode materials used in electroanalyticalmethodologies, the boron-doped diamond electrode (BDDE)has received much attention in recent years,34−37 due to itsquasi-metallic conductivity, corrosion and electrochemicalstability, very low and stable background current, high responsesensitivity, very wide working potential window (larger than 3.5V), inertness considering the adsorptions of chemical species,and easiness of surface cleanup, when compared to otherelectrode surfaces.38−40 These characteristics make it one of themore 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. Thispoisoning of the electrode surface interferes with theelectroanalytical determination of the DCL. Moreover, mostof the used electrodes were modified in the composite formand, thus, the reproducibility in the preparation is not suitable
for application to an industrial analysis routine. Thesecharacteristics encouraged us in choosing the BDDE aselectrode. To the best of our knowledge, there is no reportregarding the determination of the DCL using square-wavevoltammetry (SWV) coupling with BDDE.The electrooxidation mechanism of the DCL has been
investigated by some authors (Scheme S1 in the SupportingInformation).26,30 This mechanism was suggested based on theidentification of the product obtained after exhaustiveelectrolysis. However, the experimental conditions reportedthere led us to believe that the identified product is in fact aresult of the hydrolysis of the major electrooxidation product. Itis known41 that the electrooxidation of aniline derivatives canlead to the formation of dimers, easily hydrolyzed. Here, wealso prove dimers formation by the electrooxidation of theDCL.The aim of this paper is to describe the development of
simple, inexpensive, and reliable electrochemical methodologyfor determination of the DCL using BDDE coupled withsquare-wave voltammetry. Moreover, as far as the authorsknow, information about the oxidation process employingcomputational simulations and liquid chromatography coupledto electrospray ionization high-resolution mass spectrometry(HPLC-ESI/HRMS) is inexistent. In order to obtain practicalapplications, studies dealing with pharmaceuticals (tablets) andbiological fluids (synthetic urine) were also evaluated.
2. EXPERIMENTAL SECTION2.1. Chemicals. All the reagents were of analytical grade, and they
were used directly without further purification. The aqueous solutionswere prepared with water purified via a Milli-Q system from MilliporeCorporation (resistivity greater than 18 MΩ cm). The supportingelectrolyte used in most of the electrochemical experiments was 0.04mol L−1 Britton-Robinson (BR) buffer, prepared as described in aprevious paper.42 When it was necessary to adjust pH solution, a pHmeter (Micronal B474) was used and appropriate amounts of standard1.0 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 (forelectrochemical experiments) or methanol (HPLC solvent J.T.Baker, for chromatography experiments) and stored in a dark flask.
2.2. Apparatus and Software. The voltammetric experimentswere performed using a potentiostat/galvanostat (Autolab PGSTAT-30, Metrohm - Ecochemie) controlled by GPES 4.9 software. A three-electrode electrochemical cell with a volume of 50 mL was used. Aplate of boron-doped diamond (8,000 ppm) was used as workingelectrode, Pt-wire as auxiliary electrode, and Ag/AgCl/Cl− (saturatedKCl) as reference electrode. The working electrode material wasobtained from the Centre Suisse de Electronique et de MicrotechniqueS.A., Neuchatel, Switzerland, and the electrode was prepared asdescribed elsewhere.43 Periodically, the BDDE (0.25 cm2 geometricarea) was anodically (3.0 V) and cathodically (−2.0 V) pretreated in0.50 mol L−1 H2SO4 solution during 120 s.
The analytical chromatographic experiments were accomplishedusing high performance liquid chromatography (HPLC), using aninstrument from Shimadzu (model LC-20A), with a ultraviolet-visible(UV-vis) photodiode detector (SPD M20A/Shimadzu) and columnLC-18 (250 mm × 4.6 mm, 5.0 μm) from KNAUER Company. Thevolume injection was 20 μL. Instrument operation and data processingwere carried out with Labsolution software (LCsolution, release 5.3).
For the mechanistic studies, it was performed UV-vis spectroscopyanalysis with a ultraviolet-visible-near-infrared spectrophotometer(Cary 5G, Varian), and the HPLC-ESI/HRMS analyses were carriedout on a LTQ-Orbitrap Thermo Fisher Scientific mass spectrometrysystem (Bremen, Germany), with the resolution set at 60 K. HPLCwas fitted with a 5 μ phenyl-hexyl column (4.6 × 250 mm,Phenomenex), and samples were eluted using a linear gradient elution
Figure 1. Chemical structure of the diclofenac. Carbon (dark grayballs), hydrogen (light gray balls), oxygen (red balls), nitrogen (blueball), chlorine (green balls).
with acetonitrile and water from 50% to 90% over 30 min at a flow rateof 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 WhiteMartins Praxair Inc., Brazil) on the electrode surface for 2 min, thusensuring 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 adsorptivecharacter of the DCL and of its electrooxidation products on BDDE.Using SWV technique for detection and quantification, theoptimization of the analytical procedure was carried out following asystematic study of the experimental parameters that affect theresponses, such as pH, supporting electrolyte, pulse potentialfrequency ( f), amplitude of the pulse (a), and height of the potentialstep (ΔEs). All parameters were properly optimized in relation to themaximum value of peak current and the maximum selectivity (half-peak width, ΔEp/2), since their values exert high influence on thesensitivity of voltammetric analysis.After optimization of the voltammetric parameters, analytical curves
were obtained in supporting electrolyte from the correlation betweenpeak current (Ip) and DCL concentration ([DCL]) by the standardaddition method.The standard deviation of the mean current measured at the
oxidation potential of the DCL compound for 10 voltammograms ofthe blank solutions (Sb) in supporting electrolyte was used in thedetermination of the detection and quantification limits (DL and QL,respectively) together with the slope of the straight line(s) of theanalytical curves. The procedures used for determination of the LDand LQ were established by IUPAC.44 Recovery experiments werecarried out by adding a certain volume from the solution of thepharmaceutical formulations, with a predetermined concentration ofthe drug, to the supporting electrolyte, followed by standard additionsof the DCL stock solution. All measurements were taken in triplicateat a 95% level of confidence, and the value [DCL]found refers to theconcentration obtained by extrapolation of the analytical curves of thecorresponding artificially spiked samples. The precision of theproposed procedure was evaluated based on reproducibility experi-ments conducted with different standard solutions of DCL on differentdays (intraday). The accuracy was evaluated from experiments of therepeatability obtained in ten replicated determinations with the sameDCL solution (interday).2.4. Analytical Application in Pharmaceutical Preparation
and Synthetic Urine. The validation of the proposed method wasperformed from of a comparative statistical analysis with the standardchromatograph method in the determination of the DCL inpharmaceutical preparation (tablets). For this analysis, the averagemass of 10 tablets was determined. These tablets were finely powderedand homogenized in a mortar. An appropriate accurately weighedamount of the homogenized powder was transferred into a flaskcontaining 10 mL of methanol. The contents of the flask weresonicated for 30 min, and the undissolved excipients were removed byfiltration, using a 0.45 μm membrane (Millipore Schleicher & Schuell).Then, this solution was transferred into a 50 mL calibrated flask and itwas completed with the same supporting electrolyte used in theelectrochemical experiments or with methanol for the chromato-graphic measurements. The tablet samples obtained from a localpharmacy were analyzed by the standard addition method.45
After validation, the proposed method was applied to determine ofDCL in synthetic urine. Synthetic urine had been used in someprevious studies.46−49 The characteristics of the synthetic urine used inthis study was described by Xu et al.49 Synthetic urine was fortifiedwith 4.34 μmol L−1 of DCL and the content of DCL in the sample wasanalyzed by the standard addition method.45
2.5. Computational Simulations. For the computationalcalculations, we have employed the Adsorption Locator module ofMaterials Studio package50,51 to obtain adsorption configurations.Besides that, all structural and electronic properties for DCL wereobtained with density functional theory (DFT) calculations, using theB3LYP functional and the 6-311G(d) basis set as implemented in
Gaussian 09 package.52 Atomic charges derived from electrostaticsurface potential, in the ChelpG scheme, were obtained in aqueoussolution simulated by the continuum model IEFPCM, using Gaussian03 defaults.
2.6. Mechanistic Study. A possible pathway for DCL electro-oxidation was proposed with the aid of computational simulations,UV-vis experiments, and HPLC-ESI/HRMS characterization of theproducts to prove the reliability of the mechanism. For theidentification of the oxidation products, 1.0 × 10−3 mol L−1 DCLwas exhaustively electrolyzed by applying a constant potential equal tothe oxidation peak potential determined in the electrochemicalexperiments. The exhaustively electrolyzed solution was removedfrom the electrochemical cell and immediately taken for chromato-graphic analysis by HPLC-ESI/HRMS operating in scan mode. Toprevent hydrolysis of the products, the sample was immediately cooledand, protected from light, taken for chromatographic analysis. Allchromatographic peaks were characterized by high-resolution massspectrometer.
3. RESULTS AND DISCUSSION
3.1. Investigation of the Electrochemical Behavior.3.1.1. Preliminary Studies. CV and SWV techniques were usedto study the electrochemical behavior of DCL on BDDE and allthe studies were performed in triplicate. The DCL electro-activity on the BDDE surface was evaluated by CV experimentsat 50 mV s−1 in the potential range between −0.90 and 1.60 V.During the forward scan, for more positive potentials, it wasonly observed an oxidative electrochemical process around 0.95V, assigned to irreversible oxidation of the DCL. In thebackward scan, an electrochemical process was observed at 0.62V (with cathodic current), with redox couple at 0.65 V (withanodic current). These last redox processes were attributed toelectroactive products, since they were formed after electro-oxidation of the DCL and the peak currents increase with thenumber of potential scans. Similar electrochemical behavior ofthe DCL was observed by other authors.24,26,30,31 Aiming toobserve the adsorptive process of the molecule or of itsproducts, potential scans were carried out for 10 consecutivecycles, with 30 s delay between each cycle for maintenance ofdiffusion layer, in the potential range from 0.55 to 1.55 V. Thedecrease in the Ip at 0.95 V with the number of cycles can beassociated with adsorption of the products of reaction on theelectrode 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 theDCL, the influence of the scan rate (ν) was studied. Therelationship between the logarithm of the Ip and the logarithmof the ν showed a linear response with linear correlationcoefficient (r2) of 0.9974 and slope of 0.78. This slope is inbetween the theoretical values of 0.5 and 1.0 for diffusion- andadsorption-controlled electrode process, respectively,53 clearlyindicating that the process had more than one step. From this,it can be concluded that the electrochemical reaction of theDCL had adsorptive and diffusive determining steps. Thisbehavior was also observed for others electrochemicalsystems.54−56
Also, it was observed that the Ep values shifted to morepositive 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 estimatedthe number of electrons (n) and the value of the charge transfercoefficient (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
about 0.5 and two electrons (n = 2) involved in the DCLelectrooxidation. This value is in accordance with that observedby 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 theDCL 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 dependencebetween potential peak with the pH is shown in Figure S1(Supporting Information). It was observed a linear relationshipaccording to
= ± − ±
=
E
r
(V) 1.00 ( 0.01) 0.042 ( 0.002) pH
0.9985
p
2 (1)
showing that the molecule protonation was the determiningrate step of the electrooxidation mechanism of the DCL and, bythe slope value measured, it can be concluded that this stepinvolved equal number of protons and electrons, since that therelationship between the peak potential and the pH isproportional to 0.059 V/pH unit,53 at 298 K, multiplied byration between the number of protons and electrons involvedin the electrochemical reaction. This slope value was a littlelower than 0.059 V/pH unit, probably due the variation of theactivity of the DCL with the pH. Values lower than 0.059 V/pHunit also were obtained by other authors, like Yang et al.29 andArvand et al.,26 that obtained a slope of 0.045 V/pH unit. Thus,considering the number of electrons estimated by CV, it wasconcluded that the electrochemical oxidation of the DCLinvolved also the participation of two protons. Higher Ip valueswere observed at pH 2, and this pH was chosen as andoptimized condition in the subsequent experiments.The effect of the supporting electrolyte in the electro-
chemical 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, theelectrochemical process exhibited higher intensity of the Ip andlower 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 valuesincreased with f values from 5 to 140 s−1. Then 140 s−1 waschosen as optimized condition. It was also observed that, withincreasing frequency, Ep values shifted to more positivepotentials. There was a linear behavior of the Ep with log f,according to
= ± + ±
=
−E f
r
(V) 0.81 ( 0.005) 0.06 ( 0.002) log( /s )
0.9974
p1
2 (2)
Applying the SWV diagnostic criteria53,58 for irreversiblesystems, αn = 0.98, from slope, it was seen that the process canbe associated with a charge transfer coefficient of about 0.5 andtwo electrons (n = 2) involved for the electrooxidation of theDCL, which is in accordance with the relationship previousobtained by CV and mentioned in previous works.24,26,30
Evaluating the behavior for Ip and ΔEp/2 with a and ΔEs, itwas possible to choose the optimum values of these parameters,resulting in higher analytical sensitivity and resolution.58 Thevalues chosen for amplitude and increment potential were 20and 4 mV, respectively. Thus, the optimized parameters of theSWV 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
electrolyte and SWV parameters, the analytical curves wereobtained in triplicate for concentrations of DCL ranging from4.94 × 10−7 to 5.41 × 10−6 mol L−1. The voltammetric profilesand the linear correlation between peak currents and the[DCL] are shown in Figure 2.
The corresponding analytical equation is
= × ± ×
+ ±
=
− −
−
I A
r
/ 3.15 10 ( 2.69 10 )
1.28 ( 0.01)[DCL/mol L ]
0.9998
p7 8
1
2 (3)
The evaluation of possible random errors was conducted by atest of significance,45 whose results suggested that the value ofintercept was statistically zero.Detection and quantification limits (DL and QL) for DCL
determination were obtained by using the procedurerecommended by International Union of Pure and AppliedChemistry (IUPAC), as described in the subsection 2.3 of theExperimental Section.44 Interday and intraday experimentswere performed, with relative standard deviation (RSD) valuesindicating good repeatability and reproducibility. Also, therecovery percentages in supporting electrolyte showed that thespecies present do not interfere in the determination of theDCL, since excellent values were obtained. All the figures ofmerit can be seen in Table 1.The DL values were also in the same magnitude order and
even better than those obtained by others electrochemicalmethods (Table S1). However, most of the electrodes used inthe referred methodologies were modified in the form ofcomposite and, thus, it was difficult to obtain reproducibility inroutine analysis, reinforcing that BDDE was a more suitableelectrode material to detect DCL. DL values between squarebrackets shown in Table S1 were calculated from the standarddeviation of only three analytical blanks, which is not inaccordance with the procedure valid for any statistical analyticalcalculation of DL.
3.3. DCL Determination in Pharmaceutical Prepara-tion and Synthetic Urine. DCL contents in the analyzedtablets were of 49.32 ± 0.07 mg of DCL/tablet, according thestandard chromatographic method, and 49.32 ± 2.12 mg of
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 ofDCL in the interval from 4.94 × 10−7 to 5.41 × 10−6 mol L−1. Insetcorresponds to the analytical curve obtained from voltammograms.
DCL/tablet, from the proposed electroanalytical method, at a95% level of confidence. These results are organized in TableS2 and the recovery curves can be seen in Figure S2 in theSupporting Information. Using the statistical Student’s t testprocedure45 it can be concluded that the obtained results byelectroanalytical method were statistically equal to the obtainedby standard method. Thus, the proposed electrochemicalmethod can be efficiently used for determination of DCL withaccuracy and precision. These contents of DCL per tablet werein agreement with the DCL nominal written on the tablet label(50 mg DCL/tablet), since the Brazilian Pharmacopoeia allowsa range of ±10% of nominal value.59
The determination of DCL in synthetic urine showedrecovery of 103.0 ± 5.8%, at a 95% level of confidence. Theresults of the individual samples are organized in Table S2, andthe recovery curves can be seen in Figure S3 in the SupportingInformation. The recovery experiments were carried out toevaluate matrix effects. The added nominal content of DCL was4.34 μmol L−1. The good average recovery indicates that therewere no important matrix interferences and the novel proposedmethod may be effectively and advantageously used for DCLdetermination in pharmaceutical preparation and syntheticurine, since it was very simple, inexpensive, and rapid.3.4. Mechanistic Studies. The electrooxidation reaction
was followed using UV-vis absorption spectroscopy. It wasdetected a band at 450 nm (Figure S4) which disappears after30 min due to the hydrolysis of the generated product. Goyal etal.24 attributed the yellow coloration to the N-cation-radicalformed at the first step of DCL electrooxidation (structure ofthis compound can be seen in the Scheme S1, SupportingInformation). However, we believe that this coloration is
associated with the formation of dimers, since dimers present amore extend conjugated system and are more stable than N-cation-radical. So, we propose that radicals formed byelectrooxidation process can combine, forming the dimers(true electrooxidation product of the DCL), whose sufferedhydrolysis to 5-OH DCL, compound identified by Goyal co-workers.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 ensureit is a minimum in the energy surface potential) was obtainedand its adsorption on BDDE was evaluated. The concentrationof boron on BDDE was 8000 ppm (one boron atom for 104carbon atoms). Since we were interested in verify thepreferential adsorption structure, we have modeled theelectrode with two minima clusters, both with 110 atoms,one without boron atoms and other with one boron atoms. Inthis approach, these models furnished an evaluation of thedependence of the adsorption with boron concentration, at lowcomputational costs. In our experiments, electrode surfacereceived a final cathodic treatment, so its surface should be H-terminated. As shown in Supporting Information (Figures S5−S7), this type of termination has no important effects onadsorption structures, since we are using classical simulationsand the surface is hydrophobic in both models. Twentyadsorption structures were obtained that can be grouped inthree types, as displayed in Figure 3. Independent of thepresence of boron atoms or H-termination, adsorption ontoboth clusters was very similar; higher adsorption energy (inabsolute values) was obtained for the same geometry of
Table 1. Figures of Merit for DCL Determination Using SWV on BDDE in BR Buffer pH 2 and HPLC-UVa
parameter SWV HPLC-UV
linearity range (mol L−1) 4.94 × 10−7 to 4.43 × 10−6
%RSD (repeatability) 3.05 (n = 10)%RSD (reproducibility) 1.27 (n = 5)%recovery in SE 97.25 ± 0.70
aAll the statistical treatment was expressed at a 95% level of confidence. r2, linear correlation coefficient; Sb, standard deviation from the arithmeticmean of 10 blank solutions; DL, detection limit; QL, quantification limit; RSD, relative standard deviation; recovery in SE = recovery in supportingelectrolyte; 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.
adsorption (structure “a” of Figure 3), with the chlorinated ringand 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
character, but the radical character is predominately in the 2C.So, we have three possibilities for cation-radical, as proposed inScheme 1, but the mainly specie is the C2-structure. Goyal etal.24 proposed that the nitrogen atom had cation-radicalcharacter (N-cation-radical).Energy surface potential (ESP) atomic charges can be used
to assist the mechanistic studies. Charges obtained in aqueoussolution are presented for the heaviest atoms in Table S3. Fromour calculations, it was possible to verify that the positivecharge is distributed along the whole molecule. Comparing theESP atomic charges of the atoms of the DCL and of the DCLcation-radical, nitrogen atom has its negative charge diminishedin 0.152, but 2C was more affected, and its negative chargediminished 0.175. Then, by the analysis of atomic charges, theradical predominantly formed in the first oxidation step shouldbe represented as C2-structure, in Scheme 1.Other important electronic property that supports the
mechanistic studies was the frontier molecular orbitals. Thehighest occupied molecular orbital (HOMO) for DCL andDCL 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 thesame atoms, but HOMO orbitals are very different. The radicalform has HOMO concentrated on chlorinated ring, indicatingthat the radical formed in the first oxidation step should be alsorepresented as C1-structure, in Scheme 1.So, based on all electronic properties obtained, both
structures C1 and C2 in Scheme 1 could be representative
structures, and if so three combinations are possible to formdimers, as indicated in Scheme 2. Spin density and ESP atomiccharges indicated that the C2-structure is more reliable thanC1, and thus, based only on this point, dimer A should be themain product, followed by dimer B.Electrochemical studies indicated that the oxidation of DCL
involved two electrons and two protons. First, DCL lose oneelectron, forming the cation-radical C2 (or C1) and enablingthe dimerization. In the next step, protons bonded to bridgecarbons (carbons whose bonding forms the dimer) leave themolecule as indicated in Scheme 2.Thus, we performed DFT calculations to evaluate the
stability of dimers A, B, and C and also time-dependent DFTcalculations to simulate each electronic absorption spectrum. Ascan be seen from Table 2, dimer C (formed by the junction oftwo C1 radicals) was more stable than the other two proposedstructures, 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-radicaland 5-OH DCL proposed by Goyal et al. (the structures ofthese compounds can be seen in the Scheme S1).30 Even ifabsorption spectra calculated with TD-DFT/B3LYP presentederrors about 20% in relation to experimental spectra,60 this is animportant tool to compare different molecules. Dimers A, B,and C absorb in higher wavelength than DCL and productsproposed in the literature. The lines around 350 nm (FigureS8) were in good agreement with the band observed inexperimental spectrum (Figure S4), indicating that theformation of dimers is the pathway more likely of the DCLelectrooxidation reaction.To verify the results obtained by UV-vis spectroscopy and
computational simulations, the DCL electrooxidation productswere separated and identified by HPLC-ESI/HRMS. It waspossible to identify the formation of only three products, whichshowed retention times of 15.53 (9.2%), 21.44 (19.8%), and22.39 min (71%), as can be seen in Figure 5. The mass spectra(MS2 and MS3) and high-resolution MS1 spectra of molecularion 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 patternswith different intensities peaks. This can be predicted based onthe abundance of each isotope in nature and the relative peakheights can also be used to assist in the deduction of theempirical formula of the molecule being analyzed. In thenegative ionization mode, the molecular ion is presented withone proton less ([M − H]−). Thus, three major products of theelectrooxidation of the DCL were identified from its high-resolution mass spectrum (Figure S10). For all products, it wasobserved the same HRMS profile for the [M − H]−, showingm/z values of 587.01093, 588.01520, 589.00719, and590.01053.
Figure 4. Spin density (purple surface) calculated for DCL cation-radical (isosurface = 0.005 electrons Å−3). Dark gray balls are carbonatoms, green balls are chlorine atoms, light gray balls are hydrogenatoms, red balls are oxygen atoms, and blue ball is nitrogen atom.
Comparing the characteristic isotopic pattern seen in thisexperimentally detected peak cluster with the pattern simulatedusing MassLynx 4.1 software, the identity of the molecularformulas was confirmed with match of 100%. These m/z valueswere related to the molecular formulas C28H19N2Cl4O4(1.36914 ppm error), C27(
which have the same elemental composition of the dimersshown in Scheme 2. MS2 and MS3 experiments were alsoperformed for the DCL dimers in order to confirm their
chemical structure. The mass spectra (MS2 and MS3) for eachproduct are shown in Figure 6.
Scheme 2. Dimerization Reaction of the Radical DCL and Deprotonating of the Dimerization Products
Table 2. Total Electronic (Eele) and Free Energies ofFormation (ΔGform) and Differences (ΔEestab and ΔGestab)for Dimers A, B, and Ca
The CID fragmentation of m/z 587 produced by the dimerswith retention time (RT) of 15.53 and 21.44 min showed lossesof two CO2 molecules (44 Da each) as neutral fragments toform the peaks at m/z 543 and m/z 499, respectively. Incontrast, the dimer with RT of 22.39 min showed only one lossof 44 units, but it was possible to detect a fragment ion at m/z339 (C20H15NCl2) in MS3 experiments. This fragmentation canbe explained by an N−Ar homolytic cleavage after the CO2losses, which somehow works very well for dimer A, but it isnot a productive path for dimer B (see Scheme S2). This samepath applied to dimer C structure would result in an ion withm/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 thedimers. As can be seen in Figure 6, the dimers with RT of 21.44and 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 DFTcalculations (Table 2), we can infer that the dimer with RT of21.44 min was the dimer C and with RT of 15.53 min wasdimer B, and consequently the dimer A had RT of 22.39 min.To check these hypotheses, we have evaluated the dipole-moment obtained with DFT calculations to predict thechromatographic elution order, since the HPLC experimentswere performed with a reversed-phase (nonpolar) column andalso using a hydrophilic polar eluent. The calculations showedthat 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 thatdimer B has really shorter RT than dimers A and C, whichshould have similar RT. The sequence predicted by dipolemoment describes well the behavior observed in the chromato-gram of Figure 5. Thus, as inferred from comparison of thestabilities of the molecular ions, the compounds that showedRT of 15.53, 21.44, and 22.39 min were identified as the dimersB, C, and A, respectively.As predicted by the stability of cation-radical, the majority
dimer was A (71%), and C (formed in the proportion of19.8%) should be the minority dimer. However, the minoritywas dimer B, which was formed in the proportion of 9.2%. Thisinversion between the formation proportion of dimer B anddimer C can be explained by preferential conformation of thecation-radicals on the surface.As can be seen in Figure 3, the more energetically favorable
monomer conformation is where the chlorinated ring is parallelto the surface, increasing the effectiveness of the collisionbetween two monomer with C1-structure (in Scheme 1,monomer with radical portion on chlorined aromatic ring) andpromoting the inversion between the formation proportion ofdimer B and dimer C. Thus, we can conclude that both stabilityand the preferential conformation of the cation-radicals on thesurface directed the proportion between the formed dimers.
4. CONCLUSIONSThe electrooxidation of the DCL on the BDD is an irreversiblereaction that shows an adsorption/diffusion mixed control,involving two electrons and two protons, where the productsare colorful and easily hydrolyzable. The electroanalyticalprocedure for determination and quantification of thisnonsteroidal anti-inflammatory drug using SWV and BDDEwas validated, with results statistically equal to those obtainedby chromatographic standard method, showing a linear range of
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 speciespresent in the supporting electrolyte (BR buffer pH 2.0) didnot interfere in the accuracy and precision of the assay, showinga recovery value of 97.25 ± 0.70% at a 95% level of confidence,and a BIAS value of 2.74%. The applications of the proposedmethodology for determining DCL in tablets and syntheticurine were efficient. The content of DCL in the analyzed tabletswas statistically equal to that obtained by chromatographicstandard method and in agreement with the nominal contentwritten on the label. The average recovery value for DCLdetermination in synthetic urine was 103.0 ± 5.8% at a 95%level of confidence and BIAS of 2.99%. UV-vis spectroscopystudy and computational simulations indicated that theelectrochemical oxidation of the DCL leads to the formationof dimers. With the aid of the HPLC-ESI/HRMS andcomputational simulations, it was possible to identify thethree formed dimers A, B, and C (dimer C was more stablethan the other two proposed structures, ca. 4 kcal mol−1). Bycomparison of the stabilities of the molecular ions with dimersstabilities, the compounds that showed RT of 15.53, 21.44, and22.39 min were identified as dimers B, C, and A, respectively.Corroborating the observed chromatographic profile, dimer Bhad a dipole moment almost twice higher than that of dimers Aand C. As expected, dimer B has really shorter RT than that ofdimers A and C. The majority dimer was A (71%), and C(19.8%) should be the minority dimer, since it was formed bytwo less stable intermediaries. However, the minority was dimerB, which was formed in the proportion of 9.2%. This inversionbetween the formation proportion of dimer B and dimer C canbe explained by preferential conformation of the cation-radicalson the surface, which increases the effectiveness of the collisionbetween two less stable intermediaries.
■ ASSOCIATED CONTENT*S Supporting InformationComparison of the DL values for different electrochemicalmethods of DCL determination, data of the recovery studies forDCL determination in tablets and synthetic urine, atomiccharges for selected atoms of DCL and DCL cation-radical,incomplete DCL electrooxidation mechanism proposed in theliterature, the CID fragmentation pathway, DCL electro-chemical behavior as pH function, recovery curves for DCLdetermination in tablet and synthetic urine, monitoring of theDCL electrooxidation by UV-vis absorption spectroscopy,adsorption studies of the dimer on pure and H-terminateddiamond, simulated electronic absorption spectra, frontiersmolecular orbitals diagrams for DCL and DCL cation-radical,and EIS/HR mass spectrum profile of the dimers. This materialis available free of charge via the Internet at http://pubs.acs.org.
F.W.S.L., L.H.M., E.F.-J., and P.H.d.M. wish to thank the SaoPaulo Research Foundation (FAPESP), Grants 2012/10947-2and 2012/20653-6.
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