Investigation Of Electrochemical Oxidation Mechanism, Rapid And Low‑level Determination For Whitening Cosmetic: Arbutin in Aqueous Solution By Nano Sepiolite Clay

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Sevda Aydar Barutçu1 · Dilek Eskiköy Bayraktepe2 · Zehra Yazan2 · Kamran Polat2 · Hayati Filik1

Abstract

Arbutin (AR) is one of the important chemical agents that has the lack of adverse effects in cosmetic applications, which has still biological importance and the increasing interest toward arbutin in the cosmetic industry. Electrochemical sensors have received much attention because of their high sensitivity, simplicity, and fast. In this work, an electroanalytical method has been developed and validated for the quantification of AR on nano sepiolite-clay carbon paste electrodes. The electrochemical oxidation mechanism of AR was also investigated in the aqueous medium. Nano-sepiolite clay modified carbon paste electrode was used as rapid and the low-level electrochemical sensor for determination of AR. The electrochemical responses of AR were compared on the surfaces of the bare and nano sepiolite modified carbon paste electrode using the cyclic voltammetric method in the BR buffer solution. The results showed superior electrocatalytic performance on the peak current of AR at the modified carbon electrode. The stripping conditions and experimental parameters (pH, the effect of modifier content, accumulation potential, and time) were optimized to obtain the best oxidation signal of AR. Under the optimized conditions, linear calibration curves were obtained in the concentration range of 0.0362–80.0 µM (with the detection limit of 10.8 nM) with square-wave adsorptive stripping voltammetry. The method was successfully applied for the determination of AR in the cosmetic Tritone cream sample. This work confirms thus that electrochemical sensors may be the potential future candidate for rapid, sensitive, low-level, and reproducible analysis.

Keywords: Arbutin, Sepiolite clay, Carbon paste electrode, Cosmetic cream, Voltammetry

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Introduction

Arbutin (AR) is a hydroquinone-β-d-glucopyranoside extracted from the bearberry plant. It is found in high concentrations in many medicinal plant species, particularly in the Ericaceae family (Pavlović et al. 2009). Arbutin can prevent melanin’s formation via the inhibition of the essential tyrosinase enzyme (Parvez et al. 2007). AR has been used in many skin whitening cosmetics. The cosmetic effect of this glycosidic structure is less than hydroquinone, but its toxicity is relatively low, and its water solubility is high compared to hydroquinone when not completely removed. Due to these properties, many skin whitening and pigment removal cosmetic products are frequently used (Libánský et al. 2011; Mehrabi et al. 2021; Shih and Zen 2000).

The depigmenting effect of arbutin has been reported to reduce tyrosine conversion to melanin by inhibiting tyrosinase and thus decreasing melanin biosynthesis. Melanin is a dark biological pigment that is generally found in the skin, hair, eye membranes, some parts of the brain, and in some products called melanic. In melanocytes, which are melanin-producing cells, inhibition of the tyrosinase enzyme catalyzing tyrosine conversion to melanin suppresses the production of melanin pigment. AR, one of the few compounds that exhibit the desired whitening effect, is preferred because of the carcinogenic effect of hydroquinone, the most effective whitener (Libánský et al. 2011). As the concentrations of the whitening agents that are slowly absorbed into the skin’s blood were limited in cosmetic products, they may not reach the threshold concentrations (Degen 2016). Therefore, the determination of whitening agents, such as arbutin, hydroquinone, and kojic acid, in cosmetics is important for quality control and safety.

Cosmetics are complex samples, and many techniques may be utilized to determine the cosmetic analytes, including gas chromatography-mass spectrometry (Chisvert et al. 2010), capillary electrophoresis (Lin et al. 2007), liquid chromatography-mass spectrometry (Kim et al. 2018), electrochemistry (Shahamirifard and Ghaedi 2019; Shih and Zen 2000), and high performance liquid chromatographic methods (Huang et al. 2004). Compared with these methods, electrochemical methods are simple, fast, precise, economical, and the consumption of organic solvents is lower (Gupta et al. 2011; Hoyos-Arbeláez et al. 2017). Besides, electrochemical sensors have great potential for selective and sensitive analysis of chemical and biological analytes due to easy operation, economical, and high diversity of electrode materials (Karimi-Maleh et al. 2020, 2019).

Modern-day electrochemical sensors are operated in a system that contains three electrodes (reference, auxiliary, and working). Among them, the working electrode is the most important one because the redox reactions occur between the solution and the working electrode interface. Various types of working electrodes such as mercury (Christie et al. 1977), metal-based (Masek et al. 2011), and carbon-based (Yazan et al. 2018; Erden and Yazan 2018) electrodes have been used in the voltammetric analysis for years. Carbon-based, particularly carbon paste electrodes, have a wide potential range, long-term stability, low background current, reproducibility, surface renewal procedure, and ease of modification (Bayraktepe et al. 2016; Švancara et al. 2001). To improve sensitivity and selectivity of the voltammetric signal on carbon paste electrode, some materials, for instance, nanoparticles (Ardakani et al. 2008), carbon nanotubes (Afkhami et al. 2014), ionic liquids (Shabani-Nooshabadi and Roostaee 2016), some clay minerals (Bayraktepe et al. 2015; Sathisha et al. 2012), etc., have been used. Sepiolite, one of the clay minerals, imparts the adsorption of organic species onto the electrode surface and increases the electrical conductivity of the working electrode (Pekin et al. 2017).

The presented work aims to develop a sensitive, selective, simple adsorptive stripping voltammetric method (AdsSWV) for the determination of arbutin in cosmetic products. For this purpose, a sepiolite clay modified carbon paste electrode (Clay/CPE) was used using favorable features such as low background current, wide potential window, high adsorptive properties, easy handling, and surface renovation. Although there have been six voltammetric studies about AR in the literature reports (Blasco et al. 2004; Butwong et al. 2020; Libánský et al. 2011; Liu et al. 2008; Shahamirifard and Ghaedi 2019; Shih and Zen 2000), in this study, the developed Clay/CPE sensor offers the lowest limits of detection and the widest linear working range for AR.

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Experimental

Reagents and apparatus

All solvents, AR, sepiolite clay, graphite powder, and mineral oil were supplied from Sigma-Aldrich, and other chemicals used were analytical grade. The stock solution of AR (1.0× 10–3 mol L−1) was prepared by dissolving solid AR in water. The prepared stock solution was stored in the refrigerator at+4 °C. 0.04 mol L−1 Britton-Robinson buffer was used as the supporting electrolyte.

All electrochemical measurements [cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS)] were performed by using CHI 660C (from the USA, Texas) and C3 cell stand (Bioanalytical Systems, Inc., USA, BASi). Ag/AgCl (in 3.0 mol L−1 NaCl, BAS MF-2052) was used as a reference electrode, Clay/CPE, and CPE sensors were used as working electrodes, and as an auxiliary electrode, platinum wire (BAS MW-1032) was used. Before all assay, pH was measured with a HANNA Instruments HI2211 pH/ORPmeter. The buffers at the pH values of 4.0, 7.0, and 10.0 were used for calibration of the pH meter. Double-distilled water was supplied mpMINIpure system. All assays were carried out at 25 °C.

Sensor preparation procedure

30 mg of graphite powder and 10 µL of mineral oil were mixed in a petri dish with a spatula to prepare CPE. As for the preparation of sepiolite clay modified CPEs, sepiolite clay and graphite powder were mixed in different rations, then mineral oil (10 µL) was added. The mass ratios of the sepiolite clay in the mixture were changed between 3.3 and 10%. The electrical connection is provided by a copper wire. The surface of the prepared sensors is smoothened with smooth paper. Before all experiments, surface cleaning of modified CPE sensors was performed by washing with a water-ethanol mixture (1:1) (Aydar et al. 2018).

Analytical procedure

AR (1.0× 10–3 mol L−1) stock solution was used in all experimental studies. In all voltammetric methods, electrolyte (0.04 mol L−1 BR buffer (pH 2.0) and AR stock solution were added to the electrochemical cell with a total volume of 10.0 mL.

The work, reference, and counter electrodes were immersed in the electrochemical cell. Ultra-pure nitrogen gas (99.99% purity) is~1 min before each analysis and~30 s between each measurement. Lastly, voltammograms were recorded in the 0.5–1.0 V potential window using AdsSWV. The device parameters for AdsSWV: amplitude: 0.025 V, frequency 20 Hz, potential range 0.5–1.0 V, and for EIS: amplitude: 0.005 V, frequency range: 0.05–105 Hz and the Nyquist plots were recorded under open circuit potential.

Cream sample preparation

100.0 mg of the Tritone cream sample (cream includes 2% arbutin, 2% kojic acid, 2% ascorbic acid, 6%glycolic acid, and 0.1% glabridin) was weighed and dissolved in an ultrasonic bath for 15 min with some distilled water, and the total volume was completed to 10 ml with distilled water. A solution containing 7.4× 10–4 mol L−1 AR was prepared. This solution was kept overnight at+4 °C and taken from the clear part of the solution in appropriate volumes and diluted in the electrochemical cell with pH 2.0 BR buffer.

Results and discussion

Characterization of the sepiolite modified carbon paste electrode

The electrochemical characterization of bare and sepiolite clay modified CPEs were carried out using CV and EIS methods. Fig. S1a and b show the CV curves (ʋ: 0.050 V/s) and Nyquist plots of 5.0 mM Fe(CN)6 3−/4− in 0.1 M KCl solution using unmodified and sepiolite clay modified CPE electrodes, respectively. According to Fig. S1a, it is seen that, when CPE modified with sepiolite clay, both anodic and cathodic peak currents of Fe(CN)6 3−/4− increased (about 1.2-fold for anodic and 1.1-fold for cathodic peak currents), and the peak to peak separation potential (∆Ep) is decreased (about 0.22 V) dramatically. Accordingly, the Nyquist plots in Fig. S1b represent the lower Rct value for Clay/CPE (about 2050 Ω) than the unmodified CPE (about 4082 Ω), indicating the fast electron transfer on the sepiolite clay modified carbon paste electrode (Aydar et al. 2018). Moreover, the active surface areas of CPE and Clay/CPE electrodes were calculated using the Randles–Sevcik equation by CV measurements of 5.0 mM Fe(CN)6 3−/4− in 0.1 M KCl at different scan rates and found to be 0.08 (± 0.002) cm2 and 0.09 (± 0.001) cm2 for CPE and Clay/CPE, respectively (Bayraktepe et al. 2016; Elyasi et al. 2013; Aydar et al. 2018). The results indicated that active surface area was increased after electrode modification, leading to the higher anodic peak current response of the developed electrochemical sensor.

Electrochemical behavior of AR

Electrochemical behavior of 5.0× 10–5 µmol L−1 AR at bare and clay modified electrodes surface at 0.1 V/s scan rate and in (+0.5)–(+1.2) potential range was investigated using the CV method. As shown in Fig. 1, one oxidation peak of arbutin occurred at approximately 0.834 V. When compared with CPE, it was observed that the peak potential of AR (0.834 V) shifted to more negative values at Clay/CPE (0.790 V) surface, and the peak current of AR increased about 1.4-fold. The results indicated that sepiolite clay had an electrocatalytic effect on the electro-oxidation of AR.

The optimization of the sepiolite electrode content

The optimum content was determined for sepiolite clay to be used in the preparation of the modified electrode. For this purpose, clay-modified electrodes were prepared with a clay content of 3.3; 5.0; 6.7; 8.3; 10.0% (Pekin et al. 2017; Bayraktepe et al. 2019). Voltammograms were recorded by the CV method with a scan rate of 0.1 V s −1 in pH 2.0 BR buffer solution containing 1.0× 10–5 mol L−1 AR. As seen in Fig. 2, the peak current of AR decreased after the percentage of sepiolite clay 5.0%. For this reason, the optimum sepiolite clay amount used in the preparation of clay-modified CPE was selected as 5.0%.

Influence of pH

To investigate the electrochemical behavior of AR on the Clay/CPE surface, peak current and peak potentials were measured at different pH values. pH Optimization was performed using Britton-Robinson (BR) buffer (pH 2.0–6.0). For this purpose, a 0.04 M BR buffer was prepared, and peak current measurements of AR were recorded using the CV method (Fig. 3).

When the pH—ip graph was examined, it was seen that the peak current at the pH 2.0 value was the highest, and the peak currents gradually decreased with increasing pH at pH 6.0. For this reason, the most suitable pH to be used in method development studies for AR determination was chosen as 2.0.

To study the electrochemical oxidation mechanism of AR, the CV technique was used. The number of electrons transferred (n) in electro-oxidation of AR was calculated by using the following Eq. (1):

Here, Ea p is the anodic peak potential, Ea p/2 is the half-peak potential, α is the electron transfer coefficient. For an irreversible process, α is taken as 0.5. The number of electrons transferred in this study (n) was calculated as 2.32 (n=2.32).

The relationship between peak potential and the logarithm of scan rate can be given with the following Eq. (2), which is expressed by Laviron (Bukkitgar et al. 2015; Shetti et al. 2018),

where E0 is the formal redox potential, α is the transfer coefficient, F is the Faraday constant, n is the transferred electron number, and k0 is the standard rate constant. E0 was determined from the intercept of the Ep vs ʋ plot by extrapolating the line ʋ=0, and E0 was 0.738 V. Then, the k0 was calculated as 2.6× 104 s −1. The relationship between anodic peak potential, Ea p, and pH was found as follows: Ea p = −0.0229 pH + 0.8444 (R2 = 0.995).

The slope of 0.0229 for the oxidation peak is close to half the theoretical Nernstian value of 0.059 (David et al. 2016). With this result, it can be thought that the number of electrons transferred in the oxidation mechanism of AR is equal to twice the number of protons.

4-methoxy phenol organic compound with a chemical structure like AR (Table S1) was studied in 0.04 M BR (pH 2.0) solution at the Clay/CPE electrode in the same range potential. As shown in Fig. S2, in terms of the electrochemical shape and properties of 4-methoxy phenol and AR showed similar responses with a well-defined peak in 0.04 M BR solution (pH 2.0) in the potentials 0.698 and 0.811 V, respectively. Moreover, the difference in oxidation potential of AR was likely observed more positive potential due to the steric hindrance caused by its geometric structure than the 4-methoxy phenol compound (Pavitt et al. 2017; Ohkatsu and Suzuki 2011). In considering the results obtained, we can propose that our possible oxidation reaction (given in Scheme 1) may occur from phenol moiety at AR like 4-methoxy phenol (Enache and Oliveira-Brett 2011; Nady et al. 2017).

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Influence of scan rate

The electrochemical properties of the oxidation peak of AR were investigated. For this purpose, the voltammograms were recorded in the range of 0.005–0.5 V/s scan rate and the presence of 1.0× 10–5 M AR (pH 2.0 BR buffer) using the CV method (Fig. 4). As shown in Fig. 4, no reduction peak of AR was found; only one anodic peak at about 0.8 V was observed. Therefore, it is possible to say that the oxidation peak of AR on the Clay/CPE electrode surface exhibits an irreversible redox behavior (Nady et al. 2017). To analyze the electrochemical process of AR, the logip–logʋ graph was plotted using the CV technique, and the slope value of the logip–logʋ graph was obtained about 0.43 for the oxidation peak of AR, it can be said that arbutin is carried to the electrode surface by diffusion (Allen and Larry 2001).

Analytical method development

For the determination of AR, SWV–AdsSWV method was compared on Clay/CPE electrode surface. The voltammograms obtained were given in Fig. S3. It was observed that the peak flow of AR increased in the adsorptive stripping method. It is possible to say that although the arbutin is carried to the modified electrode surface by diffusion, it is attached to the electrode surface by adsorption.

In addition, in the presence of AR on the Clay/CPE surface, five cycles were taken using the CV method. It was observed that the first oxidation peak was considerably higher than the others, and the peak height decreased gradually with the second cycle. This decrease observed in the peaks supports the idea that AR attachment to the surface of the Clay/CPE electrode by diffusion is carried out by adsorption (Fig. S3B) (Nady et al. 2017). In this context, method development studies for the determination of AR on the Clay/CPE surface were carried out using adsorptive stripping methods.

Optimization of experimental conditions

Deposition potential and deposition time values were optimized under these device parameters for AdsSWV: amplitude: 0.025 V, frequency 20 Hz, AR solutions of 1.0× 10–5 mol L−1 in the potential range of (0.5)–(+1.0) V.

For AdsSWV, deposition potential was changed in the range of 0.0–1.0 V. It can be seen in Fig. 5a, the optimum value of deposition potential was chosen as 0.3 V. Similar trials for deposition time were done at the potential value of 0.3, and deposition time ranged from 0.0 to 80 s. The results are given in Fig. 5b for AdsSWV. The optimum value of deposition time was chosen as 15 s.

Calibration studies and validation of optimized methods

To develop the AdsSWV method for AR determination, calibration graphs were created under optimum conditions determined on the surface of Clay/CPE. In the method development studies on Clay/CPE surface, AR concentration was changed, and peak currents were measured for each concentration. The obtained voltammograms and the generated calibration graphs are given in Fig. 6.

The given results in Fig. 6 show a perfect linear relationship among i a p and CAR. The linear equations for AdsSWV are given below: i a p(휇A) = 0.0499CAR − 0.0027, R2 = 0.9994 (Inset of Fig. 6)

To calculate LOD and LOQ values, the following equations were used: LOD = 3s/m, LOQ=10s/m (3)

Here, s is the standard deviation for the AR concentration studied (3.0× 10–7 mol L−1), and m is the slope of the calibration graph. According to these equations, LOD and LOQ values were found as 0.0108 and 0.0362 µmol L−1 for AdsSWV, respectively (Table 1). According to our literature knowledge, these LOD and LOQ values are the lowest results found up to now (Table 2).

The validation parameters of proposed new methods (Table 1) and a comparison table of other literature reports about AR (Table 2) with our new voltammetric sensor (Clay/ CPE) are given upwards:

To determine the stability of the electrode, the shelf life of the Clay/CPE was investigated. The signals of AR were recorded on different days, and ten days later, the sensor signal was found to have retained 96.4% of its initial value for AdsSWV. During all experiments, the newly developed sensor was kept at+4 °C.

Interferences

The interference effects of some electroactive species that can be found in cosmetic creams were investigated on the voltammetric method developed using Clay/CPE electrode for AR determination. When the concentration of Na+, Mg2+, K+, Co2+, Fe3+, Cu2+, Ni2+ ions was added 100 times more than the concentration of AR, no significant change was observed in current values. However, when the uric acid, ascorbic acid, phenol, and resorcinol concentrations were added ten times, the oxidation peak of these species coincided with the arbutin peak, resulting in too much difference in current values and interference-effect.

Real sample analysis and recovery

To determine the accuracy of the developed method for the determination of AR on Clay/CPE, a recovery study was performed with Tritone cream containing 2.0% arbutin using the direct calibration method. Table 3 shows the data obtained as a result of the recovery study conducted at different concentrations. According to the results of the analysis, the recovery values for the AdsSWV method ranged from 98.68 to 104.06%. These results show that the developed method can be successfully applied in samples containing AR without the interference of other ingredients in the Tritone cream sample.

Conclusions

The electrochemical oxidation mechanism and AR behavior, a skin whitening cosmetic, were investigated by cyclic voltammetry. A low level, the rapid, low-cost electroanalytical method was developed to determine AR in the cosmetic cream sample. Nano sepiolite clay/CPE electrode was used to determine AR in a 0.04 M BR (pH 2.0) solution by the proposed AdsSW voltammetric method. Clay/CPE nano-sensor showed noble sensitivity and good electrocatalytic activity toward AR electro-oxidation. The LOD and LOQ values were found to be 10.8 and 36.2 nM at modified Clay/CPE nano-sensor, respectively. The method proposed exhibits not only the lowest ultra-trace level detection of AR but also applied for the quantification of AR in the cosmetic cream sample with a satisfactory recovery (101.47%).

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