Electrochemical oxidation mechanism of eugenol on

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May 22, 2017 - behavior of eugenol in acetate buffer solution at pH 8 on a glassy ... acid, and acetic acid as 0.2 M in a volumetric flask. pH values were ... The graphene modified carbon paste was prepared by mixing 90% .... during the second scan at 0.42 V (inset Fig. 2B), which increased at the. -0.2. 0.0. 0.2. 0.4. 0.6. 0.8.
Talanta 173 (2017) 1–8

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Electrochemical oxidation mechanism of eugenol on graphene modified carbon paste electrode and its analytical application to pharmaceutical analysis ⁎

Gulcemal Yildiza, , Zeynep Aydogmusb, M. Emin Cinara,c, Filiz Senkala, Turan Ozturka,d,

MARK

⁎

a

Department of Chemistry, Faculty of Science and Letters, Istanbul Technical University, Maslak, Istanbul 34469, Turkey Department of Analytical Chemistry, Faculty of Pharmacy, Istanbul University, Beyazıt, 34116 Istanbul, Turkey c Department Chemie-Biologie, OC1, Universität Siegen, Adolf-Reichwein-Str., 57068 Siegen, Germany d Chemistry Group Laboratories, TUBITAK UME, PO Box 54, 41470 Gebze, Kocaeli, Turkey b

A R T I C L E I N F O

A BS T RAC T

Keywords: Eugenol Electrochemical properties Graphene modified carbon paste electrode (CPE) Differential pulse voltammetry (DPV) DFT

Electrochemical properties of eugenol were investigated on a graphene modified carbon paste electrode (CPE) by using voltammetric methods, which exhibited a well-defined irreversible peak at about 0.7 V vs Ag/AgCl, NaCl (3 M) in Britton-Robinson buffer at pH 2.0. Mechanism of the electrochemical reaction of eugenol was studied by performing density functional theory (DFT) computations and mass spectroscopic analysis. (CPCM:water)-wB97XD/aug-cc-PVTZ//(CPCM:water)-wB97XD/6-31G(d) level calculations predicted that the formation of product P2, possessing a para-quinoid structure, is preferred rather than the product P1, suggested in the literature, having an ortho-quinoid system. Determination of eugenol in a pharmaceutical sample was realized in the light of the electrochemical findings, and a validated voltammetric method for quantitative analysis of eugenol in a pharmaceutical formulation was proposed. The differential pulse voltammogram (DPV) peak currents were found to be linear in the concentration range of 1.0 × 10−7 to 1.7 × 10−5 M. The limit of detection (LOD) and the limit of quantification (LOQ) were obtained to be 7.0 × 10−9 and 2.3 × 10−8, respectively.

1. Introduction Eugenol (4-(H2C=CHCH2)C6H3-2-(OCH3)OH) is a natural compound obtained from various plants, particularly from black cloves. Although its consumption in small amount provide anti-microbial, anti-bacterial and anti-fungal benefits, exceeding the limit may cause various harmful effects. Eugenol rich cloves have been used for food preservation and in various medical areas, owing to its anti-bacterial, anti-septic and anti-inflammatory properties [1,2]. Eugenol sources such as clove oil, cinnamon, basil and nutmeg oil are some of the basic ingredients in mouth washes, soaps, tooth pastes, perfumes and in various veterinary medications. Moreover, eugenol antibacterial and antifungal activities are very helpful in treating many digestive problems such as diarrhea, gas bloating, ulcers and candida. Eugenol rich black paper and clove can increase the release of gastric acid, which helps in digesting food and kills intestinal bacteria in stomach [3]. Clove tea can be used to expand blood vessels, increase circulation and body temperature. As an antioxidant, eugenol can prevent or decrease harmful oxidations in body [4].

⁎

Although eugenol has been nominated to be safe to consume in suggested limits by FDA (Food and Drug Administration), exceeding the limits may cause serious and fatal results such as arrhythmia, kidney damage, digestive problems, increase in heart rate and blood pressure, dizziness, vomiting and liver failure [5]. Eugenol is quickly absorbed and digested in liver, and 95% of the dose is excreted within one day. However, it is reported that ingesting clove oil as small as 8 mL can cause fatal sickness by damaging liver and nervous system [6]. Thus, investigation of the properties and quantitative analysis of eugenol is of great interest. Voltammetry is a useful technique applied in different areas especially for determination of electrochemical properties and quantitative analyses of organic and inorganic substances. Compared to the conventional methods, such as high performance liquid chromatography, gas chromatography and mass spectrometry, electrochemical analysis has certain advantages for detecting eugenol, owing to its rapid analysis, low cost, high sensitivity and simplicity [7–10]. Modification of bare electrodes has considerable benefits for the construction of electrochemical sensors. The modified layers can

Corresponding author at: Department of Chemistry, Faculty of Science and Letters, Istanbul Technical University, Maslak, Istanbul 34469, Turkey. E-mail addresses: [email protected] (G. Yildiz), [email protected] (T. Ozturk).

http://dx.doi.org/10.1016/j.talanta.2017.05.056 Received 24 February 2017; Received in revised form 15 May 2017; Accepted 20 May 2017 Available online 22 May 2017 0039-9140/ © 2017 Elsevier B.V. All rights reserved.

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2. Experimental

accelerate the electron transfer reaction and reduce the excess potential significantly [10]. Determination of eugenol, using a sensitive electrochemical sensor such as carbon paste electrode (CPE), having peak potential of 285 mV vs. Ag/AgCl and modified with gold nanoparticles in a phosphate buffer solution at pH 8, was reported [11]. In a separate study, it was oxidized on a glassy carbon electrode at 700 mV in a micellar media, created using Triton X100 and Brij 35 to provide eugenol with water solubility [12]. Analysis of eugenol in food samples was performed applying a glassy carbon electrode, which was modified with Cu doped gold nanoparticles. Cyclic voltammetry and differential pulse stripping voltammetry were used to study the electrochemical behavior of eugenol in acetate buffer solution at pH 8 on a glassy carbon electrode [13]. Antioxidant activities of eugenol in spices have been investigated by electrochemical methods and radical scavenging measurements [14]. Electrochemical behavior of eugenol and its determination in real samples were investigated using a pencil graphite electrode [15]. Its electrochemical detection by a NiCo2O4 modified electrode [16] and complexation with β- and HP-β-CDs [17] were conducted in separate studies. Moreover, chemically modified electrodes for electrochemically determination of eugenol have been constructed by using various modifying agents such as TiO2 nanotubes improved with Cu2O clusters, poly(diallyldimethylammonium chloride) functionalized graphene-MoS2, three-dimensional molecularly imprinted poly(p-aminothiophenol-co-p-aminobenzoic-aminothiophenol-co-p-aminobenzoic acid films, CeO2 nanoparticles and surfactants and 1-butyl-3-methylimidazolium hexafluorophosphate [18–22]. However, the reported methods have some disadvantages such as poor sensitivity and setup complexity of experimental conditions. On the other hand, our proposed method has advantageous in terms of simplicity, accuracy and cost effectiveness compared to the previous works (Table 1). Recently, graphene has been the most intensively explored carbon allotrope in material science because of its extraordinary properties like electrical and thermal conductivities. Its mechanical properties provide some applications in electrochemistry and electronics [23]. As graphene is also a useful modifying agent for working electrode due to its excellent electrical conductivity, in this study, electrochemical properties of eugenol were investigated on a graphene modified CPE in Britton Robinson buffer solution at pH 2. For the first time, the mechanism of its electro-oxidation was unveiled using theoretical calculations and mass spectrometric analysis. Moreover, a sensitive differential pulse voltammetric method was developed for its analysis in pharmaceutical samples.

2.1. Instruments Voltammetric measurements were performed on Autolab PGSTAT 30 (Eco Chemie) potentiostat/galvanostat using a conventional threeelectrode system (BASi, C-3 Cell Stand). While Ag/AgCl (3 M NaCl) with (BASi MF 2052) was used as a reference electrode, platinum wire (BASi MW 1032) was chosen as a counter electrode. The graphene modified CPE as a working electrode was employed in all measurement. The pH levels of the solutions were adjusted using a pH meter (WTW Inolab pH 720). Mass spectra were recorded on Bruker MICROTOFQ and Thermo LCQ-Deca ion trap mass instruments. Spectrophotometric analyses were measured by Shimadzu UV-160A (Kyoto, Japan) UV–vis spectrophotometer. 2.2. Reagents All chemicals used were of the analytical grade. Eugenol (purity of 99%) was obtained from Sigma Aldrich (Germany). A stock solution of 1.0 × 10−2 M eugenol was prepared in ethanol and kept in a refrigerator. Boric acid, phosphoric acid, acetic acid, and sodium hydroxide were purchased from Fluka (Fluka AG, Buchs, Switzerland) for the preparation of Britton-Robinson buffer solution as supporting electrolyte. Potassium permanganate was obtained from Merck. The supporting electrolyte composed of boric acid, phosphoric acid, and acetic acid as 0.2 M in a volumetric flask. pH values were adjusted by addition of NaOH. All solutions were prepared using ultrapure water from Millipore Milli Q System (resistivity equal to 18 MX). Desired concentration of working eugenol solutions was achieved by diluting the stock solution with a preferred pH of supporting electrolyte. Graphite powder and graphene (nanoplatelet, 99.5%, 1.5 µm in diameter) were purchased from Merck (Germany) and Nanografi Company (Turkey), respectively. 2.3. Preparation of graphene modified CPE The graphene modified carbon paste was prepared by mixing 90% of graphite powder and 10% of graphene with 700 µL silicon oil in a mortar for fifteen minutes until a uniform wetted paste was obtained. Before each measurement, a portion of the paste was placed into the electrode (Basi-electrode, MF 2010) cavity and polished on a smooth paper and then rinsed with deionized water. The prepared paste was kept as a closed pack, which had a lifetime at least 2 months. The electroactive surface areas of the CPE and graphene modified CPE were calculated by using CV technique in 0.1 M KCl solution, containing 1.0 mM K3Fe(CN)6. The CVs obtained for CPE and graphene modified CPE are shown in Fig. 1. For a reversible process, Randles-Sevcik equation was applied [24]:

. Chemical structure of eugenol.

Table 1 Comparison of this work with the previous methods. Electrode system

Linear range (μM)

LOD (μM)

Remarks

Ref. No

PEDOT:PSS-SWCNTs-PVP

0.150–122

0.048

[10]

Gold nanoparticles modified CPE

5.00–250

2.00

Cu@AuNPs/GCE Cu2O-TiNTs/GCE

0.30–50 4.60–450

0.085 1.30

Au/PDDA-G-MoS2/GCE GN-CNTs-IL CeO2-CPB/GCE MWCNT-IL-Gel/GC Graphene modified CPE

0.10–440 0.50–20 0.075–75 0.25–4.0 0.10–17

0.036 0.10 0.019 0.088 0.0070

phosphate buffer solution (pH 6) phosphate buffer solution (pH 8) BR buffer solution (pH 2) NaClO4/Acetonitrile (pH 7.0) NaAc-HAc (pH 5.5) Bu4NClO4/EtOH solution 0.1 M phosphate buffer solution BR buffer solution (pH 10.5) BR buffer solution (pH 2)

2

[11] [15] [18] [19] [20] [21] [22] This work

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8

2.5. Procedure

b

6 4

The BR buffer solution (pH 2.0) was transferred into ten milliliter of the electrochemical cell using a three-electrode system containing graphene modified CPE. The DPVs were measured between 0.5 V and 1.0 V. After the background measurement, aliquot of the sample solution containing eugenol was added into the cell. The DPVs were then recorded to obtain the sample peak current. The electrode was immersed into the solution and then measurements were conducted after 90 s at open circuit voltage. Before performing each measurement, the performance of the graphene modified CPE was tested in fresh 10 mL of BR buffer solution lacking of any analyte and then required quantity of analyte was added into the electrochemical cell, which was purged with nitrogen firstly for 5 min. DPV pulses were obtained at 50 mV amplitude with a pulse width of 50 ms, and scan rate of 25 mV s-1. CVs were recorded between −0.2 V and 1.1 V. All electrochemical measurements were performed in a non-agitating electrochemical cell at room temperatures (25 ± 2 °C).

a

2

I / µA

0 -2 -4 a

-6 -8

b

-10 -12

-0.2

0.0

0.2

0.4 E/V

0.6

0.8

1.0

Fig. 1. The cyclic voltammograms of 1.0 × 10−3 M solution of K3Fe(CN)6 in 0.1 M KCl at a scan rate of 0.05 V s−1 a) at the surface of bare CPE b) graphene modified CPE.

2.6. Sample preparation for mass spectroscopic analysis

ip = (2.69 × 10 5) n3/2 D01/2 C0 A0 v1/2

After the electro-oxidation of 1 × 10−4 M of eugenol in pH 2.0 BR buffer solution applying DPV between 0.0 V and 1.0 V, the electrode was washed with deionized water and immersed in tetrahydrofuran (THF) solution to dissolve the electrochemical reaction product adsorbed on the electrode surface. Its mass spectroscopic analysis was then conducted.

where ip is the anodic peak current, n is the number of electron transfers in the reaction equal to 1. A0 is the active surface area (cm2), C0 is the concentration of K3Fe(CN)6 (mol cm-3), D0 is the diffusion coefficient equal to 7.6 × 10−6 (cm2 s−1) [25], v is the scan rate. From the slope of the plot of Ip vs v1/2, the areas of the electrode surface were calculated to be 0.0423 ( ± 0.0021) and 0.0259 ( ± 0.0013) cm2 for graphene modified CPE and CPE, respectively. It is obvious that modification of the electrode ensures the increase in the real surface area of the electrode.

3. Results and discussion 3.1. Electrochemical behavior of eugenol on graphene modified CPE Electrochemical behavior of eugenol was studied by CV and DPV, using a graphene modified CPE. Cyclic voltammetry studies were performed in 0.1 M BR buffer solutions, having the pH range of 2– 10. After the first scan of 5.0 × 10−5 M eugenol at pH 2.0, a welldefined anodic peak (peak 1) at 0.70 V and a cathodic peak (peak 2) at 0.38 V were observed (Fig. 2A). On the other hand, after the second scan, a new broader anodic peak (peak 3) appeared at about 0.45 V, while the main anodic peak decreased, indicating a more easily oxidized intermediate product on the electrode surface with respect to eugenol (Fig. 2B). DPV of eugenol, obtained in BR buffer solution of pH 2, is depicted in (inset Fig. 2A). While only one anodic peak was observed at the first scan at 0.70 V, a second anodic peak appeared during the second scan at 0.42 V (inset Fig. 2B), which increased at the

2.4. Preparation of the pastille sample Pastisin(R) clove – lemon herbal pastilles were purchased from a local pharmacy (Kurtsan pharmaceuticals. Ingredients: ascorbic acid, clove, clove flavor, lemon flavor, menthol, eugenol, aspartame, acesulfame K, citric acid, glyceryl monostearate (90%), vitamin B2 and isomalt). Six pastilles were weighed accurately, thoroughly crushed and powdered in a glass mortar. The powder, equivalent to a pastille (2.56 g) was placed into a 50 mL volumetric flask, filled with 20 mL of ethanol and extracted in an ultrasonic bath for 1 h. The volume was completed with ethanol and centrifuged prior to filtration through a 0.45 µm nylon filter. Propak Oral Spray (15 mL, Salus Drug, Turkey) was purchased from a local pharmacy.

Fig. 2. CVs of 5.0 × 10−5 M eugenol obtained on graphene modified GCE in 0.1 M BR buffer solution at pH 2 (scanning rate 50 mV s−1). A) First cycle B) Second cycle. Inset figures: DPVs of 7.0 × 10−6 M eugenol obtained on graphene modified GCE in 0.1 M BR buffer solution at pH 2. A) First cycle B) Second cycle.

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3.3. Effect of pH

expense of decrease of the main anodic peak. After the fourth scan, however, peak heights remained unchanged. Thus, the oxidation peaks in the first scan were considered. It is important to have a fix accumulation time since the analyte is adsorbed to the electrode surface and the peak currents can greatly be affected. Open circuit accumulation time on the peak current was investigated and 90 s, resulting in the highest peak current, was employed as an optimum accumulation time. Formation mechanism of the probable products in two oxidations is discussed in the computational section.

The effect of pH on the anodic peak current was studied in the range of pH 2–8 by using DPV (Fig. 4), which showed a maximum at pH 2.0. The plot of Ipa vs pH provided the equation Ipa (µA) = 0.7851 (pH) – 0.9567 (R2 = 0.9994) (inset Fig. 4). In order to observe the effect of pH on the anodic peak potential, an Ep vs pH graph was constructed, using the CVs data, and the equation of Epa (V) = −0.0528 pH + 0.8672 (R2 = 0.9985) was obtained The increase in pH of the solution caused a shift to less positive value in the peak potential. The slope, obtained from the data for the main oxidation peak (peak 1), was found to be 52.8 mV/pH (the theoretical value was close to 59 mV), indicating the number of electrons transferred is equal to the number of protons in the electrochemical reaction.

3.2. Electrochemical reaction mechanism of eugenol on graphene modified CPE 3.2.1. Computational studies As all the mechanisms predicted in the literature are based on assumptions, a DFT computational study was conducted using Gaussian 09 program [26] to understand the electrochemical reaction mechanism of eugenol in depth. While wB97XD [27] method with a conventional all-electron basis set 6–31G(d) was applied on closed shell molecules, the radicals were located at unrestricted wB97XD/6– 31G(d) level. Solvent effects were incorporated by using self-consistent reaction field (SCRF) with a UAHF (united atom Hartree-Fock) parametrization [28] of the conductor-like polarizable continuum model (CPCM) [29,30] as implemented in the Gaussian 09 package. Water (ε = 78.3553) was employed as a solvent to mimic the experimental conditions. The minima were confirmed by evaluating the harmonic vibrational frequencies using analytical second derivatives providing NImag of 0. Single point calculations with (U)wB97XD functional, including solvent effects (CPCM:water), were performed using aug-cc-PVTZ [31] basis set on (CPCM:water)-(U)wB97XD/6– 31 G(d) level optimized structures. Computational studies were initiated with simultaneous removal of two proton/electron couples, leading to two possible mechanisms “A” and “B”. Through the mechanism A, homolytic cleavage of hydrogen A led to the formation of the corresponding phenoxy radical (E1-R) with a relative Gibbs free energy of 12.3 eV, major two resonance structures of which were found to be E1-R-o (ortho) and E1-R-p (para) in 1.2:1.0:1.0 ratio, respectively, based on spin densities (Scheme 1). According to the literature [12], the second electron leaves the system resulting in the formation of the carbocation E1-C, which is stabilized by the lone pairs of methoxy oxygen. Upon reaction with water, E1-C liberates a proton and a MeOH group to give the product P1 in accordance with the literature with a relative energy of 23.9 eV. As allylic proton in E1-C is labile to the cleavage, formation of P2, having 0.2 eV less energy than P1, by releasing H+ from E1-C should also be considered. Indeed, removal of the allylic hydrogen, which is also a benzylic hydrogen, in E, following the mechanism B, led to the formation of E2-R, possessing less energy than E1-R by 0.1 eV. Moreover, the corresponding carbocation E2-C, generated from E2R by releasing one electron, is more stable than E1-C by 1.0 eV. In the last step, deprotonation renders again P2. Based on these computations, unlike the literature reports, formation of the product P2 is preferred over P1 by a relative energy of 0.2 eV through the mechanism B (Scheme 1).

3.4. Effect of scan rate The effect of scan rate (ν) on the main peak current at 0.1 M, pH 2.0 BR buffer at 0.70 V, in the presence of 5.0 × 10−5 M eugenol, using the graphene modified carbon paste electrode was examined in the range of 20–700 mV s–1. The plot of the logarithm of the peak currents (log Ip) against logarithm of scanning rate (log v) exhibited a linear relationship between 20 mV and 700 mV scan rates, resulting in an equation of log Ip (µA) = 0.841 log v (mV s−1) – 1.029 (R2 = 0.9994). The dependence indicated that the current is adsorption controlled. The slope of 0.841 is close to the theoretical value for an adsorption controlled process. When the scan rate was increased, the peak potential slightly shifted to positive values (Fig. 5). The linear relationship between the peak potential and the logarithm of the scan rate was obtained as Ep (V) = 0.0483 log v (V s−1) + 0.8241; R2 = 0.9988 (inset Fig. 5). According to Laviron, for the irreversible electrode process, the following equation is given [32]:

⎛ 2. 303RT ⎞ ⎛ RTk 0 ⎞ ⎛ 2. 303RT ⎞ Ep = E 0 + ⎜ ⎟ log v ⎟ log ⎜ ⎟+⎜ ⎝ αnF ⎠ ⎝ αnF ⎠ ⎝ αnF ⎠

(1)

where, α is the electron transfer coefficient, k° is the standard heterogeneous rate constant of the reaction, n is the number of electrons transferred, v is the scan rate and E° is the formal redox potential, T = 298 K, R = 8.314 J K−1 mol−1 and F = 96485 C mol−1. Considering the slope as 0.0483, the value of αn was calculated to be 1.224. According to Bard and Faulkner [24], α is calculated using the following equation:

α=

47. 7 mV Ep − Ep/2

(2)

where Ep is the oxidation peak potential and Ep/2 is the potential, where the current is at half of the peak value. By using Eq. (2), the value of α was obtained as 0.6446. The number of electrons transferred in the electro-oxidation of eugenol was calculated to be 1.899 ≈ 2. Hence, eugenol could be assumed to undergo two-proton and two-electron transfer in the electro-oxidation reaction. The E° value of 0.7328 was obtained from the intercept of Ep vs v curve by extrapolating to the vertical axis at v = 0 [33]. By using the intercept (0.8241) and slope (0.0483), obtained from Ep vs. log v plot, k°, was calculated to be 3.696 × 102 s−1 in pH 2.0 BR buffer solution.

3.2.2. Mass spectroscopic analysis Mass spectroscopy unambiguously demonstrated the presence of both products P1 and P2, the molecular weights of which are 149 (M+ + 1) and 163 (M+ + 1) g mol−1, respectively (Fig. 3). This result is well in alignment with the DFT outcome, suggesting the possibility of formation of both products. Moreover, it should be noted that P1 can also emerge from defragmentation of P2 under the spectroscopic condition since DFT level calculations provided P2 with less energy than P1 by 0.1 eV.

3.5. Electrochemical determination of eugenol 3.5.1. Calibration curve Eugenol standard solution showed that the DPV between 0.4 V and 1.0 V was suitable for the measurements. Oxidation peak currents (0.7 V) of the DPVs, obtained for different eugenol concentrations, were utilized to generate a calibration graph in the concentration range of eugenol from 1.0 × 10−7 to 1.7 × 10−5 M (inset Fig. 6). The calibration curve obtained with the standard solution was characterized by the linear equation Ip = 4

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Scheme 1. (CPCM:water)–wB97XD/aug-cc-PVTZ//(CPCM:water)–wB97XD/6–31G(d) level calculations (ΔGrel with unscaled zpe are in eV).

0.702 C – 10−7 with R2 = 0.9991 (Ip: µA, C: µM). LOD and LOQ were calculated using the equation LOD = 3 SD/m and LOQ=10 SD/m, where SD and m stand for the standard deviation of the peak currents (N = 5) and the slope of the calibration curve, respectively. LOD and LOQ were calculated to be 7.0 × 10−9 M and 2.3 × 10−8 M, respectively.

3.5.2. Repeatability Seven replicate samples (10−6 M standard solution) were run for repeatability control. The resulting mean concentration was found to be 1.04 × 10−6 M with 3.9% RSD value, which indicated a good repeatability.

Fig. 3. Mass spectrum indicating the existence of P1 and P2.

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Table 2 The recovery results of eugenol. Added eugenol concentration (M)

Mean % eugenol recovered ( ± SD)

1.0 × 10−6 5.0 × 10−6 1.0 × 10−5

101 ( ± 0.55) 99 ( ± 0.70) 99 ( ± 0.34)

3.5.3. Recovery test Recovery tests were performed by adding 3 different concentrations of the standard solution of eugenol in a pre-analyzed known amount of tablet sample solutions, obtained from extracting the powdered tablets (Table 2). The results suggested that the proposed method has sufficient precision and accuracy for determination of eugenol (Table 2). 3.5.4. Analysis of the pharmaceutical samples In order to evaluate the applicability of the proposed method, the amount of eugenol in a commercially available eugenol containing pastille (Pastisin(R) clove – lemon herbal pastille) and Propak Sprey were determined. Propak Oral Spray is available as a food supplements, which contains propolis extract, sodium bicarbonate and clove oil. The procedure, given in Section 2.4 was followed to prepare the pastille sample solution and then it (0.4 mL) was diluted with 0.1 M BR buffer solution in a 10 mL voltammetric cell. The spray sample (0.1 mL in 10 mL BR buffer solution) was run without any treatment in a voltammetric cell. The eugenol contents of the samples were analyzed under specified voltammetric conditions, using the standard addition method. Fig. 7 shows the DPVs obtained by the standard addition method. Evaluation by the standard addition method is shown in inset Fig. 7A and B. Eugenol contents of these samples were found to be 0.198 ( ± 0.01) mg g−1 for pastille sample and 0.133 ( ± 0.007) mg mL−1 for spray sample. The pastil and spray results obtained by the proposed method were compared with UV measurements reported by Backheet [34]. Briefly, 1.0 mL of the standard or sample methanoic solutions was transferred to a volumetric flask (10 mL), to which potassium permanganate solution (0.5 mg mL-1; 1.0 mL) was added, the mixture was allowed to stand for 5 min and then it was mixed well. Then, the volume was completed with distilled water, and its absorbance was recorded at 526 nm against a blank, prepared using potassium permanganate solution. Initially, a calibration curve was prepared using standard eugenol solution, which was linear in the range of 0.5−10 µg mL−1 concentration (at 5 different concentrations, N = 3). Regression equation was y = 0.0345x + 0.0054 (R2 = 0.9944). There was no difference between the results of pastil and spray content, collected by both methods using Student's t-test and F-test at 95% confidence level (Table 3).

Fig. 4. DPVs of 5.0 × 10−6 M eugenol in 0.1 M BR buffer of different pH on graphene modified CPE a) pH 2.0 b) pH 3.0 c) pH 5.0 d) pH 8.0. Inset: Plot of peak current vs. pH.

Fig. 5. CVs of 5.0 × 10−5 M eugenol in BR buffer solution of pH 2.0 (0.1 M) at scan rates of a) 0.02 b) 0.05 c) 0.10 d) 0.20 e) 0.30 f) 0.40 g) 0.50 h) 0.70 V s−1. Inset figure: Plot of variation of peak potential with logarithm of scan rate.

y = 0.702 x - 10 R = 0.9991

3.6. Interference study Interference studies were performed in order to investigate possible interferents, i.e. ascorbic acid, citric acid, vitamin B2 and sodium bicarbonate, in the samples. DPVs for 5.0 × 10−5 M eugenol in the presence of 2.0 × 10−4 M of each interferent indicated that the response of eugenol was not affected by the presence of these substances. On the other hand, acetaminophen (2.0 × 10−4 M) caused ca. 26% of signal enhancement. 4. Conclusion

Fig. 6. DPVs of eugenol in different concentrations. (From bottom to top: 1.0 × 10−7 M, 1.0 × 10−6 M, 2.0 × 10−6 M, 4.0 × 10−6 M, 1.0 × 10−5 M, 1.7 × 10−5 M. Inset figure: Calibration graph constructed using peak currents obtained from DPVs at various eugenol concentrations.

In this study, electro-oxidation of eugenol on graphene modified CPE was investigated for the first time. Its electro-oxidation reaction mechanism was elaborated through DFT level computations and 6

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Fig. 7. Determination of eugenol by DPV in real samples in 0.1 M BR buffer of pH 2.0. A) DPVs obtained by addition of (from bottom to top) 0.00 mg mL-1, 1.64 × 10−3 mg mL-1, 3.28 × 10−3 mg mL-1, 4.92 × 10−3 mg mL-1 on pastille sample. B) DPVs obtained by addition of (from bottom to top) 0.00 mg/mL, 1.64 × 10−3 mg mL-1, 3.28 × 10−3 mg mL-1, 4.92 × 10−3 mg mL-1 on pastille sample. (Inset figures: Evaluation by the standard addition method for pastille sample (inset A) and spray sample (inset B).

References

Table 3 Comparison of the results obtained by the proposed method and the UV method for the analysis of eugenol in pastil and in oral spray. Statistical values

Mean mg for per pastil or mL of spreya RSD%a SD t-calculated F-calculated Confidence limitsb a b

Proposed method

Comparison UV method [34]

Pastil

Spray

Pastil

Spray

0.20

0.13

0.21

0.14

2.20 0.004 1.30 1.73 0.19–0.20

6.48 0.009 1.94 0.26 0.13–0.15

1.44 0.003

2.37 0.003

0.21–0.22

0.13–0.16

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Each value is the average of six determinations. Theoretical values at 95% confidence limit, t=2.23 and F=5.05.

experimental findings, which suggested the preferred formation of product P2, possessing a para-quinoid structure rather than the literature suggested product P1, having an ortho-quinoid system. This demonstrated the fact that the observed P1 might emerge from the homolytic cleavage of methyl radical from P2 under measurement conditions. The second anodic peak appeared during the second scan at 0.42 V might be attributed to the oxidation of P2 product. A differential pulse voltammetric method, based on the oxidation peak of eugenol appeared at 0.70 V in BR buffer at pH 2 on graphene modified CPE, was evaluated. The method successfully applied for eugenol determination in a pharmaceutical sample. Compared to the other known methods in the literature, the proposed method offers the advantage of easy electrode fabrication, rapid applicability and low cost.

Acknowledgements M.E. Cinar thanks TUBITAK for Postdoctoral Research Fellowships BIDEB 2216 program. Z. Aydogmus thanks Istanbul University, Scientific Project Coordination Unit for financial support (Project no: 37460). We are indebted to National Center for High Performance Computing (UYBHM) (tvddvb) and the High-Performance-Computing (HPC) Linux Cluster HorUS of University of Siegen for the computer time provided. Unsped Global Logistic is gratefully acknowledged for financial support. 7

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