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Simultaneous determination of salbutamol and propranolol in biological fluid samples using an electrochemical sensor based on functionalized-graphene, ionic liquid and silver nanoparticles
Journal of Electroanalytical Chemistry 824 (2018) 1–8
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Simultaneous determination of salbutamol and propranolol in biological fluid samples using an electrochemical sensor based on functionalizedgraphene, ionic liquid and silver nanoparticles Anderson Martin Santos, Ademar Wong, Orlando Fatibello-Filho
T
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Department of Chemistry, Federal University of São Carlos, 13560-970 São Carlos, SP, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Functionalized-graphene Simultaneous detection Salbutamol Propranolol, ionic liquid Silver nanoparticles
A simple, rapid, and low-cost electroanalytical method was developed using a glassy carbon electrode modified with functionalized-graphene (FG), ionic liquid (IL) and silver nanoparticles (AgNPs) for simultaneous detection of salbutamol (SAL) and propranolol (PRO). The electrochemical behaviour of the electrodes was investigated by cyclic voltammetry and square wave voltammetry (SWV) under optimised conditions. Using SWV, the AgNP-ILFG-NF/GCE sensor showed a linear response from 79 nmol L−1 to 2.9 μmol L−1 for SAL and from 0.1 to 2.9 μmol L−1 for PRO with limits of detection of 13 and 17 nmol L−1 for SAL and PRO, respectively. The proposed sensor showed good stability, repeatability and reproducibility, and was applied successfully to the simultaneous determination of SAL and PRO in biological fluid samples. The proposed method proved to be excellent, being therefore a reliable alternative method for the simultaneous determination of SAL and PRO.
1. Introduction Salbutamol (SAL) also known as albuterol ((RS)-4-[2-(tert-butylamino)-1-hydroxyethyl]-2-(hydroxymethyl)phenol – Fig. 1A) is a drug widely used in the treatment of asthma, chronic pulmonary disease and in the control of blood potassium levels. SAL can cause side effects, such as headache, dizziness, shakiness, and fast heart rate [1–3], and if administered in excess or if consumed improperly can cause serious health problems, including aggravating bronchospasm, irregular heartbeat, and low blood potassium levels [1, 4, 5]. Propranolol (PRO) (1-isopropylamino-3-(naphthalen-1-yloxy) propan-2-ol – Fig. 1B) is a beta-adrenergic blocking agent [6–8]. This drug is most frequently prescribed for the treatment of high blood pressure, chronic angina pectoris, prophylaxis and treatment of cardiac arrhythmias, prophylaxis of myocardial reinfarction and treatment of tremors [7, 8]. PRO may cause adverse reactions, such as congestive heart failure, aggravation of atrioventricular conduction disorders, bronchospasm, severe bradycardia and hypotension [7, 8]. Although the simultaneous use of these drugs (SAL and PRO) is not common because PRO is a non-cardioselective beta-blocking agent and the risk of its use together with SAL may outweigh the benefits for patients with asthma, their consumption should be avoided or supervised by a doctor [9–11]. In the literature, some cases of symptomatic overdose with SAL are reported, where PRO was used as an ⁎
antidote and not as an anti-asthmatic drug [12]. Ramoska et al. [13] reported the use of PRO in the treatment of SAL poisoning in two asthmatic patients, in which case PRO was used to minimise the effect caused by SAL. In another study, Kupeli [14] reported the use of PRO for infantile haemangiomas, but only 3 out of 14 patients presented bronchospasm and were treated with SAL. Thus, even though SAL and PRO are not found in together in pharmaceutical formulations, they can be co-administered in clinical treatments [12, 13] and in other applications [15–17]. Therefore, the simultaneous determination of SAL and PRO in biological fluids is very important for physiological pharmacokinetics and clinical diagnosis [12, 13]. In view of the applicability of these drugs in extreme cases, whether due to misuse or in poisoning, their quantification in biological fluid samples is of paramount importance. In the literature, some procedures have been reported for the simultaneous determination of these classes of drugs [18, 19]. Among these analytical procedures, electroanalytical techniques have shown great advantages compared to other traditional techniques such as chromatography and spectrophotometry in the analysis of biological fluid samples [20–22]. Their advantages are greater simplicity, analysis in real time, low cost and short analysis time. Considering the necessity for increasingly sensitive and selective electrochemical sensors, an analytical method that has been highlighted is the use of modifiers immobilised on conventional electrode surfaces, such as carbon nanostructures, e.g. carbon black, carbon nanotubes and graphene [23,
Corresponding author. E-mail address: [email protected] (O. Fatibello-Filho).
https://doi.org/10.1016/j.jelechem.2018.07.018 Received 3 May 2018; Received in revised form 23 June 2018; Accepted 12 July 2018 Available online 21 July 2018 1572-6657/ © 2018 Elsevier B.V. All rights reserved.
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Fig. 1. Chemical structures of (A) salbutamol and (B) propranolol.
treatment using a concentrated solution of 1:1 (v/v) H2SO4/HNO3 and, this suspension was stirred for 12 h at 25 °C. The obtained FG suspension was filtered and washed with ultrapure water to about pH 7.0 and dried at 100 °C.
24]; polymers, e.g. PDDA, chitosan and nafion (NF) [24, 25]; ionic liquids (ILs) [26]; and metallic nanoparticles, e.g. Au, Cu, Pt and Ag [23, 27], which can be used to improve the performance of the sensor and induce catalytic and/or electrocatalytic effects [28–30]. Functionalised carbon nanomaterials such as functionalized-graphene (FG) have shown to be been promising due to their properties of better dispersibility in aqueous media, which facilitates their use in the preparation of films, and also that they interact with other modifiers, thus increasing the performance of electrochemical sensors for the determination of analytes of interest [30, 31]. In addition, the use of metallic nanoparticles incorporated on the surface of an electrode can provide several advantages such as catalytic effects and greater effective sensor surface area [23, 32]. In the present work, we prepared and characterised a glassy carbon electrode (GCE) modified with FG, IL (1-butyl-3-methylimidazolium tetrafluoroborate) and silver nanoparticles (AgNPs) for the simultaneous determination of SAL and PRO in biological fluid samples.
2.4. Synthesis of the AgNPs Preparation of the AgNPs was performed as previously reported by Wong et al. [23]. Initially, solutions of 5.0 × 10−3 mol L−1 NaBH4 and 1.0 × 10−4 mol L−1 AgNO3 were prepared. Then, 1 mL of the NaBH4 solution and 49 mL of the AgNO3 solution were transferred individually to two flasks, and left in an ice bath for 30 min. Finally, the NaBH4 solution was added dropwise under stirring, thereby forming a yellowish colloidal suspension, which was stirred for 30 min and stored in an amber flask.
2.5. Preparation of the AgNP-IL-FG-NF/GCE sensor
2. Experimental
The surface of the GCE was initially cleaned with a polishing cloth and 0.5 μm alumina slurry, followed by ultrasonic cleaning with ethyl alcohol and ultrapure water for 2 min. Under optimised conditions, the dispersion of AgNP-IL-FG-NF was prepared using 1.0 mg of FG, 1.0 mg of IL (1-butyl-3-methylimidazolium tetrafluoroborate), 10 μL of 0.25% (v/v) NF solution, 250 μL of AgNPs, and 740 μL of ultrapure water. The reagents were subjected to ultrasonic agitation for 40 min in order to obtain a homogeneous dispersion. Next, an 8 μL aliquot of the dispersion was dropped onto the GCE surface and dried at room temperature for 3 h, to obtain the GCE modified with AgNP-IL-FG-NF film.
2.1. Reagents and solutions SAL, PRO, bovine serum, nafion (NF) and 1-butyl-3-methylimidazolium tetrafluoroborate (IL) standards were purchased from SigmaAldrich (São Paulo, Brazil); NaOH, reagents for phosphate buffer (H3PO4, KH2PO4, K2HPO4, and K3PO4) and KCl were acquired from Acros. Graphene (GR) was acquired from Graphene Supermarket (New York, USA). The solutions were prepared using ultrapure water with resistivity not less than 18 MΩ cm obtained from a Millipore Milli-Q system (Billerica, USA). Stock solutions of SAL and PRO, both at 0.1 mol L−1 were prepared directly in ultrapure water.
2.6. Apparatus and reference method 2.2. Preparation of urine and serum samples Electrochemical experiments were performed by employing a Metrohm/Eco Chemie Autolab PGSTAT12 potentiostat/galvanostat electrochemical system controlled by GPES 4.9. Voltammetric measurements were carried out in a three-electrode cell, with an Ag/AgCl (3.0 mol L−1 KCl) electrode as the reference electrode, a platinum foil (0.5 cm2) as the auxiliary electrode and a GCE (Ø = 3 mm) or an AgNPIL-FG-NF/GCE working electrode. The morphological characterisations of the FG and AgNPs were evaluated by images acquired by field emission gun scanning electron microscopy (FEG/SEM, FEI Magellan 400 L). The film thickness on the electrode surface was evaluated using a Hirox, model KH-7700 digital microscope. UV–Vis spectrophotometer (Shimadzu model UV 2550) with a quartz cuvette (optical path length of 10 mm) was used as comparative spectrophotometric method [34, 35].
Synthetic urine samples were prepared as previously reported by Laube et al. [33] with the addition of 0.20 mmol L−1 KCl, 0.18 mmol L−1 NH4Cl, 0.10 mmol L−1 NaCl, 0.10 mmol L−1 CaCl2, 0.15 mmol L−1 KH2PO4 and 0.18 mmol L−1 urea in a 25 mL volumetric flask. The serum (bovine serum) samples were diluted 10 times with ultrapure water, and these samples were evaluated at two known concentrations of SAL and PRO (0.60 and 2.0 μmol L−1, respectively), and analysed by recovery percentage. 2.3. Synthesis of the FG Synthesis of the FG was performed as previously reported by Santos et al. [24]. In short, 100 mg of graphene was submitted to chemical 2
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2.7. Analytical procedure
Ip = ± (2.69 × 105) n3/2 A D1/2C v1/2
First, the proportions of nanomaterial (FG), metallic nanoparticles (AgNPs) and ionic liquid (IL) used to obtain the film in the GCE were investigated. After this step, the electrochemical characterisations of the electrodes were performed by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Based in the results, the electrochemical behaviour of SAL and PRO were analysed using CV, followed by the optimisation of the experimental conditions, such as supporting electrolyte (composition and pH), and the instrumental parameters for SWV (amplitude, frequency and increment). Next, calibration curves were constructed using successive additions of SAL and PRO standard solutions. All measurements were carried out in triplicate (n = 3), and the SAL and PRO concentrations were determined simultaneously under optimised conditions. The limit of detection (LOD) was calculated as three times the standard deviation for the blank solution (n = 10) divided by the slope of the analytical curve. The precision of the proposed method was verified from intra-day (n = 5) and inter-day (n = 3) repeatability studies.
where Ip is the peak current (A), n the number of electrons transferred, A the electroactive area (cm2), D the diffusion coefficient of [Fe (CN)6]3− in a 0.10 mol L−1 KCl solution (7.6 × 10−6 cm2 s−1), C the [Fe(CN)6]3− concentration (mol cm−3) and v the potential scan rate (V s−1). The obtained slopes of the Ip vs. v1/2 plots for the [Fe (CN)6]3− oxidation process were 2.17 × 10−5 A V−1/2 s1/2 for the GCE, 4.53 × 10−5 A V−1/2 s1/2 for the FG-NF/GCE, 7.16 × 10−5 A V−1/2 s1/ 2 for the IL-FG-NF/GCE, 7.72 × 10−5 A V−1/2 s1/2 for the AgNP-FG-NF/ GCE and 9.40 × 10−5 A V−1/2 s1/2 for the AgNP-IL-FG-NF/GCE. The corresponding electroactive areas were 0.065, 0.14, 0.21, 0.23 and 0.28 cm2 for the GCE, FG-NF/GCE, IL-FG-NF/GCE, AgNP-FG-NF/GCE and AgNP-IL-FG-NF/GCE, respectively. Thus, from these results is possible to conclude that the combination of the AgNP-IL-FG-NF/GCE sensor resulted in a 4.3-fold increase in electroactive area compared to GCE. In addition, EIS was used to characterise the electrodes in experiments using 2.5 mmol L−1 [Fe(CN)6]3−/4– as redox probe in 0.10 mol L−1 KCl solution, and a frequency range 100 kHz at 0.1 Hz. The curves generally had two parts, a linear part at lower frequencies and a semicircular part at higher frequencies. This semicircle corresponds to the limited transfer of electrons, and its diameter is equal the charge transfer resistance (Rct). Therefore, the larger the size of the semicircle, the greater the Rct. Fig. 3 shows the Nyquist curves obtained for the different electrodes, CGE, FG-NF/GCE, IL-FG-NF/GCE, AgNPFG-NF/GCE and AgNP-IL-FG-NF/GCE, where the values of Rct were 1054, 668, 242, 222 and 95 Ω, respectively. Thus, by analysing the results it is possible to observe a large semicircle for the CGE, which implies a high Rct. With the progressive modification of the electrode (FG-NF/GCE < IL-FG-NF/GCE < AgNP-FG-NF/GCE < AgNP-IL-FGNF/GCE), there was a decrease in the size of the semicircle, so indicating an increase in conductivity of the electrode and a reduction in Rct. The EIS was also used to calculate the standard heterogeneous rate constant (k0) for each modification of the electrode according to equation (Eq. 2):
3. Results and discussion Initially, to evaluate the effect of the functionalization of GR to FG, a dispersion of both materials in ultra-pure water was obtained by sonification. As can be seen in Fig. S1 (Supplementary Material), the oxidation of GR to FG allow the formation of a stable dispersion of FG due to its hydrophilic character (insertion of oxygenated functional groups into the material). Then, to obtain the film on the GCE, the amount of nanomaterial (FG) in the FG/solution ratios of 0.5:1.0, 1.0:1.0, 1.5:1.0 (mg/mL), IL in the IL/solution ratios of 0.5:1.0, 1.0:1.0, 1.5:1.0 (mg/mL) and the nanoparticles (AgNPs) in the AgNP/solution ratios of 0.25:0.75, 0.5:0.5 and 0.75:0.25 (mL/mL) were investigated. The highest current signals for the analytes (SAL and PRO) were obtained using 1.0 mg of FG, 1.0 mg of IL, 10 μL of NF, 250 μL of AgNPs, and 740 μL of ultrapure water. 3.1. Morphological characterisation of the materials
k0 =
The morphological characterisation of the electrode modification was performed by FEG-SEM. Fig. 2 shows the obtained images of (A) FG, (B) IL-FG and (C) AgNP-IL-FG, all on the surface of GCE. Fig. 2A shows that FG consists of thin wrinkled sheet structures disposed in stacked blocks of different sizes, which is characteristic of this type of material. It can also be seen in Fig. 2B, where the surface morphology of FG with IL presented a small change, where the graphene sheets were arranged more closely together and homogeneous. Finally, the morphology of FG in presence of AgNPs is presented in Fig. 2C, in which it is possible to observe highly dispersed multiple grains of AgNPs (white spheres) on the FG sheets with a average diameter of 20 nm, as shown in the histogram of Fig. 2D. The thickness of the film (AgNP-IL-FG-NF) was estimated using a digital microscope, where it was possible to visualize a 3D image of these materials on the GCE surface. In Fig. S2 (Supplementary material) is shown the surface aspect of film which presented an average film thickness of 3.725 μm.
RT F 2R ct AC
(1)
(2)
where k0 is the standard heterogeneous electron transfer rate constant (cm s−1), R the universal gas constant (8.314 J K−1 mol−1), T the thermodynamic temperature (298.15 K), F the Faraday constant (96,485C mol−1), Rct is the electron transfer resistance (Ω), A the electrode surface area (cm2) and C the concentration of the [Fe (CN)6]3−/4– solution (2.5 mmol cm−3). The obtained k0 values for the CGE, FG-NF/GCE, IL-FG-NF/GCE, AgNP-FG-NF/GCE and AgNP-IL-FG-NF/GCE were 1.4 × 10−3, 2.3 × 10−3, 6.3 × 10−3, 6.8 × 10−3 and 1.6 × 10−2 cm s−1, respectively. Considering that the k0 values are a measure of the kinetic facility of the redox pair, in which a system with high k0 value will achieve equilibrium in a shorter time than a system with low k0 value, which in this case will achieve equilibrium in a longer time. Thus, with the AgNP-IL-FG-NF/GCE sensor, the k0 value is greater than with AgNPFG-NF/GCE > IL-FG-NF/GCE > FG-NF/GCE > CGE, indicating in this case, a faster transfer of electrons in comparison with the other electrodes.
3.2. Electrochemical characterisation of electrodes 3.3. Electrochemical behaviour of SAL and PRO Initially, the determination of electroactive area of the working electrodes (GCE; FG-NF/GCE; IL-FG-NF/GCE; AgNP-FG-NF/GCE and AgNP-IL-FG-NF/GCE) were estimated by the Randles–Sevcik [36] equation (Eq. 1) in CV experiments at a potential scan rate range of 10–400 mV using a 0.5 mmol L−1 [Fe(CN)6]3− solution in aqueous 0.10 mol L−1 KCl (see Fig. S3 in Supplementary material).
For this study, CV was used to evaluate the electrochemical behaviour of the SAL (0.20 mmol L−1) and PRO (0.20 mmol L−1) solutions, using the different electrodes (CGE, FG-NF/GCE, IL-FG-NF/GCE, AgNPFG-NF/GCE and AgNP-IL-FG-NF/GCE) in a potential range of 0.05–1.25 V (v = 50 mV s−1) and 0.20 mol L−1 phosphate buffer 3
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Fig. 2. SEM image of (A) FG, (B) IL-FG, (C) AgNP-IL-FG and (D) corresponding histogram of AgNPs diameters.
Fig. 4. Cyclic voltammograms for 0.20 mmol L−1 SAL and 0.20 mmol L−1 PRO in 0.20 mol L−1 phosphate buffer solution (pH 7.0) at a v = 50 mV s−1. Fig. 3. EIS diagrams for 2.5 mmol L−1 [Fe(CN)6]3−/4– in 0.10 mol L−1 KCl. Frequency range 100 KHz to 0.1 Hz.
3.4. Optimisation of analytical parameters As can be seen in Fig. 5, the influence of the effect of hydrogen ion concentration on the oxidation reactions of the SAL and PRO (0.10 mmol L−1) were investigated by SWV using 0.20 mol L−1 phosphate buffer solutions with pH ranging between 5.0 and 9.0. The optimum pH of the supporting electrolyte found was 7.0, and this pH was selected for further studies. The graphic inserted in Fig. 5 represents the dependency of peak potential (Ep) with the pH for both SAL and PRO. The Ep value for SAL electro-oxidation varied linearly with pH with a slope of −0.057 V pH −1. This slope is close to the theoretical Nernstian slope of −0.0592 V pH −1 typical for redox reactions involving the same number of electrons and protons. This result is in accordance with previous works, which reported the electro-oxidation of SAL involving the loss of one electron and one proton [37, 38]. On the other hand, although the Ep value for the PRO electro-oxidation varied linearly with
solution (pH 7.0). As can be observed in Fig. 4, the cyclic voltammograms obtained for both drugs showed well-defined oxidation peaks at potentials of 0.65 and 1.0 V for SAL and PRO, respectively. In addition, the absence of reduction peaks during the cathodic potential scanning for SAL and PRO characterised an irreversible oxidation reaction. That is, there is an irreversible process of charge transfer for both compounds in the potential range employed. As observed in Fig. 4, the final modification (AgNP-IL-FG-NF/GCE), compared with the other modifications showed higher oxidation peak current (Ipox) values for both drugs, where Ipox is increased by factors of up to 2.4 and 3.2 for SAL and PRO, respectively. Additionally, there was a displacement of the oxidation peak potential to less positive potentials, caused mainly by an electrocatalytic effect of SAL and PRO oxidation brought on by modification of the GCE surface. 4
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electrons and two protons [39, 40]. The effect of different compositions of supporting electrolytes (pH 7.0), such as PIPES, BR and phosphate buffers was tested. The phosphate buffer solution provided the highest peak current in the SWV experiments and was selected for the electrochemical studies. Next, the parameters that affected the SWV response – frequency (f), amplitude (a) and potential increment (ΔEs) – were optimised. The optimum values obtained for these parameters using 0.10 mmol L−1 SAL and PRO in 0.20 mol L−1 phosphate buffer (pH 7.0) solution with a AgNP-IL-FG-NF/GCE sensor, were f = 30 Hz, a = 50 mV and ΔEs = 9 mV (see Table S1). 3.5. Effect of potential scan rate The effect of potential scan rate on the electrochemical behaviour of the AgNP-IL-FG-NF/GCE sensor was investigated by CV, ranging the scan rate from 10 to 400 mV s−1 in the presence of 0.20 mmol L−1 of SAL or PRO and 0.20 mol L−1 phosphate buffer solution (pH 7.0). As shown in Fig. 6A and B, with increasing potential scan rate, a linear increase in the peak current was verified for both drugs. Furthermore, the results showed good linearity of the plots of peak current versus the square root of the scan rate (ΔIpa vs. v1/2) (graphic (i) inserted in Fig. 6A and B), indicating that the mass transport of SAL and PRO on the electrode surface occurred by a diffusion processes. This process was proved by plotting the logarithm of the anodic peak current versus the
Fig. 5. SW voltammograms recorded for 0.10 mmol L−1 SAL and PRO in 0.20 mol L−1 phosphate buffer solutions with pH ranging of 5.0 to 9.0 using the AgNP-IL-FG-NF/GCE sensor. Parameters: f = 70 Hz, a = 50 mV ΔEs = 4 mV s−1. Insets: graphics of Ep vs. pH.
the pH, a slope of −0.028 V pH −1 was obtained. This slope is also in agreement with the theoretical Nernstian slope of −0.029 V pH −1 for redox reactions involving different numbers of electrons and protons in a ratio of 2:1, that is, the number of electrons is twice the number of protons. These results were also verified recently in another work reported by the group [25]. However, more studies in the literature have reported the electro-oxidation of PRO involving the transfer of two
Fig. 7. SW voltammograms obtained using the AgNP-IL-FG-NF/GCE sensor in 0.20 mol L−1 phosphate buffer (pH 7.0) containing: (A) 1.0 μmol L−1 of PRO and different concentrations of SAL: 0.079; 0.30; 0.59; 0.97; 1.9 and 2.9 μmol L−1, (B) 2.0 μmol L−1 of SAL and different concentrations PRO: 0.10; 0.30; 0.59; 0.97; 1.9 and 2.9 μmol L−1. Analytical curves (inserted).
Fig. 6. Cyclic voltammograms for different scan rates (10–400 mV s−1) using the AgNP-IL-FG-NF/GCE sensor, for (A) 0.20 mmol L−1 SAL and (B) 0.20 mmol L−1 PRO in 0.20 mol L−1 phosphate buffer solution (pH 7.0). Graphics of (i) Ip vs. v1/2 and (ii) log Ip vs. log v. 5
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concentration at 1.0 μmol L−1 and varying the SAL concentration from 0.079 to 2.9 μmol L−1. Therefore, from the obtained SW voltammograms (Fig. 7A), the value of Ipa for SAL increased linearly with its concentration (r2 = 0.997), while the value of Ipa for PRO remained constant, with a relative standard deviation (RSD) of 2.7%. Similarly, when the concentration of SAL was fixed at 2.0 μmol L−1, and that of PRO was varied in the range of 0.10 to 2.9 μmol L−1 (Fig. 7B), the value of Ipa for PRO increased progressively with its concentration (r2 = 0.998), while the value of Ipa for SAL remained constant (RSD = 3.3%). From these results it can be concluded that neither drug (SAL or PRO) interferes in the simultaneous determination of one in the presence of the other. Finally, the analytical curve for the simultaneous determination of these drugs was performed with the AgNP-IL-FG-NF/GCE sensor by SWV using different concentrations of SAL and PRO. As can be seen in the voltammograms shown in Fig. 8, a proportional increase of the anodic peak current was obtained with the increase of analytes concentrations in the electrochemical cell. The analytical curves were linear in the concentrations ranges from 0.079 to 2.9 μmol L−1 for SAL and, from 0.10 to 2.9 μmol L−1 for PRO, with a LOD of 13 nmol L−1 and 17 nmol L−1 for SAL and PRO, respectively. The graphs of Fig. 8 (i) and (ii) correspond to the analytical curves obtained, conforming to the following Eqs. (3) and (4):
Fig. 8. (A) SW voltammograms obtained using the AgNP-IL-FG-NF/GCE sensor in 0.20 mol L−1 phosphate buffer (pH 7.0) containing different concentrations of SAL: (a) 0.079; (b) 0.30, (c) 0.59, (d) 0.97, (e) 1.9, (f) 2.9 μmol L−1. PRO: (g) 0.10; (h) 0.30, (i) 0.59, (j) 0.97, (k) 1.9 and (l) 2.9 μmol L−1. Analytical curves from SAL (i) and PRO (ii).
logarithm of the scan rate (log ΔIp vs. log v), which showed slopes of 0.45 and 0.56 for SAL and PRO, respectively (see the inset (ii) of Fig. 6A and B). As can be observed, these slope values are around the theoretical value of 0.5, characteristic of a system controlled by diffusion.
3.6. Simultaneous determination of SAL and PRO by SWV
Ipa /A = −1.85 × 10−7 + 3.94 [SAL/(mol L−1)] (r 2 = 0.997)
(3)
Ipa /A = −6.42 × 10−7 + 6.82 [PRO/(mol L−1)] (r 2 = 0.992)
(4)
The analytical parameters of the proposed sensor were compared with those previously reported in the literature [25, 38, 41–46] (see Table 1), and it was possible to see clearly that the use of the AgNP-FGNF/GCE sensor led to excellent linear concentration ranges and LODs for SAL and PRO, being similar to, or better than, those reported by other authors. In addition, the sensor proposed here is the first to report
Firstly, to confirm the possibility of simultaneous determination of SAL and PRO by SWV, analytical curves of one analyte in the presence of the other were obtained. As can be seen in Fig. 7A, the analytical curve for the determination of SAL was constructed by fixing the PRO
Table 1 Comparison of analytical parameters in the determination of SAL and PRO at various modified electrodes. Analyte
Linear range/mol L−1
Electrode
SAL
MWNT-DHP/GCE Poly(AHNSA)/GCE SMWCNT-NF
8.0 2.0 1.0 3.0 3.3 9.0 7.9 6.0 2.0 5.0 7.4 1.0
Nano-Au/L-cys/MWNTs-NF/GCE AgNPs-IL-GO-NF/GCE Carbon paste BDD CuNPs-GO-CB-PEDOT:PSS/GCE MWCNTs–PAH/GCE AgNPs-IL-GO-NF/GCE
PRO
× × × × × × × × × × × ×
−7
10 10−7 10−7 10−7 10−6 10−8 10−8 10−7 10−6 10−7 10−8 10−7
LOD/mol L−1 −5
– 1.0 × 10 – 8.0 × 10−6 – 3.0 × 10−7 – 3.3 × 10−6 – 33.3 × 10−6 – 7.0× 10−6 − 2.9 × 10−6 – 5.0 × 10−5 – 4.1 × 10−5 – 2.9 × 10−6 – 6.3 × 10−7 – 2.9 × 10−6
Ref.
−7
2.0 × 10 6.8 × 10−8
25 36
1.0 × 10−7
39
5.0 1.3 2.0 9.3 1.8 2.6 1.7
× × × × × × ×
10−8 10−8 10−7 10−7 10−7 10−8 10−8
40 This work 41 42 43 44 This work
Table 2 Results obtained from analysis urine and serum samples. Samples
SAL
PRO
Urine A Urine B Serum A Serum B Urine A Urine B Serum A Serum B
Proposed method / μmol L−1
Comparative method / μmol L−1
Added
Found⁎
Found⁎
0.60 2.00 0.60 2.00 0.60 2.00 0.60 2.00
0.59 ± 0.03 1.92 ± 0.04 0.58 ± 0.03 1.97 ± 0.02 0.62 ± 0.03 2.01 ± 0.03 0.59 ± 0.05 2.07 ± 0.04
0.57 ± 0.02 1.97 ± 0.03 0.61 ± 0.01 2.05 ± 0.02 0.59 ± 0.02 2.11 ± 0.03 0.63 ± 0.04 1.96 ± 0.03
⁎
Average of 3 measured concentrations. Recovery percentage = [Found / Added] × 100. ⁎⁎⁎ RSD = [(Proposed method − Comparative method) / (Comparative method)] × 100. ⁎⁎
6
Recovery⁎⁎ (sensor, %)
RSD⁎⁎⁎ %
98 96 97 98 103 101 98 104
3.5 -2.5 -4.9 -3.9 5.1 -4.7 -6.3 5.6
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the simultaneous determination of these analytes, including high stability, reproducibility, repeatability and low cost.
(1994) 213–216. [2] S. Keir, C. Page, D. Spina, Bronchial hyperresponsiveness induced by chronic treatment with albuterol: role of sensory nerves, J. Allergy Clin. Immunol. 110 (2002) 388–394. [3] L.K.A. Lundblad, L.M. Rinaldi, M.E. Poynter, E.P. Riesenfeld, M. Wu, S. Aimi, L.M. Barone, J.H.T. Bates, C.G. Irvin, Detrimental effects of albuterol on airway responsiveness requires airway inflammation and is independent of β-receptor affinity in murine models of asthma, Respir. Res. 12 (2011) 27. [4] D.R. Jarvie, A.M. Thompson, E.H. Dyson, Laboratory and clinical features of selfpoisoning with salbutamol and terbutaline, Clin. Chim. Acta 168 (1987) 313–322. [5] S.M. Barkiya, V. Kumari, N. Venugopal, A. Aseez, Effects of aerosolized levosalbutamol verses salbutamol on serum potassium level and heart rate in children with acute exacerbation of asthma, Int. J. Sci. Study 3 (2016) 223–227. [6] A. Scriabine, B.V. Clineschmidt, C.S. Sweet, Central noradrenergic control of blood pressure, Annu. Rev. Pharmacol. Toxicol. 16 (1976) 113–123. [7] N. Tuross, R.L. Patrick, Effects of propranolol on catecholamine synthesis and uptake in the central-nervous-system of the rat, J. Pharmacol. Exp. Ther. 237 (1986) 739–745. [8] D.G. Shand, Pharmacokinetics of propranolol: a review, Postgrad. Med. J. 52 (Suppl 4) (1976) 22–25. [9] D.J. Spitz, An unusual death in an asthmatic patient, Am J Forensic Med Pathol 24 (2003) 271–272. [10] J.M. Fallowfield, H.F. Marlow, Propranolol is contraindicated in asthma, BMJ 313 (1996) 1486. [11] K. Albouaini, M. Andron, A. Alahmar, M. Egred, Beta-blockers use in patients with chronic obstructive pulmonary disease and concomitant cardiovascular conditions, Int. J. Chron. Obstruct. Pulmon. Dis. 2 (2007) 535–540. [12] N.A. Minton, A.R. Baird, J.A. Henry, Modulation of the effects of salbutamol by propranolol and atenolol, Eur. J. Clin. Pharmacol. 36 (1989) 449–453. [13] E.A. Ramoska, F. Henretig, M. Joffe, H.A. Spiller, Propranolol treatment of albuterol poisoning in 2 asthmatic-patients, Ann. Emerg. Med. 22 (1993) 1474–1476. [14] S. Küpeli, Use of propranolol for infantile hemangiomas, Pediatr. Hematol. Oncol. 29 (2012) 293–298. [15] D. Jack, D. Poynter, N.W. Spurling, Beta-adrenoceptor stimulants and mesovarian leiomyomas in the rat, Toxicology 27 (1983) 315–320. [16] A.M.A. Gader, J. da Costa, J.D. Cash, The effect of propranolol, alprenolol and practolol on the fibrinolytic and factor VIII responses to adrenaline and salbutamol in man, Thromb. Res. 4 (1974) 25–33. [17] C. Gopinath, W.A. Gibson, Mesovarian leiomyomas in the rat, Environ. Health Perspect. 73 (1987) 107–113. [18] S.S. Zhou, Y.Q. Wang, T. de Beer, W.R.G. Baeyens, G.T. Fei, M. Dilinuer, J. Ouyang, Simultaneous separation of eight beta-adrenergic drugs using titanium dioxide nanoparticles as additive in capillary electrophoresis, Electrophoresis 29 (2008) 2321–2329. [19] L. Liu, Y. Wen, K. Liu, L. Sun, Y. Lu, Z. Yin, Simultaneous determination of a broad range of cardiovascular drugs in plasma with a simple and efficient extraction/clean up procedure and chromatography-mass spectrometry analysis, RSC Adv. 4 (2014) 19629–19639. [20] A.M. Santos, F.C. Vicentini, P.B. Deroco, R.C. Rocha, O. Fatibello, Square-wave voltammetric determination of paracetamol and codeine in pharmaceutical and human body fluid samples using a cathodically pretreated boron-doped diamond electrode, J. Braz. Chem. Soc. 26 (2015) 2159–2168. [21] H. Parham, B. Zargar, Determination of isosorbide dinitrate in arterial plasma, synthetic serum and pharmaceutical formulations by linear sweep voltammetry on a gold electrode, Talanta 55 (2001) 255–262. [22] R. Ghavami, A. Navaee, Determination of nimesulide in human serum using a glassy carbon electrode modified with SiC nanoparticles, Microchim. Acta 176 (2012) 493–499. [23] A. Wong, A.M. Santos, O. Fatibello-Filho, Simultaneous determination of paracetamol and levofloxacin using a glassy carbon electrode modified with carbon black, silver nanoparticles and PEDOT:PSS film, Sensors Actuators B Chem. 255 (2018) 2264–2273. [24] A.M. Santos, A. Wong, A. Araújo Almeida, O. Fatibello-Filho, Simultaneous determination of paracetamol and ciprofloxacin in biological fluid samples using a glassy carbon electrode modified with graphene oxide and nickel oxide nanoparticles, Talanta 174 (2017) 610–618. [25] A. Wong, A.M. Santos, T.A. Silva, O. Fatibello-Filho, Simultaneous determination of isoproterenol, acetaminophen, folic acid, propranolol and caffeine using a sensor platform based on carbon black, graphene oxide, copper nanoparticles and PEDOT:PSS, Talanta 183 (2018) 329–338. [26] A. Wong, T.A. Silva, F.C. Vicentini, O. Fatibello-Filho, Electrochemical sensor based on graphene oxide and ionic liquid for ofloxacin determination at nanomolar levels, Talanta 161 (2016) 333–341. [27] M. Wang, Z.X. Zheng, J.J. Liu, C.M. Wang, Pt-Pd bimetallic nanoparticles decorated nanoporous graphene as a catalytic amplification platform for electrochemical detection of xanthine, Electroanalysis 29 (2017) 1258–1266. [28] M.E. Uddin, N.H. Kim, T. Kuila, S.H. Lee, D. Hui, J.H. Lee, Preparation of reduced graphene oxide-NiFe2O4 nanocomposites for the electrocatalytic oxidation of hydrazine, Compos. Part B Eng. 79 (2015) 649–659. [29] W.M. Si, W. Lei, Y.H. Zhang, M.Z. Xia, F.Y. Wang, Q.L. Hao, Electrodeposition of graphene oxide doped poly(3,4-ethylenedioxythiophene) film and its electrochemical sensing of catechol and hydroquinone, Electrochim. Acta 85 (2012) 295–301. [30] Y. Shao, J. Wang, H. Wu, J. Liu, I.A. Aksay, Y. Lin, Graphene based electrochemical sensors and biosensors: a review, Electroanalysis 22 (2010) 1027–1036. [31] Y. Yao, X. Chen, J. Zhu, B. Zeng, Z. Wu, X. Li, The effect of ambient humidity on the
3.7. Repeatability study The repeatability of the AgNP-IL-FG-NF/GCE sensor was evaluated by SWV in intra-day (n = 5) and inter-day (n = 3) analyses. For this study, two different of concentrations of SAL (0.30 and 2.0 μmol L−1) and PRO (0.60 and 2.0 μmol L−1) in a 0.20 mol L−1 phosphate buffer (pH 7.0) solution were evaluated. For the intra-day analysis, the procedure presented RSDs of 2.7% and 1.8% for SAL and 1.6% and 2.9% for PRO. Moreover, the inter-day measures presented RSDs of 4.3% and 3.1% for SAL and 2.8% and 3.6% for PRO, respectively. These results demonstrated that the proposed analytical method exhibits excellent precision in electrochemical measurements. 3.8. Simultaneous determination of SAL and PRO in biological fluid samples The AgNP-IL-FG-NF/GCE sensor was applied to the simultaneous determination of SAL and PRO in biological fluid (urine and serum) samples. For this, the serum and synthetic urine samples were initially analysed by SWV, and it was verified that no electrochemical reactions were identified in the potential range studied. Based on this, the samples were doped using two different known concentration levels of SAL and PRO (0.60 and 2.00 μmol L−1) and analysed with the proposed sensor using SWV. The recovery percentages obtained (n = 3) were in the range of 96 to 104% (Table 2), indicating that the matrixes analysed did not cause any interference. These samples were also analysed by a comparative method, and the results from both methods are also presented in Table 2. As can be seen, the relative errors ranged from −6.3% to +5.6%, demonstrating the agreement between the methods and the potential of the proposed procedure for the simultaneous determination of SAL and PRO in biological fluid samples. 4. Conclusions The present work showed that the used of the proposed AgNP-IL-FGNF/GCE sensor combined with SWV led to the development of a method with excellent detectability, selectivity and sensitivity for the simultaneous determination of SAL and PRO, offering an alternative electroanalytical procedure that is reliable and inexpensive. In addition, with the optimisation of the experimental parameters, linear analytical curves were obtained in the concentration ranges of 0.079 to 2.9 μmol L−1 for SAL, and of 0.10 to 2.9 μmol L−1 for PRO, with LODs of 13 nmol L−1 and 17 nmol L−1 for SAL and PRO, respectively. The applicability of the proposed sensor was demonstrated in the simultaneous determination of the drugs in serum and urine samples, with recoveries close to 100%. The method proposed is an analytical alternative for the simultaneous determination of SAL and PRO in different matrix samples. Acknowledgements The authors gratefully acknowledge the financial support granted by CNPq (Grant numbers 150162/2016-2, 160150/2015-9 and 444150/2014-5) and FAPESP (Grant number 2011/13312-5). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jelechem.2018.07.018. References [1] S.E. Libretto, A review of the toxicology of salbutamol (albuterol), Arch. Toxicol. 68
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A.M. Santos et al. electrical properties of graphene oxide films, Nanoscale Res. Lett. 7 (2012) 363. [32] B. Rafiee, A.R. Fakhari, Electrocatalytic oxidation and determination of insulin at nickel oxide nanoparticles-multiwalled carbon nanotube modified screen printed electrode, Biosens. Bioelectron. 46 (2013) 130–135. [33] N. Laube, B. Mohr, A. Hesse, Laser-probe-based investigation of the evolution of particle size distributions of calcium oxalate particles formed in artificial urines, J. Cryst. Growth 233 (2001) 367–374. [34] B.C. Iacob, I. Tiuca, E. Bodoki, R. Oprean, Multivariate calibration and modeling of uv-vis spectra of guest-host complexes for the determination of the enantiomeric ratio of propranolol, Farmacia 61 (2013) 79–87. [35] L. Kalyani, C.V.N. Rao, Simultaneous spectrophotometric estimation of salbutamol, theophylline and ambroxol three component tablet formulation using simultaneous equation methods, Karbala Int. J. Mod. Sci. 4 (2018) 171–179. [36] A.J. Bard, L.R. Faulkner, Electrochemical Methods: Fundamentals and Applications, John Wiley & Sons Inc, New York, 2001. [37] R.N. Goyal, M. Oyama, S.P. Singh, Fast determination of salbutamol, abused by athletes for doping, in pharmaceuticals and human biological fluids by square wave voltammetry, J. Electroanal. Chem. 611 (2007) 140–148. [38] Y. Wei, Q. Zhang, C. Shao, C. Li, L. Zhang, X. Li, Voltammetric Determination of Salbutamol on a Glassy Carbon Electrode Coated with a Nanomaterial Thin Film, 65 (2010), pp. 398–403. [39] N. Shadjou, M. Hasanzadeh, L. Saghatforoush, R. Mehdizadeh, A. Jouyban, Electrochemical behavior of atenolol, carvedilol and propranolol on copper-oxide
nanoparticles, Electrochim. Acta 58 (2011) 336–347. [40] I. Baranowska, M. Koper, Electrochemical behavior of propranolol and its major metabolites, 4′-hydroxypropranolol and 4′-hydroxypropranolol sulfate, on glassy carbon electrode, J. Braz. Chem. Soc. 22 (2011) 1601–1609. [41] M. Amare, G. Menkir, Differential pulse voltammetric determination of salbutamol sulfate in syrup pharmaceutical formulation using poly(4-amino-3-hydroxynaphthalene sulfonic acid) modified glassy carbon electrode, Heliyon 3 (2017) e00417. [42] K.-C. Lin, C.-P. Hong, S.-M. Chen, Simultaneous determination for toxic ractopamine and salbutamol in pork sample using hybrid carbon nanotubes, Sensors Actuators B Chem. 177 (2013) 428–436. [43] Y.F. Li, Z. Ye, P.L. Luo, Y. Li, B.X. Ye, A sensitive voltammetric sensor for salbutamol based on MWNTs composite nano-Au film modified electrode, Anal. Methods 6 (2014) 1928–1935. [44] A. Radi, A.A. Wassel, M.A. El Ries, Adsorptive behaviour and voltammetric analysis of propranolol at carbon paste electrode, Chem. Anal. 49 (2004) 51–58. [45] E.R. Sartori, R.A. Medeiros, R.C. Rocha-Filho, O. Fatibello-Filho, Square-wave voltammetric determination of propranolol and atenolol in pharmaceuticals using a boron-doped diamond electrode, Talanta 81 (2010) 1418–1424. [46] G.G. Oliveira, D.C. Azzi, F.C. Vicentini, E.R. Sartori, O. Fatibello-Filho, Voltammetric determination of verapamil and propranolol using a glassy carbon electrode modified with functionalized multiwalled carbon nanotubes within a poly (allylamine hydrochloride) film, J. Electroanal. Chem. 708 (2013) 73–79.
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Simultaneous determination of salbutamol and propranolol in biological fluid samples using an electrochemical sensor based on functionalizedgraphene, ionic liquid and silver nanoparticles Anderson Martin Santos, Ademar Wong, Orlando Fatibello-Filho
T
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Department of Chemistry, Federal University of São Carlos, 13560-970 São Carlos, SP, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Functionalized-graphene Simultaneous detection Salbutamol Propranolol, ionic liquid Silver nanoparticles
A simple, rapid, and low-cost electroanalytical method was developed using a glassy carbon electrode modified with functionalized-graphene (FG), ionic liquid (IL) and silver nanoparticles (AgNPs) for simultaneous detection of salbutamol (SAL) and propranolol (PRO). The electrochemical behaviour of the electrodes was investigated by cyclic voltammetry and square wave voltammetry (SWV) under optimised conditions. Using SWV, the AgNP-ILFG-NF/GCE sensor showed a linear response from 79 nmol L−1 to 2.9 μmol L−1 for SAL and from 0.1 to 2.9 μmol L−1 for PRO with limits of detection of 13 and 17 nmol L−1 for SAL and PRO, respectively. The proposed sensor showed good stability, repeatability and reproducibility, and was applied successfully to the simultaneous determination of SAL and PRO in biological fluid samples. The proposed method proved to be excellent, being therefore a reliable alternative method for the simultaneous determination of SAL and PRO.
1. Introduction Salbutamol (SAL) also known as albuterol ((RS)-4-[2-(tert-butylamino)-1-hydroxyethyl]-2-(hydroxymethyl)phenol – Fig. 1A) is a drug widely used in the treatment of asthma, chronic pulmonary disease and in the control of blood potassium levels. SAL can cause side effects, such as headache, dizziness, shakiness, and fast heart rate [1–3], and if administered in excess or if consumed improperly can cause serious health problems, including aggravating bronchospasm, irregular heartbeat, and low blood potassium levels [1, 4, 5]. Propranolol (PRO) (1-isopropylamino-3-(naphthalen-1-yloxy) propan-2-ol – Fig. 1B) is a beta-adrenergic blocking agent [6–8]. This drug is most frequently prescribed for the treatment of high blood pressure, chronic angina pectoris, prophylaxis and treatment of cardiac arrhythmias, prophylaxis of myocardial reinfarction and treatment of tremors [7, 8]. PRO may cause adverse reactions, such as congestive heart failure, aggravation of atrioventricular conduction disorders, bronchospasm, severe bradycardia and hypotension [7, 8]. Although the simultaneous use of these drugs (SAL and PRO) is not common because PRO is a non-cardioselective beta-blocking agent and the risk of its use together with SAL may outweigh the benefits for patients with asthma, their consumption should be avoided or supervised by a doctor [9–11]. In the literature, some cases of symptomatic overdose with SAL are reported, where PRO was used as an ⁎
antidote and not as an anti-asthmatic drug [12]. Ramoska et al. [13] reported the use of PRO in the treatment of SAL poisoning in two asthmatic patients, in which case PRO was used to minimise the effect caused by SAL. In another study, Kupeli [14] reported the use of PRO for infantile haemangiomas, but only 3 out of 14 patients presented bronchospasm and were treated with SAL. Thus, even though SAL and PRO are not found in together in pharmaceutical formulations, they can be co-administered in clinical treatments [12, 13] and in other applications [15–17]. Therefore, the simultaneous determination of SAL and PRO in biological fluids is very important for physiological pharmacokinetics and clinical diagnosis [12, 13]. In view of the applicability of these drugs in extreme cases, whether due to misuse or in poisoning, their quantification in biological fluid samples is of paramount importance. In the literature, some procedures have been reported for the simultaneous determination of these classes of drugs [18, 19]. Among these analytical procedures, electroanalytical techniques have shown great advantages compared to other traditional techniques such as chromatography and spectrophotometry in the analysis of biological fluid samples [20–22]. Their advantages are greater simplicity, analysis in real time, low cost and short analysis time. Considering the necessity for increasingly sensitive and selective electrochemical sensors, an analytical method that has been highlighted is the use of modifiers immobilised on conventional electrode surfaces, such as carbon nanostructures, e.g. carbon black, carbon nanotubes and graphene [23,
Corresponding author. E-mail address: [email protected] (O. Fatibello-Filho).
https://doi.org/10.1016/j.jelechem.2018.07.018 Received 3 May 2018; Received in revised form 23 June 2018; Accepted 12 July 2018 Available online 21 July 2018 1572-6657/ © 2018 Elsevier B.V. All rights reserved.
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Fig. 1. Chemical structures of (A) salbutamol and (B) propranolol.
treatment using a concentrated solution of 1:1 (v/v) H2SO4/HNO3 and, this suspension was stirred for 12 h at 25 °C. The obtained FG suspension was filtered and washed with ultrapure water to about pH 7.0 and dried at 100 °C.
24]; polymers, e.g. PDDA, chitosan and nafion (NF) [24, 25]; ionic liquids (ILs) [26]; and metallic nanoparticles, e.g. Au, Cu, Pt and Ag [23, 27], which can be used to improve the performance of the sensor and induce catalytic and/or electrocatalytic effects [28–30]. Functionalised carbon nanomaterials such as functionalized-graphene (FG) have shown to be been promising due to their properties of better dispersibility in aqueous media, which facilitates their use in the preparation of films, and also that they interact with other modifiers, thus increasing the performance of electrochemical sensors for the determination of analytes of interest [30, 31]. In addition, the use of metallic nanoparticles incorporated on the surface of an electrode can provide several advantages such as catalytic effects and greater effective sensor surface area [23, 32]. In the present work, we prepared and characterised a glassy carbon electrode (GCE) modified with FG, IL (1-butyl-3-methylimidazolium tetrafluoroborate) and silver nanoparticles (AgNPs) for the simultaneous determination of SAL and PRO in biological fluid samples.
2.4. Synthesis of the AgNPs Preparation of the AgNPs was performed as previously reported by Wong et al. [23]. Initially, solutions of 5.0 × 10−3 mol L−1 NaBH4 and 1.0 × 10−4 mol L−1 AgNO3 were prepared. Then, 1 mL of the NaBH4 solution and 49 mL of the AgNO3 solution were transferred individually to two flasks, and left in an ice bath for 30 min. Finally, the NaBH4 solution was added dropwise under stirring, thereby forming a yellowish colloidal suspension, which was stirred for 30 min and stored in an amber flask.
2.5. Preparation of the AgNP-IL-FG-NF/GCE sensor
2. Experimental
The surface of the GCE was initially cleaned with a polishing cloth and 0.5 μm alumina slurry, followed by ultrasonic cleaning with ethyl alcohol and ultrapure water for 2 min. Under optimised conditions, the dispersion of AgNP-IL-FG-NF was prepared using 1.0 mg of FG, 1.0 mg of IL (1-butyl-3-methylimidazolium tetrafluoroborate), 10 μL of 0.25% (v/v) NF solution, 250 μL of AgNPs, and 740 μL of ultrapure water. The reagents were subjected to ultrasonic agitation for 40 min in order to obtain a homogeneous dispersion. Next, an 8 μL aliquot of the dispersion was dropped onto the GCE surface and dried at room temperature for 3 h, to obtain the GCE modified with AgNP-IL-FG-NF film.
2.1. Reagents and solutions SAL, PRO, bovine serum, nafion (NF) and 1-butyl-3-methylimidazolium tetrafluoroborate (IL) standards were purchased from SigmaAldrich (São Paulo, Brazil); NaOH, reagents for phosphate buffer (H3PO4, KH2PO4, K2HPO4, and K3PO4) and KCl were acquired from Acros. Graphene (GR) was acquired from Graphene Supermarket (New York, USA). The solutions were prepared using ultrapure water with resistivity not less than 18 MΩ cm obtained from a Millipore Milli-Q system (Billerica, USA). Stock solutions of SAL and PRO, both at 0.1 mol L−1 were prepared directly in ultrapure water.
2.6. Apparatus and reference method 2.2. Preparation of urine and serum samples Electrochemical experiments were performed by employing a Metrohm/Eco Chemie Autolab PGSTAT12 potentiostat/galvanostat electrochemical system controlled by GPES 4.9. Voltammetric measurements were carried out in a three-electrode cell, with an Ag/AgCl (3.0 mol L−1 KCl) electrode as the reference electrode, a platinum foil (0.5 cm2) as the auxiliary electrode and a GCE (Ø = 3 mm) or an AgNPIL-FG-NF/GCE working electrode. The morphological characterisations of the FG and AgNPs were evaluated by images acquired by field emission gun scanning electron microscopy (FEG/SEM, FEI Magellan 400 L). The film thickness on the electrode surface was evaluated using a Hirox, model KH-7700 digital microscope. UV–Vis spectrophotometer (Shimadzu model UV 2550) with a quartz cuvette (optical path length of 10 mm) was used as comparative spectrophotometric method [34, 35].
Synthetic urine samples were prepared as previously reported by Laube et al. [33] with the addition of 0.20 mmol L−1 KCl, 0.18 mmol L−1 NH4Cl, 0.10 mmol L−1 NaCl, 0.10 mmol L−1 CaCl2, 0.15 mmol L−1 KH2PO4 and 0.18 mmol L−1 urea in a 25 mL volumetric flask. The serum (bovine serum) samples were diluted 10 times with ultrapure water, and these samples were evaluated at two known concentrations of SAL and PRO (0.60 and 2.0 μmol L−1, respectively), and analysed by recovery percentage. 2.3. Synthesis of the FG Synthesis of the FG was performed as previously reported by Santos et al. [24]. In short, 100 mg of graphene was submitted to chemical 2
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2.7. Analytical procedure
Ip = ± (2.69 × 105) n3/2 A D1/2C v1/2
First, the proportions of nanomaterial (FG), metallic nanoparticles (AgNPs) and ionic liquid (IL) used to obtain the film in the GCE were investigated. After this step, the electrochemical characterisations of the electrodes were performed by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Based in the results, the electrochemical behaviour of SAL and PRO were analysed using CV, followed by the optimisation of the experimental conditions, such as supporting electrolyte (composition and pH), and the instrumental parameters for SWV (amplitude, frequency and increment). Next, calibration curves were constructed using successive additions of SAL and PRO standard solutions. All measurements were carried out in triplicate (n = 3), and the SAL and PRO concentrations were determined simultaneously under optimised conditions. The limit of detection (LOD) was calculated as three times the standard deviation for the blank solution (n = 10) divided by the slope of the analytical curve. The precision of the proposed method was verified from intra-day (n = 5) and inter-day (n = 3) repeatability studies.
where Ip is the peak current (A), n the number of electrons transferred, A the electroactive area (cm2), D the diffusion coefficient of [Fe (CN)6]3− in a 0.10 mol L−1 KCl solution (7.6 × 10−6 cm2 s−1), C the [Fe(CN)6]3− concentration (mol cm−3) and v the potential scan rate (V s−1). The obtained slopes of the Ip vs. v1/2 plots for the [Fe (CN)6]3− oxidation process were 2.17 × 10−5 A V−1/2 s1/2 for the GCE, 4.53 × 10−5 A V−1/2 s1/2 for the FG-NF/GCE, 7.16 × 10−5 A V−1/2 s1/ 2 for the IL-FG-NF/GCE, 7.72 × 10−5 A V−1/2 s1/2 for the AgNP-FG-NF/ GCE and 9.40 × 10−5 A V−1/2 s1/2 for the AgNP-IL-FG-NF/GCE. The corresponding electroactive areas were 0.065, 0.14, 0.21, 0.23 and 0.28 cm2 for the GCE, FG-NF/GCE, IL-FG-NF/GCE, AgNP-FG-NF/GCE and AgNP-IL-FG-NF/GCE, respectively. Thus, from these results is possible to conclude that the combination of the AgNP-IL-FG-NF/GCE sensor resulted in a 4.3-fold increase in electroactive area compared to GCE. In addition, EIS was used to characterise the electrodes in experiments using 2.5 mmol L−1 [Fe(CN)6]3−/4– as redox probe in 0.10 mol L−1 KCl solution, and a frequency range 100 kHz at 0.1 Hz. The curves generally had two parts, a linear part at lower frequencies and a semicircular part at higher frequencies. This semicircle corresponds to the limited transfer of electrons, and its diameter is equal the charge transfer resistance (Rct). Therefore, the larger the size of the semicircle, the greater the Rct. Fig. 3 shows the Nyquist curves obtained for the different electrodes, CGE, FG-NF/GCE, IL-FG-NF/GCE, AgNPFG-NF/GCE and AgNP-IL-FG-NF/GCE, where the values of Rct were 1054, 668, 242, 222 and 95 Ω, respectively. Thus, by analysing the results it is possible to observe a large semicircle for the CGE, which implies a high Rct. With the progressive modification of the electrode (FG-NF/GCE < IL-FG-NF/GCE < AgNP-FG-NF/GCE < AgNP-IL-FGNF/GCE), there was a decrease in the size of the semicircle, so indicating an increase in conductivity of the electrode and a reduction in Rct. The EIS was also used to calculate the standard heterogeneous rate constant (k0) for each modification of the electrode according to equation (Eq. 2):
3. Results and discussion Initially, to evaluate the effect of the functionalization of GR to FG, a dispersion of both materials in ultra-pure water was obtained by sonification. As can be seen in Fig. S1 (Supplementary Material), the oxidation of GR to FG allow the formation of a stable dispersion of FG due to its hydrophilic character (insertion of oxygenated functional groups into the material). Then, to obtain the film on the GCE, the amount of nanomaterial (FG) in the FG/solution ratios of 0.5:1.0, 1.0:1.0, 1.5:1.0 (mg/mL), IL in the IL/solution ratios of 0.5:1.0, 1.0:1.0, 1.5:1.0 (mg/mL) and the nanoparticles (AgNPs) in the AgNP/solution ratios of 0.25:0.75, 0.5:0.5 and 0.75:0.25 (mL/mL) were investigated. The highest current signals for the analytes (SAL and PRO) were obtained using 1.0 mg of FG, 1.0 mg of IL, 10 μL of NF, 250 μL of AgNPs, and 740 μL of ultrapure water. 3.1. Morphological characterisation of the materials
k0 =
The morphological characterisation of the electrode modification was performed by FEG-SEM. Fig. 2 shows the obtained images of (A) FG, (B) IL-FG and (C) AgNP-IL-FG, all on the surface of GCE. Fig. 2A shows that FG consists of thin wrinkled sheet structures disposed in stacked blocks of different sizes, which is characteristic of this type of material. It can also be seen in Fig. 2B, where the surface morphology of FG with IL presented a small change, where the graphene sheets were arranged more closely together and homogeneous. Finally, the morphology of FG in presence of AgNPs is presented in Fig. 2C, in which it is possible to observe highly dispersed multiple grains of AgNPs (white spheres) on the FG sheets with a average diameter of 20 nm, as shown in the histogram of Fig. 2D. The thickness of the film (AgNP-IL-FG-NF) was estimated using a digital microscope, where it was possible to visualize a 3D image of these materials on the GCE surface. In Fig. S2 (Supplementary material) is shown the surface aspect of film which presented an average film thickness of 3.725 μm.
RT F 2R ct AC
(1)
(2)
where k0 is the standard heterogeneous electron transfer rate constant (cm s−1), R the universal gas constant (8.314 J K−1 mol−1), T the thermodynamic temperature (298.15 K), F the Faraday constant (96,485C mol−1), Rct is the electron transfer resistance (Ω), A the electrode surface area (cm2) and C the concentration of the [Fe (CN)6]3−/4– solution (2.5 mmol cm−3). The obtained k0 values for the CGE, FG-NF/GCE, IL-FG-NF/GCE, AgNP-FG-NF/GCE and AgNP-IL-FG-NF/GCE were 1.4 × 10−3, 2.3 × 10−3, 6.3 × 10−3, 6.8 × 10−3 and 1.6 × 10−2 cm s−1, respectively. Considering that the k0 values are a measure of the kinetic facility of the redox pair, in which a system with high k0 value will achieve equilibrium in a shorter time than a system with low k0 value, which in this case will achieve equilibrium in a longer time. Thus, with the AgNP-IL-FG-NF/GCE sensor, the k0 value is greater than with AgNPFG-NF/GCE > IL-FG-NF/GCE > FG-NF/GCE > CGE, indicating in this case, a faster transfer of electrons in comparison with the other electrodes.
3.2. Electrochemical characterisation of electrodes 3.3. Electrochemical behaviour of SAL and PRO Initially, the determination of electroactive area of the working electrodes (GCE; FG-NF/GCE; IL-FG-NF/GCE; AgNP-FG-NF/GCE and AgNP-IL-FG-NF/GCE) were estimated by the Randles–Sevcik [36] equation (Eq. 1) in CV experiments at a potential scan rate range of 10–400 mV using a 0.5 mmol L−1 [Fe(CN)6]3− solution in aqueous 0.10 mol L−1 KCl (see Fig. S3 in Supplementary material).
For this study, CV was used to evaluate the electrochemical behaviour of the SAL (0.20 mmol L−1) and PRO (0.20 mmol L−1) solutions, using the different electrodes (CGE, FG-NF/GCE, IL-FG-NF/GCE, AgNPFG-NF/GCE and AgNP-IL-FG-NF/GCE) in a potential range of 0.05–1.25 V (v = 50 mV s−1) and 0.20 mol L−1 phosphate buffer 3
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Fig. 2. SEM image of (A) FG, (B) IL-FG, (C) AgNP-IL-FG and (D) corresponding histogram of AgNPs diameters.
Fig. 4. Cyclic voltammograms for 0.20 mmol L−1 SAL and 0.20 mmol L−1 PRO in 0.20 mol L−1 phosphate buffer solution (pH 7.0) at a v = 50 mV s−1. Fig. 3. EIS diagrams for 2.5 mmol L−1 [Fe(CN)6]3−/4– in 0.10 mol L−1 KCl. Frequency range 100 KHz to 0.1 Hz.
3.4. Optimisation of analytical parameters As can be seen in Fig. 5, the influence of the effect of hydrogen ion concentration on the oxidation reactions of the SAL and PRO (0.10 mmol L−1) were investigated by SWV using 0.20 mol L−1 phosphate buffer solutions with pH ranging between 5.0 and 9.0. The optimum pH of the supporting electrolyte found was 7.0, and this pH was selected for further studies. The graphic inserted in Fig. 5 represents the dependency of peak potential (Ep) with the pH for both SAL and PRO. The Ep value for SAL electro-oxidation varied linearly with pH with a slope of −0.057 V pH −1. This slope is close to the theoretical Nernstian slope of −0.0592 V pH −1 typical for redox reactions involving the same number of electrons and protons. This result is in accordance with previous works, which reported the electro-oxidation of SAL involving the loss of one electron and one proton [37, 38]. On the other hand, although the Ep value for the PRO electro-oxidation varied linearly with
solution (pH 7.0). As can be observed in Fig. 4, the cyclic voltammograms obtained for both drugs showed well-defined oxidation peaks at potentials of 0.65 and 1.0 V for SAL and PRO, respectively. In addition, the absence of reduction peaks during the cathodic potential scanning for SAL and PRO characterised an irreversible oxidation reaction. That is, there is an irreversible process of charge transfer for both compounds in the potential range employed. As observed in Fig. 4, the final modification (AgNP-IL-FG-NF/GCE), compared with the other modifications showed higher oxidation peak current (Ipox) values for both drugs, where Ipox is increased by factors of up to 2.4 and 3.2 for SAL and PRO, respectively. Additionally, there was a displacement of the oxidation peak potential to less positive potentials, caused mainly by an electrocatalytic effect of SAL and PRO oxidation brought on by modification of the GCE surface. 4
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electrons and two protons [39, 40]. The effect of different compositions of supporting electrolytes (pH 7.0), such as PIPES, BR and phosphate buffers was tested. The phosphate buffer solution provided the highest peak current in the SWV experiments and was selected for the electrochemical studies. Next, the parameters that affected the SWV response – frequency (f), amplitude (a) and potential increment (ΔEs) – were optimised. The optimum values obtained for these parameters using 0.10 mmol L−1 SAL and PRO in 0.20 mol L−1 phosphate buffer (pH 7.0) solution with a AgNP-IL-FG-NF/GCE sensor, were f = 30 Hz, a = 50 mV and ΔEs = 9 mV (see Table S1). 3.5. Effect of potential scan rate The effect of potential scan rate on the electrochemical behaviour of the AgNP-IL-FG-NF/GCE sensor was investigated by CV, ranging the scan rate from 10 to 400 mV s−1 in the presence of 0.20 mmol L−1 of SAL or PRO and 0.20 mol L−1 phosphate buffer solution (pH 7.0). As shown in Fig. 6A and B, with increasing potential scan rate, a linear increase in the peak current was verified for both drugs. Furthermore, the results showed good linearity of the plots of peak current versus the square root of the scan rate (ΔIpa vs. v1/2) (graphic (i) inserted in Fig. 6A and B), indicating that the mass transport of SAL and PRO on the electrode surface occurred by a diffusion processes. This process was proved by plotting the logarithm of the anodic peak current versus the
Fig. 5. SW voltammograms recorded for 0.10 mmol L−1 SAL and PRO in 0.20 mol L−1 phosphate buffer solutions with pH ranging of 5.0 to 9.0 using the AgNP-IL-FG-NF/GCE sensor. Parameters: f = 70 Hz, a = 50 mV ΔEs = 4 mV s−1. Insets: graphics of Ep vs. pH.
the pH, a slope of −0.028 V pH −1 was obtained. This slope is also in agreement with the theoretical Nernstian slope of −0.029 V pH −1 for redox reactions involving different numbers of electrons and protons in a ratio of 2:1, that is, the number of electrons is twice the number of protons. These results were also verified recently in another work reported by the group [25]. However, more studies in the literature have reported the electro-oxidation of PRO involving the transfer of two
Fig. 7. SW voltammograms obtained using the AgNP-IL-FG-NF/GCE sensor in 0.20 mol L−1 phosphate buffer (pH 7.0) containing: (A) 1.0 μmol L−1 of PRO and different concentrations of SAL: 0.079; 0.30; 0.59; 0.97; 1.9 and 2.9 μmol L−1, (B) 2.0 μmol L−1 of SAL and different concentrations PRO: 0.10; 0.30; 0.59; 0.97; 1.9 and 2.9 μmol L−1. Analytical curves (inserted).
Fig. 6. Cyclic voltammograms for different scan rates (10–400 mV s−1) using the AgNP-IL-FG-NF/GCE sensor, for (A) 0.20 mmol L−1 SAL and (B) 0.20 mmol L−1 PRO in 0.20 mol L−1 phosphate buffer solution (pH 7.0). Graphics of (i) Ip vs. v1/2 and (ii) log Ip vs. log v. 5
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concentration at 1.0 μmol L−1 and varying the SAL concentration from 0.079 to 2.9 μmol L−1. Therefore, from the obtained SW voltammograms (Fig. 7A), the value of Ipa for SAL increased linearly with its concentration (r2 = 0.997), while the value of Ipa for PRO remained constant, with a relative standard deviation (RSD) of 2.7%. Similarly, when the concentration of SAL was fixed at 2.0 μmol L−1, and that of PRO was varied in the range of 0.10 to 2.9 μmol L−1 (Fig. 7B), the value of Ipa for PRO increased progressively with its concentration (r2 = 0.998), while the value of Ipa for SAL remained constant (RSD = 3.3%). From these results it can be concluded that neither drug (SAL or PRO) interferes in the simultaneous determination of one in the presence of the other. Finally, the analytical curve for the simultaneous determination of these drugs was performed with the AgNP-IL-FG-NF/GCE sensor by SWV using different concentrations of SAL and PRO. As can be seen in the voltammograms shown in Fig. 8, a proportional increase of the anodic peak current was obtained with the increase of analytes concentrations in the electrochemical cell. The analytical curves were linear in the concentrations ranges from 0.079 to 2.9 μmol L−1 for SAL and, from 0.10 to 2.9 μmol L−1 for PRO, with a LOD of 13 nmol L−1 and 17 nmol L−1 for SAL and PRO, respectively. The graphs of Fig. 8 (i) and (ii) correspond to the analytical curves obtained, conforming to the following Eqs. (3) and (4):
Fig. 8. (A) SW voltammograms obtained using the AgNP-IL-FG-NF/GCE sensor in 0.20 mol L−1 phosphate buffer (pH 7.0) containing different concentrations of SAL: (a) 0.079; (b) 0.30, (c) 0.59, (d) 0.97, (e) 1.9, (f) 2.9 μmol L−1. PRO: (g) 0.10; (h) 0.30, (i) 0.59, (j) 0.97, (k) 1.9 and (l) 2.9 μmol L−1. Analytical curves from SAL (i) and PRO (ii).
logarithm of the scan rate (log ΔIp vs. log v), which showed slopes of 0.45 and 0.56 for SAL and PRO, respectively (see the inset (ii) of Fig. 6A and B). As can be observed, these slope values are around the theoretical value of 0.5, characteristic of a system controlled by diffusion.
3.6. Simultaneous determination of SAL and PRO by SWV
Ipa /A = −1.85 × 10−7 + 3.94 [SAL/(mol L−1)] (r 2 = 0.997)
(3)
Ipa /A = −6.42 × 10−7 + 6.82 [PRO/(mol L−1)] (r 2 = 0.992)
(4)
The analytical parameters of the proposed sensor were compared with those previously reported in the literature [25, 38, 41–46] (see Table 1), and it was possible to see clearly that the use of the AgNP-FGNF/GCE sensor led to excellent linear concentration ranges and LODs for SAL and PRO, being similar to, or better than, those reported by other authors. In addition, the sensor proposed here is the first to report
Firstly, to confirm the possibility of simultaneous determination of SAL and PRO by SWV, analytical curves of one analyte in the presence of the other were obtained. As can be seen in Fig. 7A, the analytical curve for the determination of SAL was constructed by fixing the PRO
Table 1 Comparison of analytical parameters in the determination of SAL and PRO at various modified electrodes. Analyte
Linear range/mol L−1
Electrode
SAL
MWNT-DHP/GCE Poly(AHNSA)/GCE SMWCNT-NF
8.0 2.0 1.0 3.0 3.3 9.0 7.9 6.0 2.0 5.0 7.4 1.0
Nano-Au/L-cys/MWNTs-NF/GCE AgNPs-IL-GO-NF/GCE Carbon paste BDD CuNPs-GO-CB-PEDOT:PSS/GCE MWCNTs–PAH/GCE AgNPs-IL-GO-NF/GCE
PRO
× × × × × × × × × × × ×
−7
10 10−7 10−7 10−7 10−6 10−8 10−8 10−7 10−6 10−7 10−8 10−7
LOD/mol L−1 −5
– 1.0 × 10 – 8.0 × 10−6 – 3.0 × 10−7 – 3.3 × 10−6 – 33.3 × 10−6 – 7.0× 10−6 − 2.9 × 10−6 – 5.0 × 10−5 – 4.1 × 10−5 – 2.9 × 10−6 – 6.3 × 10−7 – 2.9 × 10−6
Ref.
−7
2.0 × 10 6.8 × 10−8
25 36
1.0 × 10−7
39
5.0 1.3 2.0 9.3 1.8 2.6 1.7
× × × × × × ×
10−8 10−8 10−7 10−7 10−7 10−8 10−8
40 This work 41 42 43 44 This work
Table 2 Results obtained from analysis urine and serum samples. Samples
SAL
PRO
Urine A Urine B Serum A Serum B Urine A Urine B Serum A Serum B
Proposed method / μmol L−1
Comparative method / μmol L−1
Added
Found⁎
Found⁎
0.60 2.00 0.60 2.00 0.60 2.00 0.60 2.00
0.59 ± 0.03 1.92 ± 0.04 0.58 ± 0.03 1.97 ± 0.02 0.62 ± 0.03 2.01 ± 0.03 0.59 ± 0.05 2.07 ± 0.04
0.57 ± 0.02 1.97 ± 0.03 0.61 ± 0.01 2.05 ± 0.02 0.59 ± 0.02 2.11 ± 0.03 0.63 ± 0.04 1.96 ± 0.03
⁎
Average of 3 measured concentrations. Recovery percentage = [Found / Added] × 100. ⁎⁎⁎ RSD = [(Proposed method − Comparative method) / (Comparative method)] × 100. ⁎⁎
6
Recovery⁎⁎ (sensor, %)
RSD⁎⁎⁎ %
98 96 97 98 103 101 98 104
3.5 -2.5 -4.9 -3.9 5.1 -4.7 -6.3 5.6
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the simultaneous determination of these analytes, including high stability, reproducibility, repeatability and low cost.
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3.7. Repeatability study The repeatability of the AgNP-IL-FG-NF/GCE sensor was evaluated by SWV in intra-day (n = 5) and inter-day (n = 3) analyses. For this study, two different of concentrations of SAL (0.30 and 2.0 μmol L−1) and PRO (0.60 and 2.0 μmol L−1) in a 0.20 mol L−1 phosphate buffer (pH 7.0) solution were evaluated. For the intra-day analysis, the procedure presented RSDs of 2.7% and 1.8% for SAL and 1.6% and 2.9% for PRO. Moreover, the inter-day measures presented RSDs of 4.3% and 3.1% for SAL and 2.8% and 3.6% for PRO, respectively. These results demonstrated that the proposed analytical method exhibits excellent precision in electrochemical measurements. 3.8. Simultaneous determination of SAL and PRO in biological fluid samples The AgNP-IL-FG-NF/GCE sensor was applied to the simultaneous determination of SAL and PRO in biological fluid (urine and serum) samples. For this, the serum and synthetic urine samples were initially analysed by SWV, and it was verified that no electrochemical reactions were identified in the potential range studied. Based on this, the samples were doped using two different known concentration levels of SAL and PRO (0.60 and 2.00 μmol L−1) and analysed with the proposed sensor using SWV. The recovery percentages obtained (n = 3) were in the range of 96 to 104% (Table 2), indicating that the matrixes analysed did not cause any interference. These samples were also analysed by a comparative method, and the results from both methods are also presented in Table 2. As can be seen, the relative errors ranged from −6.3% to +5.6%, demonstrating the agreement between the methods and the potential of the proposed procedure for the simultaneous determination of SAL and PRO in biological fluid samples. 4. Conclusions The present work showed that the used of the proposed AgNP-IL-FGNF/GCE sensor combined with SWV led to the development of a method with excellent detectability, selectivity and sensitivity for the simultaneous determination of SAL and PRO, offering an alternative electroanalytical procedure that is reliable and inexpensive. In addition, with the optimisation of the experimental parameters, linear analytical curves were obtained in the concentration ranges of 0.079 to 2.9 μmol L−1 for SAL, and of 0.10 to 2.9 μmol L−1 for PRO, with LODs of 13 nmol L−1 and 17 nmol L−1 for SAL and PRO, respectively. The applicability of the proposed sensor was demonstrated in the simultaneous determination of the drugs in serum and urine samples, with recoveries close to 100%. The method proposed is an analytical alternative for the simultaneous determination of SAL and PRO in different matrix samples. Acknowledgements The authors gratefully acknowledge the financial support granted by CNPq (Grant numbers 150162/2016-2, 160150/2015-9 and 444150/2014-5) and FAPESP (Grant number 2011/13312-5). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jelechem.2018.07.018. References [1] S.E. Libretto, A review of the toxicology of salbutamol (albuterol), Arch. Toxicol. 68
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