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A modified nanostructured graphene-gold nanoparticle carbon screen-printed electrode for the sensitive voltammetric detection of rutin
Measurement 114 (2018) 37–43
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A modified nanostructured graphene-gold nanoparticle carbon screenprinted electrode for the sensitive voltammetric detection of rutin I.M. Apetreia, C. Apetreib,
MARK
⁎
Department of Pharmaceutical Sciences, Medical and Pharmaceutical Research Center, Faculty of Medicine and Pharmacy, “Dunarea de Jos” University of Galati, Romania b Department of Chemistry, Physics and Environment, Faculty of Sciences and Environment, “Dunarea de Jos” University of Galati, Romania a
A R T I C L E I N F O
A B S T R A C T
Keywords: Rutin Graphene Gold nanoparticle Sensor Square-wave voltammetry Pharmaceutics
This study presents a graphene-gold nanoparticles screen-printed voltammetric sensor for the determination and quantification of rutin in pharmaceutics by means of square-wave voltammetry. The cyclic voltammetry electrochemical studies have demonstrated that the sensor has a large active surface and the transfer of electrons is facilitated by the nanostructured materials in the sensing material. To increase the performance of the sensor to rutin, the following experimental conditions were optimized: the detection method, the nature of the electrolyte solution and the pH. In optimum conditions of square-wave voltammetry in acetate buffer solution of pH 5.0, the sensor allows the detection of rutin on a potential of 0.44 V vs. Ag/AgCl. The current of the anodic peak varies linearly with the rutin concentration ranging in the domain 0.1 × 10−6 to 15 × 10−6 M, with a detection limit of 1.1 × 10−8 M. The nanomaterials-based sensor was effectively used for the quantification of rutin in the pharmaceutical products being characterized by precision, repeatability and great accuracy. Furthermore, the results obtained correspond with those obtained with the standard method and with the amounts indicated by the producer, respectively, having a 99% confidence level.
1. Introduction Rutin is a flavonoid glycoside, also known as vitamin P. It is the most common flavonoid in people’s diet and an activator factor of vitamin C. Rutin is found in Flos Sophorae buds, citrus fruits and different berries [1,2]. Rutin has the property to scavenge different radical chemical species, i.e. superoxide anion, hydroxyl and peroxyl radicals, therefore it has a noticeable antioxidant character. As a result, rutin is used as an active pharmacological substance with antibacterial, antioxidant, antiviral, antitumoral etc. properties in more than 150 pharmaceutical formulations worldwide [3–5]. Being a compound of great interest, the detection and quantification of rutin in vegetal or pharmaceutical products raises much attention. There are numerous analysis methods such as the capillary electrophoresis, chemiluminescence, HPLC, spectrophotometry, and electrochemistry, to mention only a few [6–15]. Table 1 presents a series of rutin electrochemical analysis methods, described by the technical literature, that encompass the main features of the analytical performance of the sensors used, and which are based on similar sensitive materials as the sensor developed in this work.
The electrochemical techniques can be successfully used for the detection of rutin mainly because they are simple, rapid, sensitive and cost effective. One of them, the square-wave voltammetry (SWV), evidenced to be particularly sensitive in the determination of the electroactive organic compounds due to the very low non-Faradaic current [16,17]. The advantage of SWV when compared to cyclic voltammetry (CV) and differential pulse voltammetry (DPV) lists: shorter time of analysis, reduced consumption of the electroactive compounds and minor problems with the electrode surface fouling [18]. Furthermore, with this technique, high sensitivities and very low detection and quantification limits can be obtained [3,15]. The development of novel methods of electroanalysis can be facilitated by using screen-printed electrodes (SPEs), which have a series of practical advantages when compared to the classical electrodes, such as the simplicity of use, the commercial availability, the low price and very good reproducibility in term of industrial production. Therefore, SPEs can be easily replaced after each analysis, which enhances the repeatability and reproducibility of the analyses [19,20]. The performance characteristics of SPEs can be further improved if the sensitive element is based on nanomaterials, such being the case of the sensitive element based on carbon or metallic nanoparticles.
⁎ Corresponding author at: Department of Chemistry, Physics and Environment, Faculty of Sciences and Environment, “Dunarea de Jos” University of Galati, 47 Domneasca Street, 800008 Galati, Romania. E-mail address: [email protected] (C. Apetrei).
http://dx.doi.org/10.1016/j.measurement.2017.09.020 Received 9 May 2017; Received in revised form 10 September 2017; Accepted 11 September 2017 Available online 12 September 2017 0263-2241/ © 2017 Elsevier Ltd. All rights reserved.
Measurement 114 (2018) 37–43
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Table 1 Performance characteristics of carbonaceous material based sensors used in detection of rutin. Sensitive material
Electrochemical technique
Linear range (M)
LOD (M)
Reference
Chitosan/graphene Electrodeposited graphene/gold nanoparticles Chemically reduced graphene oxide/Pt nanoparticles Reduced graphene oxide Graphene oxide/multi-walled carbon nanotube Electrochemically reduced graphene oxide Graphene/n-octylpyridinum hexafluorophosphate
DPV DPV DPV DPV DPV DPV SWV
5 × 10−7 to 1.04 × 10−5 8 × 10−8 to 8 × 10−5 0.057 × 10−6 to 102.59 × 10−6 0.1 × 10−6 to 2.0 × 10−6 0.08 × 10−6 to 80 × 10−6 4.7 × 10−7 to 1.2 × 10−5 0.05 × 10−6 to 11 × 10−6
– 2.55 × 10−8 2.0 × 10−8 2.32 × 10−8 2.0 × 10−8 1.8 × 10−8 1 × 10−8
9 10 11 12 13 14 15
2.2. The fabrication of voltammetric sensor
Graphene (GPH) have been extensively used in the development of sensors and biosensors for the distinctive properties related to two-dimensional nanostructure, for instance the high surface area, the outstanding electrical conductivity and the biocompatibility with organic molecules [21,22]. On the other hand, the incorporation of gold nanoparticles (AuNPs) with GPH could represent a promising way towards significant improvements of the electrochemical properties of the sensitive nanomaterials [10]. Due to its good compatibility and excellent electrical conductivity, chitosan is used for the better dispersion of the nanomaterials and the preservation of the nanostructures [22]. Generally, the effects of these nanomaterials are the increase of the performance characteristics, due to the facilitated transfer of electrons at the electrode-solution interface, characterized by the enhancement of the sensor response and the specific interactions with the target molecule [10,22,23]. To the best of our knowledge, the development of novel sensors using graphene and gold nanoparticles is a challenging and yet a promising way to accomplish sensitive, fast and simple detection of rutin. Therefore, the aim of this paper is to develop, optimize and validate an electroanalytical method based on the square-wave voltammetry technique for the assessment of rutin in pharmaceutical products using a sensitive voltammetric sensor based on graphene (GPH) and gold nanoparticles (AuNP).
The commercial CSPEs were modified with graphene, chitosan, and Au nanoparticles in order to achieve a novel sensor with an improved sensitivity to rutin. The GPH dispersion was prepared by mixing 1 mg GPH with 1 mL chitosan solution (0.2% in acetic acid, pH 5). To achieve a homogeneous dispersion the GPH-chitosan mixture was ultrasonicated for 2 h. The chitosan improved the dispersion of graphene. The gold nanoparticles (AuNPs) were prepared through the reduction of HAuCl4 with trisodium citrate in aqueous solution [24]. The synthesis reaction can be summarized as follows: 2HAuCl 4 + 3C6 H8O7 (citric acid) → 2Au + 3C5 H6 O5 (3-ketoglutaric acid) + 8HCl + 3CO2
After the synthesis and the separation of the gold nanoparticles, 1 mg of AuNPs was added to homogeneous dispersion GPH-chitosan and ultrasonicated for 1 h. The chemically modified carbon screen-printed electrodes were developed using the drop-and-dry method. 10 μL of the nanomaterials composite dispersion was dropped on the SPCEs using micropipette and then were dried in a desiccator at room temperature. The modified electrodes were the ones containing GPH (GPH/CSP sensor) and GPH and AuNPs (GPH-AuNP/CSP sensor) in the sensitive element. The prepared electrodes were positioned at 4 °C.
2. Materials and methods 2.1. Reagents and solutions
2.3. Equipment
All the chemicals were of the highest purity commercially available and were used without further purification. Rutin and ethanol (absolute, ≥99.8%) of analytical grades, were acquired from Sigma-Aldrich (St. Louis, USA). Chitosan, chloroauric acid, potassium ferricyanide, potassium chloride, hydrochloric acid, acetic acid, sodium acetate, sodium diphosphate, disodium phosphate, sodium hydroxide, trisodium citrate, lactic acid, ascorbic acid, lactose, sodium lauryl sulfate, starch, and sucrose were used in electrochemical studies. The carbon screen-printed electrodes (CSPE), DRP-110 working in solution (the working electrode diameter of 4 mm) were purchased from Dropsens Ltd. (Llanera, Spain). The graphene (GPH), platelet planar size of 0.3–5 μm, from Sigma-Aldrich (St. Louis, MO, USA) was used for CSPE modification. All the solutions were prepared with ultrapure water (resistivity was 18 MΩ cm) obtained from a Milli-Q water ultrapurification system (Simplicity®, Millipore, USA). In all electrochemical experiments, electrolytes buffer solutions with ionic strength 0.1 M were used. These buffer solutions were prepared with analytical grade substances and ultrapure water. The stock rutin solution (10−3 M) was prepared in ethanol, in order to facilitate the dissolving process, by means of ultrasonication. The solutions used for the spectrophotometric and the electrochemical analyses were obtained right before utilization by diluting the stock solutions with buffer solution until the desired concentration was reached. The stock solution was kept in the fridge, at a 4 °C temperature and remained stable throughout the analyses.
The cyclic voltammetry (CV) and square-wave voltammetry (SWV) experiments were performed on a Biologic SP 150 (Bio-Logic Science Instruments SAS, France) potentiostat/galvanostat controlled by a microcomputer with EC-Lab Express software. A three-electrode system was used where a silver-silver chloride (Ag/AgCl in 3 M KCl) electrode was the reference electrode, a platinum wire electrode was the auxiliary electrode and the graphene-gold nanoparticles screen-printed (GPHAuNP/CSP) sensor was the working electrode. Thus, the connections to the potentiostat/galvanostat were made through independent cables for each electrode. All the subsequent potentials indicated in this study were compared with the Ag/AgCl in 3 M KCl system. The cyclic voltammograms were obtained for different potential ranges at 0.1 V s−1, while the square wave voltammograms were obtained using a 90 mV pulse height, a 5 mV scan increment, and a 15 Hz frequency. All the electrochemical measurements were carried out at room temperature. The morphology of the carbon screen-printed electrode modified with graphene - gold nanoparticles composite was characterized using the scanning electron microscope FlexSEM 1000 (Hitachi, Japan). The S10H ultrasonic apparatus (Elmasonic, Germany) was used for the ultrasonic experiment. The pH of the buffer solutions was measured with an Inolab pH 7310 pH-meter (WTW, Germany). The spectrophotometric measurements in the ultraviolet range were carried out with a Rayleigh UV-1601 spectrophotometer (Beijing Beifen-Ruili Analytical Instrument Co. Ltd., China). 38
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2.4. Application in pharmaceutical analysis The applicability of the graphene-gold nanoparticles sensor was studied by analyzing pharmaceutical products containing rutin. Rutin S (Antibiotice SA, Iasi, Romania – 40 mg rutin per capsule), Rutin C (Medica Pro Natura, Romania - 200 mg rutin per capsule), and Rutin Forte (Organika, Canada - 500 mg rutin per capsule) which were purchased from local pharmacies in Galati, Romania. Ten capsules from each type of pharmaceutical capsules were weighed and then triturated into a mortar and pestle. An adequate quantity from this blend was weighted and dissolved into a buffer solution/ethanol (70:30 v/v) obtaining, thus, the stock solution with the desired concentration. The solutions to be analyzed were subsequently prepared through the dilution of this solution with buffer solution. For the interference studies on rutin standard stock solution, different chemical compounds and excipients used in the pharmaceutical products (potassium chloride, sodium acetate, sodium diphosphate, disodium phosphate, sodium citrate, lactic acid, ascorbic acid, lactose, sodium lauryl sulfate, starch and sucrose) were added in various concentrations. The solutions obtained were three times washed with buffer solution/ethanol (70:30 v/v). The filtrate was brought to the volumetric flask and filled up with buffer solution/ethanol (70:30 v/v) so that the rutin concentration to be equal with the initial concentration. Each solution was transferred to the electrochemical cell and the voltammetric signals were recorded in the same conditions as in the case of the standard solutions or the solutions obtained from the pharmaceutical products selected for analysis. In order to validate rutin determination by means of the voltammetric method, the spectrophotometric method mentioned in the Romanian Pharmacopeia, Edition X was used [25]. 50 mg rutin are dissolved in 20 mL ethanol through heating, and after cooling the solution is brought to 100 mL in a volumetric flask by adding the same solvent. For 3 mL of this solution 1 mL acetic acid 1.2 g L−1 is added and is filled up with alcohol up to 100 mL, in a volumetric flask (sample solution). The absorbance at 362 nm is determined for the mixture of solvents used for the sample dissolution. Simultaneously, the absorbance of a standard solution obtained in the same conditions as the sample solution from 3 mL rutin (previously dried at 130 °C until constant mass) 0.05% w/v in ethanol, 1 mL acetic acid 1.2 g L−1 and ethanol at 100 mL is determined. Also, the voltammetric method was validated determining the recovery via the method of the standard addition.
Fig. 1. High resolution SEM image of SPCE modified GPH/AuNP nanocomposite.
Fig. 2. Cyclic voltammograms of CSPE (_._._), GPH/CSP sensor (_ _ _), and GPH-AuNP/CSP sensor (___) in 10−3 M K4[Fe(CN)6] and 0.2 M KCl solution.
the chitosan coated CSPE, respectively. As shown in Fig. 2, K4[Fe(CN)6] showed redox current peaks at 0.26 V and 0.16 V. The currents of the redox peaks agreed with the order: Ip (CSPE) < Ip (GPH/CSPE) < Ip (GPH-AuNP/CSPE), both for the anodic and cathodic peaks. The special two-dimensional nanostructure of the GPH can be beneficial for the electrical conductivity, which explains the increased peak currents when compare with the unmodified electrode [23]. Additionally, the AuNPs facilitate the electron transfer at the sensor surface having a significant role in increasing the peak currents [10]. As it can be seen in the case of GPH-AuNP/CSP sensor, the peak-topeak potential separation (ΔEp = Epa − Epc) was of 0.1 V, corresponding to a quasi-reversible electron transfer process (Fig. 2). Furthermore, the ratio between the cathodic current peak and the anodic peak is sub-unitary, 0.89, but close to the ideal value, 1, for a reversible process [26,27]. From the study of the influence of the scanning rate of the potential over the anodic current peak it was determined that the electrochemical process is controlled by the diffusion process of the electroactive species on the surface of the electrode for all sensors (anodic peak current, Ipa, is proportional with the square root of scan rate, v1/2) [22]. Moreover, these studies made possible the estimation of the active surface of the sensor using the Randles-Sevcik equation [27]. The ratio between the active surface, electrochemically determined, and the geometric one (roughness factor) is of 12.64 for the GPH-AuNP/CSP sensor, 8.45 for the GPH/CSPE, and 2.28 for the CSPE, respectively. The higher value obtained in the case of GPH-AuNP/CSP sensor is determined by the GPH and the gold nanoparticles, which can increase the number of
3. Results and discussion 3.1. Morphological investigation Fig. 1 shows the high resolution scanning electron microscope (SEM) image of the GPH/AuNP-modified CSPE. The SEM image of GPH/AuNP nanocomposite shows a typical 3D morphology, a few layers nanoflakes of GPH decorated with gold nanoparticles. In addition, the micrograph presented in Fig. 1 shows the spherical morphology of the gold nanoparticles. 3.2. The electrochemical characterization of the modified graphene-gold nanoparticles screen-printed (GPH-AuNP/CSP) sensor The general information regarding the electroactivity of the compounds under study, as well as the processes at the interface of the electrode/solution, was obtained through cyclic voltammetry. Potassium ferrocyanide was chosen as reference solution in order to evaluate the electrochemical properties of the developed sensor. Fig. 2 shows the electrochemical signals of the CSPE, GPH/CSP, and GPHAuNP/CSP sensors in 10−3 M K4[Fe(CN)6] and 0.2 M KCl solution. No changes were observed in the cyclic voltammograms of the CSPE and of 39
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Fig. 3. Chemical structure of rutin.
active centers on the sensor surface because of the modified nanostructures and of the synergetic effects [20,26,28]. These studies demonstrates the improved electrochemical sensitivity of the GPH-AuNP/ CSP sensor based on nanostructured materials and recommend its use in the detection of rutin. 3.3. Electrochemical detection of rutin at GPH-AuNP/CSP based sensor The rutin electro-activity is in relation with its chemical structure that contains more hydroxyl groups, which can be oxidized electrochemically [9,16]. Rutin is 3′,4′,5,7-tetrahydroxyflavone-3b-D-rutinoside and the chemical structure is given in Fig. 3. The electrochemical behavior of rutin at GPH-AuNP/CSP sensor was firstly investigated by means of cyclic voltammetry. Fig. 4 shows the CV of 10−5 M rutin in 0.1 M acetate buffer solution of pH 5.0. In the direct scanning, two anodic peaks can be observed, IA and IIA at EpaI = +0.44 V and EpaII = +1.08 V, respectively. In the reverse scan a cathodic peak, IC, corresponding to the anodic peak IA, appears at EpcI = +0.22 V. The redox pair (EpaI/EpcI) noticed at E1/2 = 0.33 V corresponds to the catechol moiety, 3′,4′-dihydroxyl groups on B-ring, that go through a reversible process of oxido-reduction with the forming of an ortho quinonic derivative [16]. The separation of the potential peaks (ΔE) is equal with 0.22 V, a relative high value which suggests that the electrochemical processes on the active surface of the sensor, that implies the transfer of two electrons, is slow and quasi-reversible [16,19]. On the other hand, the peak IIA, at EpaII = +1.08 V corresponds to the irreversible electrochemical oxidation of the hydroxyl groups in the positions 5 and 7 (resorcinol moiety of ring A) [16,19]. The influence of the potential scan rate (v) on the peaks currents (IpaI and IpcI) of 10−5 M rutin for the GPH-AuNP/CSP sensor was studied by cyclic voltammetry at various scan rates (Fig. 5). As shown in Fig. 5(a), the peak currents of rutin grow along with the
Fig. 5. (a) CVs of GPH-AuNP/CSP sensor immersed in 10−5 M rutin (in 0.1 M acetate buffer solution with pH 5) registered with different scan rates in the range 0.05 V s−1 to 0.5 V s−1; (b) relationships between the peak currents and the potential scan rate.
increase of scan rates (v) and there are significant linear dependences among the peak currents and scan rates (Fig. 5b). The regression equation is IpaI = 86.97 × v + 21.73 (IpaI: μA, v: V s−1, R2 = 0.983); IpcI = −45.21 × v − 13.77 (IpcI: μA, v: V s−1, R2 = 0.989), indicates the redox process of 10−5 M rutin for the GPH-AuNP/CSP sensor in adsorption-controlled conditions [27]. Therefore, the limitative factor of the redox process is the transfer of electrons. The square-wave voltammetry method was investigated in order to increase the sensitivity. Fig. 6 shows the SWV (direct scan) of the GPHAuNP/CSP sensor immersed in 10−5 M rutin (0.1 M acetate buffer solution of pH 5). As it can be noticed, the two anodic peaks appears to have the same potentials as in the case of the cyclic voltammetry. Compared to the CV results, the peaks obtained via SWV are better defined, the currents are
Fig. 6. The SWV (direct scan) of the GPH-AuNP/CSP sensor immersed in 10−5 M rutin (0.1 M acetate buffer solution with pH 5).
Fig. 4. CV of 10−5 M rutin in 0.1 M acetate buffer solution of pH 5.
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higher, and the background current is lower. Furthermore, the SWV presents some advantages such as lower consumption of electroactive species and subsequent lessening of the species which block the electrode surface [18]. Therefore, the SWV technique is the most suitable for the quantitative studies. 3.4. The influence of the supporting electrolyte and pH The nature and the type of electrolytes used have a central role in the voltammetric signal of the sensor towards rutin. The currents of the responses obtained with the sensor towards a 10−5 M rutin solution were determined in many buffer solutions based on different electrolytes such as Na2HPO4 + NaH2PO4, CH3COONa + CH3COOH, sodium citrate + HCl, KCl + NaOH, KCl + HCl. The results obtained through cyclic voltammetry revealed that the most intense currents and the best peak shape were obtained in CH3COONa + CH3COOH buffer solution. As a result, the CH3COONa + CH3COOH buffer solution was used in the succeeding studies. The electrode reaction of rutin, as in the case of other polyphenol type compounds, is influenced by the pH of the medium. Therefore, the redox process of rutin was studied for a pH range 2–11, in various buffer solutions, by means of SWV (Fig. 7a). As the value of the pH rises, a linear movement towards the lower peak potential values, until the pH reaches the value of 9, can be observed. For values above 9, the potential of the peak becomes almost independent of the pH of the electrolyte solution containing rutin (Fig. 7b). The slope between the peak potential and the pH is of 62 mV per pH unit and it demonstrates that an equal number of electrons and protons are involved in the redox process [27]. Furthermore, the value of the half width of the peak is close to the theoretical value of 90 mV
Fig. 8. (a) The SWVs of GPH-AuNP/CSP sensor immersed in rutin solution with different concentrations in the range 0.1 × 10−6 to 15 × 10−6 M; (b) the relationships between the peak currents and concentration (calibration curve).
established for a redox process in which there are involved two electrons and two protons [14,27]. On the other hand, the variation of the peak currents, in relation with the pH, is nonlinear, the current of the peak being higher in a weak acid medium with pH 5. Since the best response and sensitivity were obtained for pH 5.0 (acetic acid/sodium acetate buffer solution), this condition was selected for the quantitative analysis of rutin. 3.5. The calibration curve Under the optimum experimental conditions, square-wave voltammograms of a series of rutin solutions of various concentrations were registered. These results are presented in Fig. 8a. As Fig. 8b shows, the calibration curve is linear for the concentration domain 1 × 10−7 to 1.5 × 10−5 M, with a detection limit (three times the standard deviation of the signal blank/slope) of 1.1 × 10−8 M. The equation of the linear regression is (I/μA) = 3.045 × [Rutin] + 1.12, R2 = 0.9988), where I is the voltammetric signal corresponding to the peak and [Rutin] is the concentration of rutin in μM. As the graphs and the calculations show, the method of quantitative
Fig. 7. (a) The square wave voltammograms of GPH-AuNP/CSP sensor immersed in buffer solutions with the pH ranging from 3 to 7 containing 10−5 M rutin; (b) the dependence between the peak potential and the pH values ranging from 2 to 11.
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determination is relatively simple and very sensitive. By comparing the performance characteristics of the sensor developed in this study with the main sensors used for the rutin analysis in Table 1, it can be concluded that the GPH-AuNP/CSP sensor owes its improved performance features to the nanostructured sensitive materials.
Table 2 Determination of rutin in tablets by standard addition method. No.
Sample Sample Sample Sample Sample Sample Sample
3.6. The precision of the voltammetric method The repeatability of the analysis method was determined by performing 6 quantitative determinations for a solution 5 × 10−6 M using 5 different GPH-AuNP/CSP sensors, for time intervals of 15 min between one and the next. The relative standard deviation (RSD) for all the determinations was of 2.1% which demonstrates that the analysis method has a very good repeatability. In order to establish the intermediate precision of the voltametric determination, the quantitative analysis was carried out in 5 different days, using a new GPH-AuNP/CSP sensor at all times, and new rutin standard solutions of two concentrations, 2 × 10−6 M and 5 × 10−6 M. Under these conditions, the repeatability was not affected by the concentration of the rutin solution and the RSD of the peak currents was of 3%, which proves that the sensor shows a very good precision.
1 2 3 4 5 6 7
Sampling amount (mg)
Detected amount (mg)
Recovery (%)
Average recovery (%)
40.5 40.3 39.7 39.8 40.0 40.2 40.4
39.3 39.6 40.5 40.2 40.1 38.8 41.6
97.04 98.26 102.02 101.01 100.25 96.52 102.97
99.72
Table 3 Determination of rutin in pharmaceutical products by spectrophotometric and voltammetric methods. Sample
Rutin S (Antibiotice SA, Romania) Rutin C (Medica Pro Natura, Romania) Rutin Forte (Organika, Canada)
3.7. The study of interferences In order to study the effect of various excipients used in the pharmaceutical rutin products and of other substances that can be found in the samples to be analyzed for the 5 × 10−6 M rutin determination, the decrease or the increase of the anodic peak current was determined. The tolerance limit considered was the ratio between the concentrations of the interfering species and the concentration of rutin, which determines a ± 5.0% relative error. The tolerance limit for the interfering substances for the 5 × 10−6 M rutin determination were 200 for Na+, K+, PO43−, HPO42−, H2PO4−, Cl−, citrate, acetate, 150 for lactose, sodium lauryl sulfate, lactic acid, 50 for starch and sucrose, and 5 for the ascorbic acid. Thus, the quantification of rutin is barely affected by the presence of the main excipients in the sample under study, the voltammetric signals remaining almost unmodified in the presence of the compounds previously mentioned.
Rutin concentration (mg per capsule) Label value
Spectrophotometric method
Voltammetric method
40
41 ± 1
40 ± 1
200
199 ± 3
198 ± 4
500
503 ± 8
502 ± 9
determination using the calibration curve was used for the analysis. The results obtained via this method and those indicated by the producer on the pharmaceutical product label are presented in Table 3. The statistic calculations have proven that the voltammetric method has a very good accuracy and the t-test has shown that there is no significant difference between the results at a 99% confidence level. This proves that the GPH-AuNP/CSP sensor is useful in the determination of rutin in pharmaceutical products. 4. Conclusions The GPH-AuNP/CSP sensor proved to be useful in the analysis of rutin in pharmaceutical products. The use of the square wave voltammetry as detection method allowed reaching excellent analytical performances with applicability in the laboratory practice. The results obtained with the GPH-AuNP/CSP sensor are very close to the results obtained via the standard method of analysis at a 99% confidence level. Furthermore, the developed method has a series of advantages such as good precision, reliability, simplicity, and low cost. The method is precise and has a very good accuracy, and it is versatile and feasible for large-scale analysis in the quality control of the pharmaceutical products, food and food supplements and other types of samples.
3.8. The validation of the method The validation of the method established in this work, based on the voltammetric sensor and using as detection method SWV, was performed through two methods. First, the results found in the analysis of some rutin samples of known concentration were compared with the ones obtained via the official method mentioned in the Pharmacopoeia [25]. The difference between the results was less than 1%, thus confirming the validity of the electroanalytical method proposed in this study. The second method used for validation was the standard addition method. The accuracy of the method was determined by calculating the mean of the recovery values obtained when rutin solutions were analyzed in the micromolar domain. The average value of the recovery obtained was 99.72% with a RSD of 2.49% (number of determination 7) (Table 2). Considering the results obtained, it is possible to use this voltammetric method in the quantification of rutin in pharmaceutical products.
Acknowledgment This work was supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CNCS - UEFISCDI, project number PN-II-RU-TE-2014-4-1093. Conflict of interest The authors declare no conflict of interest. References
3.9. The analysis of rutin pharmaceutical products
[1] H. Hosseinzadeh, M. Nassiri-Asl, Review of the protective effects of rutin on the metabolic function as an important dietary flavonoid, J. Endocrinol. Invest. 37 (2014) 783–788. [2] M. Medvidović-Kosanović, M. Šeruga, L. Jakobek, I. Novak, Electrochemical and
The GPH-AuNP/CSP sensor was used for the quantitative analysis of rutin in different commercial pharmaceutical products. The direct 42
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[3]
[4]
[5] [6]
[7]
[8]
[9]
[10]
[11]
[12] [13]
[14]
reduced graphene oxide modified sensor, J. Pharm. Anal. 6 (2016) 80–86. [15] H. Wang, Z. Wang, G. Liu, Z. Zhang, Electrochemical behavior of rutin on graphene and ionic liquids composite film modified electrode, Nongye Jixie Xuebao/Trans. Chinese Soc. Agric. Mach. 45 (2014) 219–224. [16] M.-E. Ghica, A.M. Oliveira Brett, Electrochemical oxidation of rutin, Electroanalysis 17 (2005) 313–318. [17] J.V. Piovesan, C.L. Jost, A. Spinelli, Electroanalytical determination of total phenolic compounds by square-wave voltammetry using a poly(vinylpyrrolidone)modified carbon-paste electrode, Sensor. Actuat. B-Chem. 216 (2015) 192–197. [18] V. Mirceski, R. Gulaboski, M. Lovric, I. Bogeski, R. Kappl, M. Hoth, Square-wave voltammetry: a review on the recent progress, Electroanalysis 25 (2013) 2411–2422. [19] A. de Oliveira-Roberth, D.I.V. Santos, D.D. Cordeiro, F.M.A. de Lino, M.T.F. Bara, E.S. de Gil, Voltammetric determination of rutin at screen-printed carbon disposable electrodes, Cent. Eur. J. Chem. 10 (2012) 1609–1616. [20] I.M. Apetrei, C. Apetrei, Study of different carbonaceous materials as modifiers of screen-printed electrodes for detection of catecholamines, IEEE Sens. J. 15 (2015) 3094–3101. [21] W. Zhang, S. Zhu, R. Luque, S. Han, L. Hu, G. Xu, Recent development of carbon electrode materials and their bioanalytical and environmental applications, Chem. Soc. Rev. 45 (2016) 715–752. [22] I.M. Apetrei, C. Apetrei, Voltammetric determination of melatonin using a graphene-based sensor in pharmaceutical products, Int. J. Nanomed. 11 (2016) 1859–1866. [23] E. Jubete, O.A. Loaiza, E. Ochoteco, J.A. Pomposo, H. Grande, J. Rodriguez, Nanotechnology: a tool for improved performance on electrochemical screenprinted (bio)sensors, J. Sensors (2009) (Article ID 842575, 13 pages). [24] G. Frens, Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions, Nat. Phys. Sci. 241 (1973) 20–22. [25] Farmacopeea Romana, Editia a X-a. Ed. Medicala, Bucuresti, 1993, pp. 830–832. [26] C. Apetrei, I.M. Apetrei, J.A. de Saja, M.L. Rodriguez-Mendez, Carbon paste electrodes made from different carbonaceous materials: application in the study of antioxidants, Sensors 11 (2011) 1328–1344. [27] A.J. Bard, L.R. Faulkner, Electrochemical Methods, John Wiley and Sons, New York, 2001. [28] S.A. Zaidi, J.H. Shin, A review on the latest developments in nanostructure-based electrochemical sensors for glutathione, Anal. Methods 8 (2016) 1745–1754.
antioxidant properties of (+)-catechin, quercetin and rutin, Croat. Chem. Acta 83 (2010) 197–207. K.H. Garcia Freitas, O. Fatibello-Filho, I.L. de Mattos, Square-wave voltammetric determination of rutin in pharmaceutical formulations using a carbon composite electrode modified with copper (II) phosphate immobilized in polyester resin, Braz. J. Pharm. Sci. 48 (2012) 639–649. I. Petkova Pencheva, V. Nikolova Maslarska, A. Christova Stoimenova, M. Metodieva Manova, L. Antonova Andonova, P. Krumova Zdraveva, Quality control optimization solutions for determination of rutin in supplements containing ginkgo biloba extract, Curr. Pharm. Anal. 12 (2016) 386–390. L.S. Chua, A review on plant-based rutin extraction methods and its pharmacological activities, J. Ethnopharmacol. 150 (2013) 805–817. L. Xiangjun, Z. Yuping, Y. Zhuobin, Separation and determination of rutin and quercetin in the flowers of Sophora japonica L. by capillary electrophoresis with electrochemical detection, Chromatographia 55 (2002) 243–246. S. Li, L. Zhang, L. Chen, Y. Zhong, Y. Ni, Determination of rutin by chemiluminescence based on a luminol–potassium periodate–ZnSe system, Anal. Methods 8 (2016) 4056–4063. D. Šatínský, K. Jägerová, L. Havlíková, P. Solich, A new and fast HPLC method for determination of rutin, troxerutin, diosmin and hesperidin in food supplements using fused-core column technology, Food Anal. Method. 6 (2013) 1353–1360. J. An, Y.-Y. Bi, C.-X. Yang, F.-D. Hu, C.-M. Wang, Electrochemical study and application on rutin at chitosan/graphene films modified glassy carbon electrode, J. Pharm. Anal. 3 (2013) 102–108. W. Sun, D. Wang, Y.-Y. Zhang, X.-M. Ju, H.-X. Yang, Y.-X. Chen, Z.-F. Sun, Electrodeposited graphene and gold nanoparticle modified carbon ionic liquid electrode for sensitive detection of rutin, Chinese J. Anal. Chem. 41 (2013) 709–713. S. Li, B. Yang, J. Wang, D. Bin, C. Wang, K. Zhang, Y. Du, Nonenzymatic electrochemical detection of rutin on Pt nanoparticles/graphene nanocomposite modified glassy carbon electrode, Anal. Methods 8 (2016) 5435–5440. Y. Lei, D. Du, L. Tang, C. Tan, K. Chen, G.-J. Zhang, Determination of rutin by a graphene-modified glassy carbon electrode, Anal. Lett. 48 (2015) 894–906. L. Yan, X.g. Niu, W. Wang, X. Li, X. Sun, C. Zheng, J. Wang, W. Sun, Electrochemical sensor for rutin detection with graphene oxide and multi-walled carbon nanotube nanocomposite modified electrode, Int. J. Electrochem. Sci. 11 (2016) 1738–1750. P. Zhang, Y.-Q. Gou, X. Gao, R.-B. Bai, W.-X. Chen, B.-L. Sun, F.-D. Hu, W.-H. Zhao, The pharmacokinetic study of rutin in rat plasma based on an electrochemically
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A modified nanostructured graphene-gold nanoparticle carbon screenprinted electrode for the sensitive voltammetric detection of rutin I.M. Apetreia, C. Apetreib,
MARK
⁎
Department of Pharmaceutical Sciences, Medical and Pharmaceutical Research Center, Faculty of Medicine and Pharmacy, “Dunarea de Jos” University of Galati, Romania b Department of Chemistry, Physics and Environment, Faculty of Sciences and Environment, “Dunarea de Jos” University of Galati, Romania a
A R T I C L E I N F O
A B S T R A C T
Keywords: Rutin Graphene Gold nanoparticle Sensor Square-wave voltammetry Pharmaceutics
This study presents a graphene-gold nanoparticles screen-printed voltammetric sensor for the determination and quantification of rutin in pharmaceutics by means of square-wave voltammetry. The cyclic voltammetry electrochemical studies have demonstrated that the sensor has a large active surface and the transfer of electrons is facilitated by the nanostructured materials in the sensing material. To increase the performance of the sensor to rutin, the following experimental conditions were optimized: the detection method, the nature of the electrolyte solution and the pH. In optimum conditions of square-wave voltammetry in acetate buffer solution of pH 5.0, the sensor allows the detection of rutin on a potential of 0.44 V vs. Ag/AgCl. The current of the anodic peak varies linearly with the rutin concentration ranging in the domain 0.1 × 10−6 to 15 × 10−6 M, with a detection limit of 1.1 × 10−8 M. The nanomaterials-based sensor was effectively used for the quantification of rutin in the pharmaceutical products being characterized by precision, repeatability and great accuracy. Furthermore, the results obtained correspond with those obtained with the standard method and with the amounts indicated by the producer, respectively, having a 99% confidence level.
1. Introduction Rutin is a flavonoid glycoside, also known as vitamin P. It is the most common flavonoid in people’s diet and an activator factor of vitamin C. Rutin is found in Flos Sophorae buds, citrus fruits and different berries [1,2]. Rutin has the property to scavenge different radical chemical species, i.e. superoxide anion, hydroxyl and peroxyl radicals, therefore it has a noticeable antioxidant character. As a result, rutin is used as an active pharmacological substance with antibacterial, antioxidant, antiviral, antitumoral etc. properties in more than 150 pharmaceutical formulations worldwide [3–5]. Being a compound of great interest, the detection and quantification of rutin in vegetal or pharmaceutical products raises much attention. There are numerous analysis methods such as the capillary electrophoresis, chemiluminescence, HPLC, spectrophotometry, and electrochemistry, to mention only a few [6–15]. Table 1 presents a series of rutin electrochemical analysis methods, described by the technical literature, that encompass the main features of the analytical performance of the sensors used, and which are based on similar sensitive materials as the sensor developed in this work.
The electrochemical techniques can be successfully used for the detection of rutin mainly because they are simple, rapid, sensitive and cost effective. One of them, the square-wave voltammetry (SWV), evidenced to be particularly sensitive in the determination of the electroactive organic compounds due to the very low non-Faradaic current [16,17]. The advantage of SWV when compared to cyclic voltammetry (CV) and differential pulse voltammetry (DPV) lists: shorter time of analysis, reduced consumption of the electroactive compounds and minor problems with the electrode surface fouling [18]. Furthermore, with this technique, high sensitivities and very low detection and quantification limits can be obtained [3,15]. The development of novel methods of electroanalysis can be facilitated by using screen-printed electrodes (SPEs), which have a series of practical advantages when compared to the classical electrodes, such as the simplicity of use, the commercial availability, the low price and very good reproducibility in term of industrial production. Therefore, SPEs can be easily replaced after each analysis, which enhances the repeatability and reproducibility of the analyses [19,20]. The performance characteristics of SPEs can be further improved if the sensitive element is based on nanomaterials, such being the case of the sensitive element based on carbon or metallic nanoparticles.
⁎ Corresponding author at: Department of Chemistry, Physics and Environment, Faculty of Sciences and Environment, “Dunarea de Jos” University of Galati, 47 Domneasca Street, 800008 Galati, Romania. E-mail address: [email protected] (C. Apetrei).
http://dx.doi.org/10.1016/j.measurement.2017.09.020 Received 9 May 2017; Received in revised form 10 September 2017; Accepted 11 September 2017 Available online 12 September 2017 0263-2241/ © 2017 Elsevier Ltd. All rights reserved.
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Table 1 Performance characteristics of carbonaceous material based sensors used in detection of rutin. Sensitive material
Electrochemical technique
Linear range (M)
LOD (M)
Reference
Chitosan/graphene Electrodeposited graphene/gold nanoparticles Chemically reduced graphene oxide/Pt nanoparticles Reduced graphene oxide Graphene oxide/multi-walled carbon nanotube Electrochemically reduced graphene oxide Graphene/n-octylpyridinum hexafluorophosphate
DPV DPV DPV DPV DPV DPV SWV
5 × 10−7 to 1.04 × 10−5 8 × 10−8 to 8 × 10−5 0.057 × 10−6 to 102.59 × 10−6 0.1 × 10−6 to 2.0 × 10−6 0.08 × 10−6 to 80 × 10−6 4.7 × 10−7 to 1.2 × 10−5 0.05 × 10−6 to 11 × 10−6
– 2.55 × 10−8 2.0 × 10−8 2.32 × 10−8 2.0 × 10−8 1.8 × 10−8 1 × 10−8
9 10 11 12 13 14 15
2.2. The fabrication of voltammetric sensor
Graphene (GPH) have been extensively used in the development of sensors and biosensors for the distinctive properties related to two-dimensional nanostructure, for instance the high surface area, the outstanding electrical conductivity and the biocompatibility with organic molecules [21,22]. On the other hand, the incorporation of gold nanoparticles (AuNPs) with GPH could represent a promising way towards significant improvements of the electrochemical properties of the sensitive nanomaterials [10]. Due to its good compatibility and excellent electrical conductivity, chitosan is used for the better dispersion of the nanomaterials and the preservation of the nanostructures [22]. Generally, the effects of these nanomaterials are the increase of the performance characteristics, due to the facilitated transfer of electrons at the electrode-solution interface, characterized by the enhancement of the sensor response and the specific interactions with the target molecule [10,22,23]. To the best of our knowledge, the development of novel sensors using graphene and gold nanoparticles is a challenging and yet a promising way to accomplish sensitive, fast and simple detection of rutin. Therefore, the aim of this paper is to develop, optimize and validate an electroanalytical method based on the square-wave voltammetry technique for the assessment of rutin in pharmaceutical products using a sensitive voltammetric sensor based on graphene (GPH) and gold nanoparticles (AuNP).
The commercial CSPEs were modified with graphene, chitosan, and Au nanoparticles in order to achieve a novel sensor with an improved sensitivity to rutin. The GPH dispersion was prepared by mixing 1 mg GPH with 1 mL chitosan solution (0.2% in acetic acid, pH 5). To achieve a homogeneous dispersion the GPH-chitosan mixture was ultrasonicated for 2 h. The chitosan improved the dispersion of graphene. The gold nanoparticles (AuNPs) were prepared through the reduction of HAuCl4 with trisodium citrate in aqueous solution [24]. The synthesis reaction can be summarized as follows: 2HAuCl 4 + 3C6 H8O7 (citric acid) → 2Au + 3C5 H6 O5 (3-ketoglutaric acid) + 8HCl + 3CO2
After the synthesis and the separation of the gold nanoparticles, 1 mg of AuNPs was added to homogeneous dispersion GPH-chitosan and ultrasonicated for 1 h. The chemically modified carbon screen-printed electrodes were developed using the drop-and-dry method. 10 μL of the nanomaterials composite dispersion was dropped on the SPCEs using micropipette and then were dried in a desiccator at room temperature. The modified electrodes were the ones containing GPH (GPH/CSP sensor) and GPH and AuNPs (GPH-AuNP/CSP sensor) in the sensitive element. The prepared electrodes were positioned at 4 °C.
2. Materials and methods 2.1. Reagents and solutions
2.3. Equipment
All the chemicals were of the highest purity commercially available and were used without further purification. Rutin and ethanol (absolute, ≥99.8%) of analytical grades, were acquired from Sigma-Aldrich (St. Louis, USA). Chitosan, chloroauric acid, potassium ferricyanide, potassium chloride, hydrochloric acid, acetic acid, sodium acetate, sodium diphosphate, disodium phosphate, sodium hydroxide, trisodium citrate, lactic acid, ascorbic acid, lactose, sodium lauryl sulfate, starch, and sucrose were used in electrochemical studies. The carbon screen-printed electrodes (CSPE), DRP-110 working in solution (the working electrode diameter of 4 mm) were purchased from Dropsens Ltd. (Llanera, Spain). The graphene (GPH), platelet planar size of 0.3–5 μm, from Sigma-Aldrich (St. Louis, MO, USA) was used for CSPE modification. All the solutions were prepared with ultrapure water (resistivity was 18 MΩ cm) obtained from a Milli-Q water ultrapurification system (Simplicity®, Millipore, USA). In all electrochemical experiments, electrolytes buffer solutions with ionic strength 0.1 M were used. These buffer solutions were prepared with analytical grade substances and ultrapure water. The stock rutin solution (10−3 M) was prepared in ethanol, in order to facilitate the dissolving process, by means of ultrasonication. The solutions used for the spectrophotometric and the electrochemical analyses were obtained right before utilization by diluting the stock solutions with buffer solution until the desired concentration was reached. The stock solution was kept in the fridge, at a 4 °C temperature and remained stable throughout the analyses.
The cyclic voltammetry (CV) and square-wave voltammetry (SWV) experiments were performed on a Biologic SP 150 (Bio-Logic Science Instruments SAS, France) potentiostat/galvanostat controlled by a microcomputer with EC-Lab Express software. A three-electrode system was used where a silver-silver chloride (Ag/AgCl in 3 M KCl) electrode was the reference electrode, a platinum wire electrode was the auxiliary electrode and the graphene-gold nanoparticles screen-printed (GPHAuNP/CSP) sensor was the working electrode. Thus, the connections to the potentiostat/galvanostat were made through independent cables for each electrode. All the subsequent potentials indicated in this study were compared with the Ag/AgCl in 3 M KCl system. The cyclic voltammograms were obtained for different potential ranges at 0.1 V s−1, while the square wave voltammograms were obtained using a 90 mV pulse height, a 5 mV scan increment, and a 15 Hz frequency. All the electrochemical measurements were carried out at room temperature. The morphology of the carbon screen-printed electrode modified with graphene - gold nanoparticles composite was characterized using the scanning electron microscope FlexSEM 1000 (Hitachi, Japan). The S10H ultrasonic apparatus (Elmasonic, Germany) was used for the ultrasonic experiment. The pH of the buffer solutions was measured with an Inolab pH 7310 pH-meter (WTW, Germany). The spectrophotometric measurements in the ultraviolet range were carried out with a Rayleigh UV-1601 spectrophotometer (Beijing Beifen-Ruili Analytical Instrument Co. Ltd., China). 38
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2.4. Application in pharmaceutical analysis The applicability of the graphene-gold nanoparticles sensor was studied by analyzing pharmaceutical products containing rutin. Rutin S (Antibiotice SA, Iasi, Romania – 40 mg rutin per capsule), Rutin C (Medica Pro Natura, Romania - 200 mg rutin per capsule), and Rutin Forte (Organika, Canada - 500 mg rutin per capsule) which were purchased from local pharmacies in Galati, Romania. Ten capsules from each type of pharmaceutical capsules were weighed and then triturated into a mortar and pestle. An adequate quantity from this blend was weighted and dissolved into a buffer solution/ethanol (70:30 v/v) obtaining, thus, the stock solution with the desired concentration. The solutions to be analyzed were subsequently prepared through the dilution of this solution with buffer solution. For the interference studies on rutin standard stock solution, different chemical compounds and excipients used in the pharmaceutical products (potassium chloride, sodium acetate, sodium diphosphate, disodium phosphate, sodium citrate, lactic acid, ascorbic acid, lactose, sodium lauryl sulfate, starch and sucrose) were added in various concentrations. The solutions obtained were three times washed with buffer solution/ethanol (70:30 v/v). The filtrate was brought to the volumetric flask and filled up with buffer solution/ethanol (70:30 v/v) so that the rutin concentration to be equal with the initial concentration. Each solution was transferred to the electrochemical cell and the voltammetric signals were recorded in the same conditions as in the case of the standard solutions or the solutions obtained from the pharmaceutical products selected for analysis. In order to validate rutin determination by means of the voltammetric method, the spectrophotometric method mentioned in the Romanian Pharmacopeia, Edition X was used [25]. 50 mg rutin are dissolved in 20 mL ethanol through heating, and after cooling the solution is brought to 100 mL in a volumetric flask by adding the same solvent. For 3 mL of this solution 1 mL acetic acid 1.2 g L−1 is added and is filled up with alcohol up to 100 mL, in a volumetric flask (sample solution). The absorbance at 362 nm is determined for the mixture of solvents used for the sample dissolution. Simultaneously, the absorbance of a standard solution obtained in the same conditions as the sample solution from 3 mL rutin (previously dried at 130 °C until constant mass) 0.05% w/v in ethanol, 1 mL acetic acid 1.2 g L−1 and ethanol at 100 mL is determined. Also, the voltammetric method was validated determining the recovery via the method of the standard addition.
Fig. 1. High resolution SEM image of SPCE modified GPH/AuNP nanocomposite.
Fig. 2. Cyclic voltammograms of CSPE (_._._), GPH/CSP sensor (_ _ _), and GPH-AuNP/CSP sensor (___) in 10−3 M K4[Fe(CN)6] and 0.2 M KCl solution.
the chitosan coated CSPE, respectively. As shown in Fig. 2, K4[Fe(CN)6] showed redox current peaks at 0.26 V and 0.16 V. The currents of the redox peaks agreed with the order: Ip (CSPE) < Ip (GPH/CSPE) < Ip (GPH-AuNP/CSPE), both for the anodic and cathodic peaks. The special two-dimensional nanostructure of the GPH can be beneficial for the electrical conductivity, which explains the increased peak currents when compare with the unmodified electrode [23]. Additionally, the AuNPs facilitate the electron transfer at the sensor surface having a significant role in increasing the peak currents [10]. As it can be seen in the case of GPH-AuNP/CSP sensor, the peak-topeak potential separation (ΔEp = Epa − Epc) was of 0.1 V, corresponding to a quasi-reversible electron transfer process (Fig. 2). Furthermore, the ratio between the cathodic current peak and the anodic peak is sub-unitary, 0.89, but close to the ideal value, 1, for a reversible process [26,27]. From the study of the influence of the scanning rate of the potential over the anodic current peak it was determined that the electrochemical process is controlled by the diffusion process of the electroactive species on the surface of the electrode for all sensors (anodic peak current, Ipa, is proportional with the square root of scan rate, v1/2) [22]. Moreover, these studies made possible the estimation of the active surface of the sensor using the Randles-Sevcik equation [27]. The ratio between the active surface, electrochemically determined, and the geometric one (roughness factor) is of 12.64 for the GPH-AuNP/CSP sensor, 8.45 for the GPH/CSPE, and 2.28 for the CSPE, respectively. The higher value obtained in the case of GPH-AuNP/CSP sensor is determined by the GPH and the gold nanoparticles, which can increase the number of
3. Results and discussion 3.1. Morphological investigation Fig. 1 shows the high resolution scanning electron microscope (SEM) image of the GPH/AuNP-modified CSPE. The SEM image of GPH/AuNP nanocomposite shows a typical 3D morphology, a few layers nanoflakes of GPH decorated with gold nanoparticles. In addition, the micrograph presented in Fig. 1 shows the spherical morphology of the gold nanoparticles. 3.2. The electrochemical characterization of the modified graphene-gold nanoparticles screen-printed (GPH-AuNP/CSP) sensor The general information regarding the electroactivity of the compounds under study, as well as the processes at the interface of the electrode/solution, was obtained through cyclic voltammetry. Potassium ferrocyanide was chosen as reference solution in order to evaluate the electrochemical properties of the developed sensor. Fig. 2 shows the electrochemical signals of the CSPE, GPH/CSP, and GPHAuNP/CSP sensors in 10−3 M K4[Fe(CN)6] and 0.2 M KCl solution. No changes were observed in the cyclic voltammograms of the CSPE and of 39
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Fig. 3. Chemical structure of rutin.
active centers on the sensor surface because of the modified nanostructures and of the synergetic effects [20,26,28]. These studies demonstrates the improved electrochemical sensitivity of the GPH-AuNP/ CSP sensor based on nanostructured materials and recommend its use in the detection of rutin. 3.3. Electrochemical detection of rutin at GPH-AuNP/CSP based sensor The rutin electro-activity is in relation with its chemical structure that contains more hydroxyl groups, which can be oxidized electrochemically [9,16]. Rutin is 3′,4′,5,7-tetrahydroxyflavone-3b-D-rutinoside and the chemical structure is given in Fig. 3. The electrochemical behavior of rutin at GPH-AuNP/CSP sensor was firstly investigated by means of cyclic voltammetry. Fig. 4 shows the CV of 10−5 M rutin in 0.1 M acetate buffer solution of pH 5.0. In the direct scanning, two anodic peaks can be observed, IA and IIA at EpaI = +0.44 V and EpaII = +1.08 V, respectively. In the reverse scan a cathodic peak, IC, corresponding to the anodic peak IA, appears at EpcI = +0.22 V. The redox pair (EpaI/EpcI) noticed at E1/2 = 0.33 V corresponds to the catechol moiety, 3′,4′-dihydroxyl groups on B-ring, that go through a reversible process of oxido-reduction with the forming of an ortho quinonic derivative [16]. The separation of the potential peaks (ΔE) is equal with 0.22 V, a relative high value which suggests that the electrochemical processes on the active surface of the sensor, that implies the transfer of two electrons, is slow and quasi-reversible [16,19]. On the other hand, the peak IIA, at EpaII = +1.08 V corresponds to the irreversible electrochemical oxidation of the hydroxyl groups in the positions 5 and 7 (resorcinol moiety of ring A) [16,19]. The influence of the potential scan rate (v) on the peaks currents (IpaI and IpcI) of 10−5 M rutin for the GPH-AuNP/CSP sensor was studied by cyclic voltammetry at various scan rates (Fig. 5). As shown in Fig. 5(a), the peak currents of rutin grow along with the
Fig. 5. (a) CVs of GPH-AuNP/CSP sensor immersed in 10−5 M rutin (in 0.1 M acetate buffer solution with pH 5) registered with different scan rates in the range 0.05 V s−1 to 0.5 V s−1; (b) relationships between the peak currents and the potential scan rate.
increase of scan rates (v) and there are significant linear dependences among the peak currents and scan rates (Fig. 5b). The regression equation is IpaI = 86.97 × v + 21.73 (IpaI: μA, v: V s−1, R2 = 0.983); IpcI = −45.21 × v − 13.77 (IpcI: μA, v: V s−1, R2 = 0.989), indicates the redox process of 10−5 M rutin for the GPH-AuNP/CSP sensor in adsorption-controlled conditions [27]. Therefore, the limitative factor of the redox process is the transfer of electrons. The square-wave voltammetry method was investigated in order to increase the sensitivity. Fig. 6 shows the SWV (direct scan) of the GPHAuNP/CSP sensor immersed in 10−5 M rutin (0.1 M acetate buffer solution of pH 5). As it can be noticed, the two anodic peaks appears to have the same potentials as in the case of the cyclic voltammetry. Compared to the CV results, the peaks obtained via SWV are better defined, the currents are
Fig. 6. The SWV (direct scan) of the GPH-AuNP/CSP sensor immersed in 10−5 M rutin (0.1 M acetate buffer solution with pH 5).
Fig. 4. CV of 10−5 M rutin in 0.1 M acetate buffer solution of pH 5.
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higher, and the background current is lower. Furthermore, the SWV presents some advantages such as lower consumption of electroactive species and subsequent lessening of the species which block the electrode surface [18]. Therefore, the SWV technique is the most suitable for the quantitative studies. 3.4. The influence of the supporting electrolyte and pH The nature and the type of electrolytes used have a central role in the voltammetric signal of the sensor towards rutin. The currents of the responses obtained with the sensor towards a 10−5 M rutin solution were determined in many buffer solutions based on different electrolytes such as Na2HPO4 + NaH2PO4, CH3COONa + CH3COOH, sodium citrate + HCl, KCl + NaOH, KCl + HCl. The results obtained through cyclic voltammetry revealed that the most intense currents and the best peak shape were obtained in CH3COONa + CH3COOH buffer solution. As a result, the CH3COONa + CH3COOH buffer solution was used in the succeeding studies. The electrode reaction of rutin, as in the case of other polyphenol type compounds, is influenced by the pH of the medium. Therefore, the redox process of rutin was studied for a pH range 2–11, in various buffer solutions, by means of SWV (Fig. 7a). As the value of the pH rises, a linear movement towards the lower peak potential values, until the pH reaches the value of 9, can be observed. For values above 9, the potential of the peak becomes almost independent of the pH of the electrolyte solution containing rutin (Fig. 7b). The slope between the peak potential and the pH is of 62 mV per pH unit and it demonstrates that an equal number of electrons and protons are involved in the redox process [27]. Furthermore, the value of the half width of the peak is close to the theoretical value of 90 mV
Fig. 8. (a) The SWVs of GPH-AuNP/CSP sensor immersed in rutin solution with different concentrations in the range 0.1 × 10−6 to 15 × 10−6 M; (b) the relationships between the peak currents and concentration (calibration curve).
established for a redox process in which there are involved two electrons and two protons [14,27]. On the other hand, the variation of the peak currents, in relation with the pH, is nonlinear, the current of the peak being higher in a weak acid medium with pH 5. Since the best response and sensitivity were obtained for pH 5.0 (acetic acid/sodium acetate buffer solution), this condition was selected for the quantitative analysis of rutin. 3.5. The calibration curve Under the optimum experimental conditions, square-wave voltammograms of a series of rutin solutions of various concentrations were registered. These results are presented in Fig. 8a. As Fig. 8b shows, the calibration curve is linear for the concentration domain 1 × 10−7 to 1.5 × 10−5 M, with a detection limit (three times the standard deviation of the signal blank/slope) of 1.1 × 10−8 M. The equation of the linear regression is (I/μA) = 3.045 × [Rutin] + 1.12, R2 = 0.9988), where I is the voltammetric signal corresponding to the peak and [Rutin] is the concentration of rutin in μM. As the graphs and the calculations show, the method of quantitative
Fig. 7. (a) The square wave voltammograms of GPH-AuNP/CSP sensor immersed in buffer solutions with the pH ranging from 3 to 7 containing 10−5 M rutin; (b) the dependence between the peak potential and the pH values ranging from 2 to 11.
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determination is relatively simple and very sensitive. By comparing the performance characteristics of the sensor developed in this study with the main sensors used for the rutin analysis in Table 1, it can be concluded that the GPH-AuNP/CSP sensor owes its improved performance features to the nanostructured sensitive materials.
Table 2 Determination of rutin in tablets by standard addition method. No.
Sample Sample Sample Sample Sample Sample Sample
3.6. The precision of the voltammetric method The repeatability of the analysis method was determined by performing 6 quantitative determinations for a solution 5 × 10−6 M using 5 different GPH-AuNP/CSP sensors, for time intervals of 15 min between one and the next. The relative standard deviation (RSD) for all the determinations was of 2.1% which demonstrates that the analysis method has a very good repeatability. In order to establish the intermediate precision of the voltametric determination, the quantitative analysis was carried out in 5 different days, using a new GPH-AuNP/CSP sensor at all times, and new rutin standard solutions of two concentrations, 2 × 10−6 M and 5 × 10−6 M. Under these conditions, the repeatability was not affected by the concentration of the rutin solution and the RSD of the peak currents was of 3%, which proves that the sensor shows a very good precision.
1 2 3 4 5 6 7
Sampling amount (mg)
Detected amount (mg)
Recovery (%)
Average recovery (%)
40.5 40.3 39.7 39.8 40.0 40.2 40.4
39.3 39.6 40.5 40.2 40.1 38.8 41.6
97.04 98.26 102.02 101.01 100.25 96.52 102.97
99.72
Table 3 Determination of rutin in pharmaceutical products by spectrophotometric and voltammetric methods. Sample
Rutin S (Antibiotice SA, Romania) Rutin C (Medica Pro Natura, Romania) Rutin Forte (Organika, Canada)
3.7. The study of interferences In order to study the effect of various excipients used in the pharmaceutical rutin products and of other substances that can be found in the samples to be analyzed for the 5 × 10−6 M rutin determination, the decrease or the increase of the anodic peak current was determined. The tolerance limit considered was the ratio between the concentrations of the interfering species and the concentration of rutin, which determines a ± 5.0% relative error. The tolerance limit for the interfering substances for the 5 × 10−6 M rutin determination were 200 for Na+, K+, PO43−, HPO42−, H2PO4−, Cl−, citrate, acetate, 150 for lactose, sodium lauryl sulfate, lactic acid, 50 for starch and sucrose, and 5 for the ascorbic acid. Thus, the quantification of rutin is barely affected by the presence of the main excipients in the sample under study, the voltammetric signals remaining almost unmodified in the presence of the compounds previously mentioned.
Rutin concentration (mg per capsule) Label value
Spectrophotometric method
Voltammetric method
40
41 ± 1
40 ± 1
200
199 ± 3
198 ± 4
500
503 ± 8
502 ± 9
determination using the calibration curve was used for the analysis. The results obtained via this method and those indicated by the producer on the pharmaceutical product label are presented in Table 3. The statistic calculations have proven that the voltammetric method has a very good accuracy and the t-test has shown that there is no significant difference between the results at a 99% confidence level. This proves that the GPH-AuNP/CSP sensor is useful in the determination of rutin in pharmaceutical products. 4. Conclusions The GPH-AuNP/CSP sensor proved to be useful in the analysis of rutin in pharmaceutical products. The use of the square wave voltammetry as detection method allowed reaching excellent analytical performances with applicability in the laboratory practice. The results obtained with the GPH-AuNP/CSP sensor are very close to the results obtained via the standard method of analysis at a 99% confidence level. Furthermore, the developed method has a series of advantages such as good precision, reliability, simplicity, and low cost. The method is precise and has a very good accuracy, and it is versatile and feasible for large-scale analysis in the quality control of the pharmaceutical products, food and food supplements and other types of samples.
3.8. The validation of the method The validation of the method established in this work, based on the voltammetric sensor and using as detection method SWV, was performed through two methods. First, the results found in the analysis of some rutin samples of known concentration were compared with the ones obtained via the official method mentioned in the Pharmacopoeia [25]. The difference between the results was less than 1%, thus confirming the validity of the electroanalytical method proposed in this study. The second method used for validation was the standard addition method. The accuracy of the method was determined by calculating the mean of the recovery values obtained when rutin solutions were analyzed in the micromolar domain. The average value of the recovery obtained was 99.72% with a RSD of 2.49% (number of determination 7) (Table 2). Considering the results obtained, it is possible to use this voltammetric method in the quantification of rutin in pharmaceutical products.
Acknowledgment This work was supported by a grant of the Romanian National Authority for Scientific Research and Innovation, CNCS - UEFISCDI, project number PN-II-RU-TE-2014-4-1093. Conflict of interest The authors declare no conflict of interest. References
3.9. The analysis of rutin pharmaceutical products
[1] H. Hosseinzadeh, M. Nassiri-Asl, Review of the protective effects of rutin on the metabolic function as an important dietary flavonoid, J. Endocrinol. Invest. 37 (2014) 783–788. [2] M. Medvidović-Kosanović, M. Šeruga, L. Jakobek, I. Novak, Electrochemical and
The GPH-AuNP/CSP sensor was used for the quantitative analysis of rutin in different commercial pharmaceutical products. The direct 42
Measurement 114 (2018) 37–43
I.M. Apetrei, C. Apetrei
[3]
[4]
[5] [6]
[7]
[8]
[9]
[10]
[11]
[12] [13]
[14]
reduced graphene oxide modified sensor, J. Pharm. Anal. 6 (2016) 80–86. [15] H. Wang, Z. Wang, G. Liu, Z. Zhang, Electrochemical behavior of rutin on graphene and ionic liquids composite film modified electrode, Nongye Jixie Xuebao/Trans. Chinese Soc. Agric. Mach. 45 (2014) 219–224. [16] M.-E. Ghica, A.M. Oliveira Brett, Electrochemical oxidation of rutin, Electroanalysis 17 (2005) 313–318. [17] J.V. Piovesan, C.L. Jost, A. Spinelli, Electroanalytical determination of total phenolic compounds by square-wave voltammetry using a poly(vinylpyrrolidone)modified carbon-paste electrode, Sensor. Actuat. B-Chem. 216 (2015) 192–197. [18] V. Mirceski, R. Gulaboski, M. Lovric, I. Bogeski, R. Kappl, M. Hoth, Square-wave voltammetry: a review on the recent progress, Electroanalysis 25 (2013) 2411–2422. [19] A. de Oliveira-Roberth, D.I.V. Santos, D.D. Cordeiro, F.M.A. de Lino, M.T.F. Bara, E.S. de Gil, Voltammetric determination of rutin at screen-printed carbon disposable electrodes, Cent. Eur. J. Chem. 10 (2012) 1609–1616. [20] I.M. Apetrei, C. Apetrei, Study of different carbonaceous materials as modifiers of screen-printed electrodes for detection of catecholamines, IEEE Sens. J. 15 (2015) 3094–3101. [21] W. Zhang, S. Zhu, R. Luque, S. Han, L. Hu, G. Xu, Recent development of carbon electrode materials and their bioanalytical and environmental applications, Chem. Soc. Rev. 45 (2016) 715–752. [22] I.M. Apetrei, C. Apetrei, Voltammetric determination of melatonin using a graphene-based sensor in pharmaceutical products, Int. J. Nanomed. 11 (2016) 1859–1866. [23] E. Jubete, O.A. Loaiza, E. Ochoteco, J.A. Pomposo, H. Grande, J. Rodriguez, Nanotechnology: a tool for improved performance on electrochemical screenprinted (bio)sensors, J. Sensors (2009) (Article ID 842575, 13 pages). [24] G. Frens, Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions, Nat. Phys. Sci. 241 (1973) 20–22. [25] Farmacopeea Romana, Editia a X-a. Ed. Medicala, Bucuresti, 1993, pp. 830–832. [26] C. Apetrei, I.M. Apetrei, J.A. de Saja, M.L. Rodriguez-Mendez, Carbon paste electrodes made from different carbonaceous materials: application in the study of antioxidants, Sensors 11 (2011) 1328–1344. [27] A.J. Bard, L.R. Faulkner, Electrochemical Methods, John Wiley and Sons, New York, 2001. [28] S.A. Zaidi, J.H. Shin, A review on the latest developments in nanostructure-based electrochemical sensors for glutathione, Anal. Methods 8 (2016) 1745–1754.
antioxidant properties of (+)-catechin, quercetin and rutin, Croat. Chem. Acta 83 (2010) 197–207. K.H. Garcia Freitas, O. Fatibello-Filho, I.L. de Mattos, Square-wave voltammetric determination of rutin in pharmaceutical formulations using a carbon composite electrode modified with copper (II) phosphate immobilized in polyester resin, Braz. J. Pharm. Sci. 48 (2012) 639–649. I. Petkova Pencheva, V. Nikolova Maslarska, A. Christova Stoimenova, M. Metodieva Manova, L. Antonova Andonova, P. Krumova Zdraveva, Quality control optimization solutions for determination of rutin in supplements containing ginkgo biloba extract, Curr. Pharm. Anal. 12 (2016) 386–390. L.S. Chua, A review on plant-based rutin extraction methods and its pharmacological activities, J. Ethnopharmacol. 150 (2013) 805–817. L. Xiangjun, Z. Yuping, Y. Zhuobin, Separation and determination of rutin and quercetin in the flowers of Sophora japonica L. by capillary electrophoresis with electrochemical detection, Chromatographia 55 (2002) 243–246. S. Li, L. Zhang, L. Chen, Y. Zhong, Y. Ni, Determination of rutin by chemiluminescence based on a luminol–potassium periodate–ZnSe system, Anal. Methods 8 (2016) 4056–4063. D. Šatínský, K. Jägerová, L. Havlíková, P. Solich, A new and fast HPLC method for determination of rutin, troxerutin, diosmin and hesperidin in food supplements using fused-core column technology, Food Anal. Method. 6 (2013) 1353–1360. J. An, Y.-Y. Bi, C.-X. Yang, F.-D. Hu, C.-M. Wang, Electrochemical study and application on rutin at chitosan/graphene films modified glassy carbon electrode, J. Pharm. Anal. 3 (2013) 102–108. W. Sun, D. Wang, Y.-Y. Zhang, X.-M. Ju, H.-X. Yang, Y.-X. Chen, Z.-F. Sun, Electrodeposited graphene and gold nanoparticle modified carbon ionic liquid electrode for sensitive detection of rutin, Chinese J. Anal. Chem. 41 (2013) 709–713. S. Li, B. Yang, J. Wang, D. Bin, C. Wang, K. Zhang, Y. Du, Nonenzymatic electrochemical detection of rutin on Pt nanoparticles/graphene nanocomposite modified glassy carbon electrode, Anal. Methods 8 (2016) 5435–5440. Y. Lei, D. Du, L. Tang, C. Tan, K. Chen, G.-J. Zhang, Determination of rutin by a graphene-modified glassy carbon electrode, Anal. Lett. 48 (2015) 894–906. L. Yan, X.g. Niu, W. Wang, X. Li, X. Sun, C. Zheng, J. Wang, W. Sun, Electrochemical sensor for rutin detection with graphene oxide and multi-walled carbon nanotube nanocomposite modified electrode, Int. J. Electrochem. Sci. 11 (2016) 1738–1750. P. Zhang, Y.-Q. Gou, X. Gao, R.-B. Bai, W.-X. Chen, B.-L. Sun, F.-D. Hu, W.-H. Zhao, The pharmacokinetic study of rutin in rat plasma based on an electrochemically
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