Voltammetric Determination of Anethole on La2O3/CPE and BDDE

In this work, DPV determination of anethole was presented using various carbon, two-diameter (1.5 and 3 mm) electrodes, that is, BDD, GC, CP, and CP doped by La2O3 and CeO2 nanoparticles. La2O3/CPE to our best knowledge was proposed first time. Cyclic voltammograms confirmed totally irreversible electrode electrooxidation process, controlled by diffusion, in which two electrons take part. The most satisfactory sensitivity 0.885 ± 0.016 µA/mg L−1 in 0.1 mol L−1 acetate buffer was obtained for La2O3/CPE with the correlation coefficient r of 0.9993, while for BDDE it was 0.135 ± 0.003 µA/mg L−1 with r of 0.9990. The lowest detection limit of 0.004 mg L−1 was reached on La2O3/CPE (3 mm), what may be compared with the most sensitive conjugate methods, but in the proposed approach, no sample preparation and analyte separation was needed. Anethole was successfully determined in specially prepared ethanol extracts of herbal mixtures of various compositions, which imitated real products. The proposed procedure was verified in analysis of commercial products, that is, anise essential oil, which contains a large concentration of anethole, and in alcohol drinks like Metaxa, Ouzo, and Rakija, in which the considered analyte occurs on trace levels. Structure and properties of the considered nanopowders and graphite pastes were investigated by EDX, SEM, and EIS.

Due to its organoleptic properties, pleasant aroma and sweet taste, essential oils containing anethole have been used for centuries in the perfume, pharmaceutical, and spirit industries [1-6, 11, 12]. In pharmaceutical applications, anethole properties such as oestrogenic action, depressive action to the central nervous system, psycholeptic, insecticide, bactericidal, anticarcinogenic, anti-in ammatory, and anesthetics activity [1-6, 11, 12] are very important. e bactericidal properties of anethole are, due to lack of free phenolic group, weaker than their natural analogue-eugenol ( Figure 1) [6]. In the spirit industry, anethole is present in various types of alcoholic beverages based on anise, fennel, or licorice-mainly Absinthe, Pastise, Ouzo, Rakija, and Metaxa [6,12]. In addition, some alcohols must contain exactly the speci ed amount of anethole; for example, Pastise contains 1.5-2.0 g L −1 of this compound [12]. So, accurate determination of anethole content is one of the important stages of drinks production.
Among the quantitative methods of anethole assays in various matrices (Table 2), chromatographic techniques are dominant. Voltammetric techniques are used to evaluate the antioxidant properties of the compounds containing anethole [13] and may be useful in the classi cation of alcoholic beverages [14]; therefore, developing the method of anethole determination seems to be justi ed and interesting.
In this work, we present the possibility of trans-anethole determination by di erential pulse voltammetry (DPV) technique. Various carbon electrodes, that is, glassy carbon electrode (GCE), boron-doped diamond electrode (BDDE), carbon paste electrode (CPE), carbon paste electrode doped by cerium(IV) oxide (CeO 2 /CPE), and carbon paste electrode doped by lanthanum(III) oxide (La 2 O 3 /CPE), were used and tested. After designation of the analytical parameters of the method and optimization, quantitative and qualitative assays of anethole were applied in four specially prepared herbal matrices similar to anethole-containing beverages and in various commercially available products of natural origin. e obtained results are very promising and can be used in determination of anethole in a variety of matrices without analyte separation or sample preparation.

Measuring Apparatus and Software. A multipurpose
Electrochemical Analyzer M161 with the electrode stand M164 (both MTM-ANKO, Poland) was used for all voltammetric measurements. e classical three-electrode quartz cell of 10 mL volume was applied. Various carbon sensors were utilized as the working electrodes, that is, glassy carbon electrode (BASi, ϕ � 3 mm and home-made, ϕ � 1.5 mm),    3 ) and a platinum wire as an auxiliary electrode were used. e ambient temperature was ca. 23°C. e MTM-Anko EAPro 1.0 software enabled electrochemical measurements, data acquisition, and processing of the results. Electrochemical impedance spectroscopy measurements were performed using a frequency analyzer (Solartron model FRA 1260) coupled with dielectric interface (model 1296). e surface morphology of electrode material was observed using ultrahigh-resolution scanning electron microscope with eld emission (FEG-Schottky emitter; Nova Nano-SEM 200, FEI Europe BV) cooperating with EDAX EDS analyzer.

Carbon Paste Electrodes Doped by La 2 O 3 and CeO 2 .
Carbon-based electrodes are useful for voltammetric determination of wide range of analytes in liquid solutions. Moreover, their applicative properties can be improved by doping with metal oxide modi ers. e usage of di erent doping oxides was reported for modi cation of carbon-based electrodes so far [18][19][20][21][22]. According to the literature, addition of controlled amounts of cerium(IV) oxide to glassy carbon electrodes material led to the signi cant enhancement of sensitivity, selectivity, reproducibility, and response time in the amperometric quanti cation of eugenol [22].
In this work, the experimental data obtained by use of lanthanum oxide-doped graphite paste electrodes applied in voltammetric analysis are presented for the rst time. e carbon pastes were prepared by hand mixing an adequate amount of graphite powder and rare earth oxide powder with para n oil using a pestle and mortar for at least 30 minutes in the case of each batch. Nanopowders of lanthanum(III) oxide (99.99%) and cerium(IV) oxide (99.9%) were provided by Acros Organics. e ratio of used para n oil and graphite powder was determined based on literature repots as well as our experience in order to get electrodes characterized by high chemical and mechanical stability during performance in liquid solutions. After standing overnight, the resulting homogenous pastes were packed into the well of the working electrodes to depth of 2 mm with two di erent diameters (1.5 and 3 mm). e body of working electrode was a Te on tube with stainless steel rode of 1.5 mm diameter serving as electric contact. To provide the required smoothness of electrodes, working surfaces the forehead of electrodes were polished on a print paper or tissue paper. e amounts of used reagents, details of prepared electrodes, and pastes are presented in Table 3.

Chemicals and Glassware.
As a supporting electrolyte, bu ers of a di erent pH were prepared in our laboratory (from reagents pure for analysis, POCH, Poland): acetate bu er-mix acetic acid and sodium acetate; Britton-Robinson bu er-mix boric acid, phosphoric acid, acetic acid, and sodium hydroxide; Sørensen phosphate bu er-mix sodium hydrogen phosphate and sodium dihydrogen phosphate; ammonia bu er-mix ammonia and ammonium chloride. As a standard solution, trans-anethole (analytical standard, Sigma-Aldrich) was used. 1 µL of solution contains 3.48 µg of trans-anethole. Reagents used to determine the impact of interferents are 99% eugenol (Reagent Plus, Sigma-Aldrich), 99% carvacrol (food grade, Sigma-Aldrich), ≥98.5% thymol (pure, Sigma-Aldrich), and zinc, lead, cadmium, bismuth, aluminum, thallium, chromium, and vanadium (all metals from Certipur, Merck). e other chemicals were 95% ethanol (food grade, Polmos, Poland) and 0.1 mol L −1 solution of sulfuric acid (pure for analysis, POCH, Poland) for activation of BDD electrode. All reagents used were prepared using quadruply distilled water (two last stages from quartz). Glassware was rst immersed in 6 M nitric acid and then rinsed repeatedly with distilled water.

Samples.
To verify the possibility to determine anethole in herbal matrices A, B, and D, three solutions were prepared. Also matrix C was tested, which did not contain anethole. e composition and preparation of the matrices imitated di erent anethole-containing beverages. Each matrix was prepared by pouring with the ethanol (95%, food grade) the appropriate herbal composition and the ve-day maceration of the mixture. After this time, each matrix was recti ed once.

Standard Procedure of Voltammetric Measurements.
Measurements were performed using di erential pulse voltammetry (DPV). Before each series of measurements, surface of the BDD electrode was activated 15 minutes in 0.1 mol L −1 sulfuric acid solution by the potential of 2400 mV. Before each calibration, the BDDE surface was additionally renewed by the potential 1500 mV and time of 30 s in supporting electrolyte. GCE was activated by polishing with polishing powder MicroPolish Alumina 0.05 μm (Buehler, USA). e investigation of anethole was performed in different supporting electrolytes depending on the working electrode used, that is, 0.1 M acetate bu er with pH 3, 4, 5, or 6; Britton-Robinson bu er with pH 2 and 3; 0.1 M Sørensen's phosphate bu er with pH 6, 7, and 8; or 0.1 M ammonia bu er with pH 9 and distilled water, giving total volume of 5 mL lling the quartz voltammetric cell. e best results were obtained in supporting electrolyte consisting 5 mL of 0.1 M acetate bu er with pH 6. e volume of added standard solution of anethole was of 1-5 µL.
e solution in cell was stirred (ca. 500 rpm) using a magnetic stirring bar. en, after a rest period of 5 s, di erential pulse voltammograms were recorded in the potential window: 0-1200 mV (BDDE), 500-1300 mV (CPE, La 2 O 3 /CPE, CeO 2 /CPE), and 600-1200 mV (GCE). e other standard experimental parameters were as follows: potential step E s � 5 mV; pulse potential dE � 50 mV; and time of potential step � 40 ms (20 ms waiting time + 20 ms sampling time).
All experiments were performed at 23°C. All experiments were carried out in triplicate.

Carbon Electrodes in Determination of Anethole.
e purpose of the study was to investigate whether carbon paste electrodes doped by two new rare earth element oxides may be useful in voltammetric determination of the anethole. Commercially available and popular sensors were used as a comparison. e well-de ned DPV anethole peak   ( Figure 2) was obtained on the various carbon electrodes, that is, glassy carbon, boron-doped diamond, carbon paste, and carbon paste doped by lanthanum(III) oxide and cerium(IV) oxide, which were considered in this work. e peak position was observed between 965 and 1155 mV ( Table 4, second column). Anodic shift (of 150-200 mV) of anethole oxidation potential has been obtained for carbon paste and two nanoparticles-modi ed electrodes compared to GCE and BDDE, con rming the lower transfer rate on CPE and nanoparticles/CPE. Quantitative analysis was preceded by especially projected procedure of baseline modeling and subtraction ( Figure 3). e rst step of the proposed approach was subtraction of the experimental baseline obtained for the supporting electrolyte. Next, the typical approximation by the polynomial of the 2nd degree was utilized. ese two steps were necessary, because the background shape was very di erent from the polynomial function.
Analytical parameters were determined and tested for two groups of electrodes (Table 4), that is, of the diameter of 3 mm (geometric area of 7.07 mm 2 ) and of the diameter of 1.5 mm (geometric area of 1.77 mm 2 ). After signal processing, the linear relation between peak current and concentration of the anethole in the range of 0.7-17.5 mg L −1 was noticed. Generally, paste electrodes were characterized by the greater sensitivity and lower detection limit in comparison to BDDE and GCE. However, the repeatability of the signal for successive analyte concentration was excellent for the latter (CV < 1%). e highest sensitivity of 0.89 µA/mg L −1 among the considered electrodes was obtained on the carbon paste doped by the 20% of lanthanum(III) oxide nanoparticles, with the correlation coe cient r of 0.9993 (for averaged signals for each concentration) and the lowest detection limit of 0.004 mg L −1 . e sensitivity for the anethole on the electrode doped by the 20% of cerium(IV) oxide nanoparticles (ϕ � 3 mm) was even lower (0.34 µA/mg L −1 ) than the reference value obtained on CPE (0.55 µA/mg L −1 ). e lowest sensitivity in the group of sensors with a diameter of 3 mm was obtained on BDDE (0.14 µA/mg L −1 ) what was ca. 6 times less than on La 2 O 3 /CPE. Considering the sensors with a diameter of 1.5 mm, the highest sensitivity of 0.45 µA/mg L −1 was obtained on CeO 2 /CPE. For CPE-and La 2 O 3 -doped CPE, the repeatability of the signal relied on the percent (w/w) of the added nanoparticles and was on the level of 5-8% (CV) when the nanopowder addition was lower than 20%. For the addition greater than 20%, the repeatability of the signal rapidly deteriorated (CV > 10%), and therefore these electrodes were not considered in further tests. CP electrodes doped by CeO 2 did not also show the satisfactory repeatability of the signals recorded for each concentration. It was also observed that increasing addition of the lanthanum(III) oxide nanoparticles decreased sensitivity for the anethole.
For further detailed analysis, La 2 O 3 /CPE (ϕ � 3 mm) as a sensor of the greatest sensitivity for the anethole was chosen and for comparison BDDE, which is reliable after appropriate activation. Voltammograms and calibration lines for the anethole in the concentration range from 1.39 to 6.96 mg L −1 prepared on the mentioned two sensors are presented in Figure 2(b).

Supporting Electrolyte E ects.
ere are several ways in which the supporting electrolytes solvent system can inuence mass transfer, the electron reaction (electron transfer), and the chemical reactions which are coupled to the electron transfer. As a supporting electrolyte, 4 di erent bu ers (acetate, Britton-Robinson, phosphate, and ammonia) were applied in examination of the analyte behavior in pH range from 2.0 (BR bu er) to pH 9.0 (ammonia bu er). e best parameters-repeatability, sensitivity, limit of detection, and the favorable relation between signal and baseline-were obtained using acetate bu er; therefore, the pH e ect was tested carefully in the pH range typical for this electrolyte, that is, from 3.0 to 6.0. In the considered Table 4: DPV anethole determination in the range of 0.7-17.5 mg L −1 in 0.1 mol L −1 acetate bu er, pH 6.0 (n � 3).

Electrode
Anethole peak position (mV) a ± SD a (µA/mg supporting electrolytes at strongly acidic pH (Britton-Robinson bu er, pH 2.0), neutral pH (phosphate bu er, pH 7.0), and basic pH (phosphate bu er, 8.0; ammonia bu er, pH 9.0), the investigated analyte did not show adequate analytical sensitivity and repeatability. Figure 4 presents the in uence of the acetate bu er pH on the anethole voltammetric signal. e well-de ned DPV peak was observed in the whole range of the considered pH, that is, 3.0-6.0. For BDDE, the peak position changed in the range from 950 to 990 mV, without a distinct maximum current change. For La 2 O 3 /CPE, the oxidation peak currents decreased to pH 5.0 and then increased. Anethole oxidation potential decreased from 1180 mV to 1080 mV as pH increased. Further experiments were done by pH 6, because less positive peak position equal to 1080 mV is more suitable for oxidation. Sensitivity in this case was also ca. 44% greater in comparison with the best variant obtained for the other pH.

Parameters of Anethole Electrooxidation on BDDE and
e voltammetric behavior of anethole on two carbon electrodes, that is, BDD and La 2 O 3 -modi ed electrodes, in 0.1 mol L −1 acetate bu er of pH 6.0 has been investigated by recording cyclic voltammograms (CV) using the scan rates of 0.025, 0.05, 0.1, 0.2, 0.25, and 0.5 V s −1 . It was observed that anethole is irreversibly oxidized on these electrodes ( Figure 5), what was con rmed by the absence of cathodic step on the backward branch of the CV. e CP electrode modi cation with La 2 O 3 nanoparticles leads to the anodic shift of anethole oxidation potential on ca. 200 mV. e e ect of potential scan rate in the range of 0.025-0.5 V s −1 on the voltammetric behavior of anethole is also presented in Figure 5. e anethole oxidation currents were proportional to the square root of the potential scan rate (1), con rming that the electrochemical process is di usion controlled [23]. Moreover, the natural logarithm of anethole peak current (lni p ) increases linearly with the natural logarithm of scan rate (lnν) in the range of potential scan rate under investigation, and the regression equation is described by  (2) e value of the slope is below the theoretical value of 0.5, what proves the di usion nature of anethole oxidation peak once again [23]. A linear relationship between the oxidation potential E p and ln] has been observed, conrming totally irreversible electrode processes: In this case, the number of electrons participating in the reaction can be calculated according to [24] where α is assumed to be 0.5 for a totally irreversible electrode process. e E p − E p/2 is 53 mV for BDDE and 59 mV for La 2 O 3 /CPE. Hence, the number of electrons participating in the anethole oxidation process equals n α to 2.22 for BDDE and 2.47 for La 2 O 3 /CPE, what agree well with the values reported earlier [25]. Figure 6 presents the proposition of the electrode reaction.

Investigation by Energy-Dispersive X-Ray Spectroscopy and Scanning Electron Microscopy.
e chemical composition of the pastes used for construction of CPE and La 2 O 3 /CPE was analyzed by EDX. e EDX spectrum for the nondoped carbon paste (Figure 7(a)) con rmed the presence of carbon, as the dominant element, and a small  e SEM test showed that the nondoped carbon paste was characterized by a surface formed by irregular graphite akes (Figures 8(a) and 8  (b)). e surface of the La 2 O 3 -doped carbon paste is more porous, heterogeneous, and irregular than the surface of nondoped carbon paste (Figures 8(c) and 8(d)). is suggests that the presence of La 2 O 3 molecules in carbon paste signi cantly increases the morphological structure of the material, which facilitates the electron transfer process in the electrode-solution interface, giving better sensitivity and higher repeatability of the voltammetric signal.

Application of Electrochemical Impedance Spectroscopy.
Electrical properties of experimental set comprised of the studied carbon paste electrode and Ag/AgCl/KCl reference electrode immersed in solution containing analyte were determined by Electrochemical Impedance Spectroscopy (EIS) method. e measurements were performed in room temperature, with the frequency range of 0.1-10 MHz and the amplitude of sinusoidal voltage signal of 20 mV. e experimental data were analyzed using the ZView software (version 2.2, Scribner Associates, Inc.), which helped in determination of equivalent circuits' optimal parameters. e comparison of Nyquist's spectra obtained for three di erent carbon paste electrodes is given in Figure 9. In each case, spectrum was comprised of semicircle visible in highfrequency range and the spur in middle and low frequencies.
Electrical equivalent circuits were tted to the experimental data sets. Spectra were analyzed by connected in series two parallel equivalent circuits consisted of resistors (R) and constant phase elements (CE) and additional constant phase element indispensable to model the spur in low frequencies.
e scheme of the used equivalent model is depicted in inset of Figure 9. e simulated spectra are plotted by solid black line and exhibit good agreement with experimental data presented by points. e semicircle parts of the spectra in high frequencies look similar in each case. e course of above mentioned part of the spectrum depends on the reference electrode and solution used during the measurements. erefore, the parameters of R1 and CE1 are of similar value (Table 5). e di erences in course of spectrum in the middle-and low-frequency parts indicate that it is attributable to carbon paste electrode properties. On the basis of conducted analysis, there is a strong relation between the applicable properties of carbon paste electrode and the value of resistance exhibited in middle-frequency fragments of spectra. In particular, the highest value of resistance R2 shows the electrode modi ed by lanthanum (III) oxide, while the electrode without rare earth oxide addition is characterized by the lowest value of this parameter. Concomitantly, the lower CE-T-2 value, the better performance of electrode. e most signi cant di erences between behaviors of studied electrodes are visible in low frequencies part of the spectra. It is re ected in particular in CE3 element. CE-T-3 value determined for undoped electrode is of order higher than for electrodes modi ed by rare earth metal oxides. Moreover, parameter n3 for CPE is somehow higher than for doped electrodes and close to 1, indicating stronger capacitive properties of undoped graphite than in the case of electrodes modi ed modi ed by lanthanum and cerium oxides.
3.6. Interferences. Such parameters as potential window, potential step, potential pulse, and time of potential step were tested to optimize the procedure of the anethole determination.
e criteria of optimization were repeatability, sensitivity of the method, and the favorable relation between signal and baseline. It was observed that starting potential does not have in uence on the anethole Table 5: Parameter values of tted equivalent circuits from Figure 9. peak. Taking into account all the criteria selected experiment conditions are potential step 5 mV, potential pulse 50 mV, and time of the potential step 40 ms (i.e., waiting time 20 ms + sampling time 20 ms). As possible interferences, metal ions such as Zn(II), Pb (II), Cd(II), V(III), Bi(III), Al(III), Tl(I), and Cr(III) and organic compounds such as eugenol, carvacrol, and thymol were tested, which may be present in plants and products of biological origin, in which anethole also occurs. e concentration of the metal ions was in the range of 1.4-14 mg L −1 by the 13.92 mg L −1 of anethole, which was present in the measured solution. e anethole peak position was not moved, and also no additional peaks were observed. However, the impact of the analyzed ions on the height of the anethole peak after addition of metals was noticeable: change for BDDE anethole signal was in the range of 93-99%, and change for La 2 O 3 /CPE was in the range from 82 to 127% (Table 6). e greater sensitivity variation in the last case may be connected with the chemical reactions between metal ions and active lanthanum(III) oxide nanoparticles, what could cause the change of the number of active centres on the electrode surface.
No additional current peaks coming from eugenol, carvacrol, and thymol were observed in the considered acetate bu er (pH 6.0) and potential area where anethole peak was recorded. e concentration of the added substances was in the range of 0.7-3.5 mg L −1 by the 13.92 mg L −1 of anethole. e presence of biological compounds in the solution caused, in the experiments with BDDE, increase of the sensitivity up to 100%. is value is related to the study of the carvacrol e ect at the 4 times excess of anethole.
e mentioned interferents may facilitate the charge transfer between the analyte and the electrode. In the case of measurements on La 2 O 3 /CPE, an addition of 3 biological compounds resulted in the change of the signal amplitude in the range of 84-126%.

Determination of Anethole in Herbal Matrices and
Commercial Products. Because anethole occurs in food products (beverages, herbal oils, and tinctures) and herbs such as anise, star anise, fennel, liquorice, and caraway, the problem of anethole determination of specially prepared herbal matrices was considered. e composition of these mixtures which mimics the real commercially available products is given in Table 7. It is important that some matrices contain anethole, while the other did not contain this analyte, and it was added at the stage of recovery studies. e concentration of the anethole in matrices A, B, and D was on the level 0.1-1.6 g L −1 (Table 7). e highest concentration was in the most complex mixture B, while the lowest in D, where only one component contained anethole. e signi cant decrease of the sensitivity of the method was observed in comparison to the measurements in only supporting electrolyte. e decrease was to 73% (matrix B) in the case of BDDE and to 66% (matrix D) in the case of La 2 O 3 /CPE. Exemplary voltammograms recorded on La 2 O 3 /CPE in the case of anethole determination in matrix D are presented in Figure 10(a).
Matrix C did not contain a detectable concentration of the anethole; therefore, this analyte was added to the herbal extract, and percent of recovery was studied (Table 8)  Further, anethole was determined in commercially available products. Some research objects were chosen in which the mentioned analyte is present on very low and very  high concentration level.
e measurements were done without sample preparation or anethole extraction. e adequate sample volume was added directly to the electrochemical cell. It was observed (Tables 9 and 10) that, in anise essential oil, the concentration of anethole was ca. 570 g L −1 , while in alcohol drinks, like Metaxa, Ouzo, and Rakija, it was ca. 0.13-0.21 g L −1 . e results obtained using both electrodes were compatible. At 95% con dence level, the calculated Student's t-values for the replicate measurements of each sample (Table 10) using both fabricated sensors did not exceed the theoretical value (2.7765), indicating that the results obtained are not signi cantly di erent. An F-test revealed no signi cant di erence between the standard deviations of the two sets of replicate measurements for each sample. Exemplary voltammograms recorded on La 2 O 3 /CPE in the case of anethole determination in Ouzo and Raki are presented in Figures 10(b) and 10(c).

Conclusions
In this work, a sensitive, rapid, and convenient DPV procedure of anethole determination was proposed, which does not require sample preparation and separation of the analyte, even in the case of complex matrices. Additionally, it was proved that various carbon electrodes, that is, BDD, GC, CP, and CP doped by La 2 O 3 , and CeO 2 nanoparticles, are sensitive for anethole, and the proposed analytical strategies ful ll typical validation criteria. Recording cyclic voltammograms, it was noticed that electrooxidation process has totally irreversible character, controlled by di usion, in which two electrons take part. e most sensitive electrode turned out to be La 2 O 3 /CPE with 20% of nanoparticles in graphite paste (w/w). According to our knowledge, it is the rst literature report about application of such a sensor. Sensitivity obtained in DPV experiments realized by optimized parameters in 0.1 mol L −1 acetate bu er was for La 2 O 3 /CPE of 3 mm diameter equal to 0.885 ± 0.016 µA/mg L −1 with the correlation coe cient r of 0.9993 and the detection limit of 0.004 mg L −1 , while for commercially available sensor BDDE it was 0.135 ± 0.003 µA/mg L −1 with r of 0.9990.
Operation of the selected electrodes was veri ed using especially prepared herbal ethanol extracts which contained and did not contain anethole. In the last case, recovery was tested applying standard addition method. Anethole was also successfully determined in commercially available products, such as anise essential oil, which contains a large concentration of anethole, and in alcohol drinks like Metaxa, Ouzo, and Rakija, in which the considered analyte occurs on trace levels. e results obtained on La 2 O 3 /CPE and BDDE were statistically consistent, at 95% con dence level.

Conflicts of Interest
e authors declare that there are no con icts of interest regarding the publication of this article.