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A novel electrochemical method for the detection of oxymetazoline drug based on MWCNTs and TiO2 nanoparticles
Journal of Electroanalytical Chemistry 844 (2019) 58–65
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A novel electrochemical method for the detection of oxymetazoline drug based on MWCNTs and TiO2 nanoparticles Azeema Munira,b, Burcin Bozal-Palabiyikb, Amjad Khanc, Afzal Shaha,d, Bengi Uslub,
T
⁎
a
Department of Chemistry, Quaid-i-Azam University, 45320 Islamabad, Pakistan Department of Analytical Chemistry, Faculty of Pharmacy, Ankara University, 06560, Ankara, Turkey c Barking, Havering and Redbridge University Hospitals NHS Trust, London, UK d Department of Chemistry, College of Science, University of Bahrain, Sakhir 32038, Bahrain b
ARTICLE INFO
ABSTRACT
Keywords: Oxymetazoline Carbon nanotubes TiO2 nanoparticles Electrochemistry Voltammetry
Oxymetazoline, a nasal decongestant drug was investigated by a sensitive, simple and reproducible electrochemical method using Titania (TiO2) nanoparticles and carboxyl group functionalized multi-walled carbon nanotubes fabricated on glassy carbon electrode using cyclic voltammetry and differential pulse voltammetry. The possible electro-oxidation mechanism of this drug was also studied in detail. The performance of designed method was determined by electrochemical impedance spectroscopy, cyclic voltammetry and anodic stripping differential pulse voltammetry. The obtained results revealed enhanced electrochemical behavior of oxymetazoline as compared to bare glassy carbon electrode. Under optimum conditions i.e. buffer pH; pH 7.0 phosphate buffer solution, accumulation potential; 0 V, and accumulation time; 180 s, the differential pulse voltammetric response of oxymetazoline was probed between the linear concentration range of 0.12 μM to 1.5 μM in aqueous medium containing pH 7.0 PBS and 20% methanol. The limit of detection of oxymetazoline was evaluated as 4.40 nM. Moreover, the developed sensing method was validated for its practical application in nasal spray with excellent recovery.
1. Introduction Topical nasal decongestants are drugs that react immediately to cure nasal congestion i.e. allergic rhinitis, common cold and sinusitis. Generally, they include inhalers, drops and sprays. They work by constriction of nasal arterioles leading to ease in drainage of secretions in nasal mucous membrane, thus resulting in elimination of nasal congestion. They can be classified into two major groups: sympathomimetic amines (cocaine, amphetamine, adrenaline, ephedrine) and imidazolines (oxymetazoline and xylometazoline) [1]. One member of imidazoline i.e. oxymetazoline (OMZ), (6-tert-butyl-3-(2-imidazolin-2ylmethyl)-2,4-dimethyl-phenol) has been used as nasal vasoconstrictor for more than forty years by influencing the α-adrenoceptor (an agonist material used as nasal mucosa decongestant) [2,3]. The use of this drug does not cause severe toxicity rather than mild side effects at cardiovascular or/and central nerve systems caused by systemic absorption of excess dose of unprescribed OMZ drug from nasal mucosa [2]. Literature survey of OMZ reveals that the determination of OMZ and its oxidation degradants has previously been performed by flow injection analysis with chemiluminescence detector [4], high performance
⁎
liquid chromatography [5] and liquid chromatography/mass spectrometry method [6]. While, no report is available about the electrochemical analysis of OMZ from pharmaceutical formulations. The electrochemical techniques are considered as efficient methods for the sensitive and selective determination of drugs as well as their redox properties. Moreover, their low cost and small size of the instrument makes them alternative classical analytical techniques for providing rapid and sensitive determination of desired organic compounds [7–10]. Therefore, in the present work electrochemical techniques were used for OMZ analysis. For the electrochemical sensing of redox components at the electrode surface, surface modifiers i.e. carbon structures, metallic nanoparticles and conductive polymers or combination of these materials are used to accelerate the transformation of irreversible oxidation processes to reversible processes due to rapid electron transfer rate. Among these nanoparticles are widely used for electrochemical analysis of various compounds owing to their distinguishing characteristics such as improved conductivity via mass transport, electro-catalytic effect, enhanced effective surface area and stability of electrode's chemical microenvironment [11]. The carbon nanomaterials are useful for
Corresponding author. E-mail address: [email protected] (B. Uslu).
https://doi.org/10.1016/j.jelechem.2019.05.017 Received 16 January 2019; Received in revised form 8 May 2019; Accepted 8 May 2019 Available online 10 May 2019 1572-6657/ © 2019 Elsevier B.V. All rights reserved.
Journal of Electroanalytical Chemistry 844 (2019) 58–65
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electrode modification owing to their large surface area, wide potential window and electrocatalytic effect [12]. Among carbon nanostructures, single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs) and graphene are extensively used to enhance the electrochemical response of analytes [13]. Similarly, metallic nanoparticles are used for the electroanalysis of electrochemical processes due to their unique physical and chemical properties. The electrodes modified with noble metallic nanoparticles show enhanced electrocatalytic effect as compared to bare electrodes. TiO2 nanoparticles find extensive use in the designing sensing devices due to their adsorption ability, chemical and thermal stability, cost affordability, bio-compatibility and environment-friendliness. Such nanomaterials can also be used as surface-charge regulators and adsorbents for modification of electrodes which allow the researcher to study at a wider potential range and improve the stability and reproducibility of the designed electrode [14,15]. Therefore, in the present study TiO2 nanoparticles were used in combination with -COOH functionalized MWCNTs for the modification of glassy carbon electrode to combine the sensing properties of both nanomaterials for the sensitive detection of OMZ from its pharmaceutical dosage form by anodic stripping differential pulse voltammetry (ASDPV).
(21 nm, Sigma Aldrich) was prepared in ultrapure water. 2.2. Preparation of modified TiO2NPs/fMWCNTs/GCE Homogenous and stable fMWCNTs suspension (0.5 mg mL−1) was prepared in DMF after sonication in ultrasonic bath for 2 h. The mechanical cleaning of GCE surface was performed with alumina slurry (0.05 μm) on a polishing pad followed by washing it with distilled water and drying in air. For the fabrication of electrode surface, 3 μL, 5 μL, 7 μL and 10 μL volume of TiO2NPs and fMWCNTs were loaded independently in order to check the distinct and the highest analytical response of OMZ. The optimum loading selected for TiO2NPs/ fMWCNTs/GCE preparation was 5 μL of both fMWCNTs and TiO2NPs. After each casting of drop, the electrode was dried at 40 °C in vacuum oven. 2.3. Nasal spray and recovery assay procedure In order to check the validity of the designed method for practical applications, 1 mM stock solution of OMZ was prepared in 20% methanol by using 7.5 μL of Iliadin® (containing 0.05% of OMZ) nasal spray. The working solutions were then prepared by diluting the stock solution with PBS (pH 7.0) and 20% methanol. This solution was subjected to electrochemical analysis. The nominal amount of OMZ in nasal spray was then calculated by corresponding regression equation of calibration curves. The recovery studies were also carried out to determine the accuracy of the developed method and to check the possible interferences from excipients present in the spray solution. For this purpose, a known amount of OMZ solution was added to pre-analyzed nasal spray solution. The recovery results were further investigated using calibration equation by DPV for five repeated measurements.
2. Experimental 2.1. Apparatus and reagents AUTOLAB-PGSTAT302 (Eco Chemie, Utrecht, The Netherlands) was used for electrochemical measurements with General Purpose Electrochemical Software (GPES) 4.9 using a conventional three electrode system in one compartment. In this system, glassy carbon electrode, Ag/AgCl electrode and platinum wire were used as working, reference and auxiliary electrodes, respectively. In differential pulse voltammetric (DPV) measurements: pulse amplitude, 0.0505 V; step potential, 0.00795 V; interval time, 0.500 s and modulation time, 0.050 s were used. The accumulation potential of 0 V and accumulation time of 180 s were further selected for adsorptive stripping differential pulse voltammetric analysis (ASDPV). While the pulse width of 0.010 V was adjusted for average baseline correction. The electrochemical impedance spectroscopy (EIS) was performed using AUTOLABPGSTAT100N (Eco Chemie, Utrecht, The Netherlands) electrochemical analyzer controlled by NOVA 2.1 software. For EIS measurements, the frequency range of 0.1 Hz to 100 kHz was used. While the amplitude and potential of AC voltage were selected as 10 mV and 0.0 V, respectively. Scanning electron microscopy (SEM) was performed by Zeiss Evo 40 instrument to characterize the fMWCNTs/GCE, TiO2NPs/GCE and TiO2NPs/fMWCNTs/GCE. For pH measurements 538 pH meter (WTW, Austria) was used. OMZ was kindly supplied from Santa Farma (Istanbul, Turkey) and its pharmaceutical dosage form (nasal spray) i.e. Iliadin® (0.05% of OMZ) was purchased from a local pharmacy. For the study of model compounds, naphazoline (NZ) and xylometazoline (XMZ) were also provided by Santa Farma (Istanbul, Turkey). Stock solutions of OMZ (2 × 10−3 M) were prepared in methanol and kept in refrigerator. For voltammetric experiments, working solutions of OMZ were prepared by dilution of a stock solution with the desired buffer solutions. The amount of methanol (20%) was kept constant in all working solutions. Acetate buffer solution (pH 3.7–5.7), phosphate buffer solution PBS (pH 2.0, 3.0 and 6.0–8.0) and borate buffer solution (pH 9.0–10.0) were used as supporting electrolytes. The reagents for preparation of buffers i.e. CH3COOH and H3PO4 were obtained from Sigma–Aldrich while NaH2PO4·2H2O and Na2HPO4 were purchased from Riedel-de Haen. The molarities of acids and bases used in the formation of these buffers are given in Table S1. For the modification of electrode, fMWCNTs were purchased from DropSens (DRP-MWCNTCOOH; 10 nm diameter approx. and 1–2 μm average length). The solution of fMWCNTs was suspended in dimethyl formamide (DMF, Merck) while that of TiO2NPs
3. Results and discussion 3.1. Voltammetric behavior of OMZ In this study, the electrochemical response of 10 μM OMZ was firstly analyzed with bare GCE in pH 7.0 PBS and 20% methanol by DPV in the potential range of 0–1.5 V. One defined oxidative peak of 0.22 μA intensity was observed at 401 mV. So, the potential range of 0–1.0 V was selected for further experimentation. The study of blank solution showed no distinct oxidative response with bare GCE in the absence of OMZ, thus, indicating the observed oxidative peak to be the result of oxidation of OMZ drug. Later, the oxidative signature of OMZ was studied by modification of GCE with different nanomaterials as shown in Fig. 1. The anodic peak current of OMZ was increased to 0.43 μA and 2.37 μA by modification of GCE with TiO2NPs and fMWCNTs, respectively. Moreover, on fabrication of GCE with fMWCNTs, the electrode surface became sensitive enough to detect one more anodic peak of 2.37 μA at 585 mV along with the previous peak. In order to get the best anodic response, the GCE was simultaneously modified with fMWCNTs and TiO2NPs. As expected, the intensity of the peak of OMZ enhanced about 11 times than that of the bare GCE. This enhancement of peak current can be attributed to the increase on the surface area of the electrode using fMWCNTs and TiO2NPs. Therefore, TiO2NPs and fMWCNTs were collectively selected as modifiers to study the oxidation mechanism of OMZ drug. 3.2. Effect of type and quantity of nanomaterials As the sensing properties of nanomaterials depend on the type and quantity of nanoparticles used, different types of metal oxide nanoparticles and CNTs were fabricated on the electrode surface in order to get the best oxidative response of OMZ. The metal oxide nanoparticles 59
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3
d Peak 2
Peak 1
c
Ip / A
2
as compared to bare GCE (2805 Ω) indicating increase in surface area of the electrode that could cause rapid electron transfer process. The increase in electronic transduction can be attributed to adsorption of fMWCNTs onto the GCE surface and sp2 hybridization of MWCNTs leading to π-π interaction between adjacent MWCNTs layers resulting in more surface roughness. These surface defects are expected to attract TiO2NPs producing even larger surface area for TiO2NPs/fMWCNTs/ GCE to detect OMZ [16]. The value of inhomogeneity factor/surface rough roughness (N) was found to vary in the range of 0.84–0.93 suggesting smoother surface for getting reproducible results. A prominent change in the EIS parameters of both unmodified and nanomaterials modified electrodes as the recognition layer suggests successful fabrication of the electrode surface with nanomaterials. The least resistive and capacitive values of TiO2NPs/fMWCNTs/GCE qualify this electrode as a preferred sensing platform for OMZ analysis. Secondly, CV was conducted to determine the effective surface area of bare, TiO2NPs, fMWCNTs and TiO2NPs/fMWCNTs modified GC electrodes in 0.1 M KCl and 5.0 mM K3Fe(CN)6 solution for finding the accuracy of surface fabrication procedure. K3Fe(CN)6 solution was taken as a redox probe. The surface areas of bare and modified electrodes were calculated by Randles-Sevcik equation. The effective surface areas of unmodified, TiO2NPs, fMWCNTs and TiO2NPs/fMWCNTs modified GC electrodes were evaluated as 0.0238 cm2, 0.0286 cm2, 0.0492 cm2 and 0.0647 cm2, respectively. The TiO2NPs/fMWCNTs/ GCE was found to have approximately three times greater surface area than that of bare GCE surface (Fig. S1B). Thus, these findings are consistent with EIS results indicating the efficiency of the designed electrode surface.
Bare GCE TiO2NPs/GCE fMWCNTs/GCE fMWCNTs/TiO2NPs/GCE
1 b a
0 0.0
0.3
0.6 0.9 Ep/ V vs cg/AgCl
1.2
Fig. 1. DPV of 10 μM OMZ in pH 7.0 PBS and 20% methanol at (a) bare GCE, (b) TiO2NPs/GCE, (c) fMWCNTs/GCE and (d) TiO2NPs/fMWCNTs/GCE.
tested for the oxidation of OMZ were ZnO, Fe2O3 and TiO2 nanoparticles (5% solution in water). TiO2NPs showed the highest current response, thus, TiO2 nanoparticles were selected as modifier among metal oxide nanoparticles. The concentration of these nanoparticles was then optimized by using 0.05%, 0.5% and 5% solution of TiO2NPs. 0.5% solution of TiO2NPs demonstrated the best results so further experiments were performed by using the same concentration of TiO2NPs. Moreover, a variety of CNTs i.e. SWCNTs, non-functionalized MWCNTs, -NH2 functionalized MWCNTs and -COOH functionalized MWCNTs were tested for improvement of the analytical response of OMZ after their fabrication at the surface of GCE. The -COOH functionalized MWCNTs/GCE registered the highest anodic current intensity so it was selected as modifier for further experimentation. The optimum concentration of 1 mM was used for -COOH functionalized MWCNTs in order to get distinct and the highest oxidative response of OMZ.
3.4. Analysis of surface morphology of modified electrodes The surface morphologies of modified electrodes were examined by SEM. The SEM image of bare GCE, fMWCNTs modified GCE and TiO2NPs modified GCE are displayed in Fig. 2A, B and C, respectively. Fig. 2D shows the SEM image of layer-by-layer modification of GCE surface with fMWCNTs and TiO2NPs simultaneously. The SEM images indicated the homogeneous modification of GCE surface which enabled the modified electrode to have much higher surface area hence resulting in the generation of clearer and repeatable electrochemical signals in the subsequent experiments [17]. The pictorial representation of electrode surface by SEM also supported the higher N value appeared in EIS.
3.3. Determination of charge transfer properties of modified electrodes The charge transfer properties of bare and modified electrodes were analyzed by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Firstly, in this study, EIS was performed using bare GCE and nanomaterials i.e. TiO2NPs, fMWCNTs and TiO2NPs/ fMWCNTs modified GCE in 0.1 M KCl and 5 mM K3[Fe(CN)6] solution. EIS data are presented in the form of Nyquist plot. A semicircle portion at high frequencies corresponds to electron transfer limited process and an oblique line like part at lower frequencies gives information about diffusion limited process. The diameter of semicircle points to charge transfer resistance (Rct). In the present study, the highest semicircular arc was obtained at bare GCE while it was appeared to decrease after each loading of TiO2NPs, fMWCNTs and TiO2NPs/fMWCNTs alternatively on GCE as shown in Fig. S1A. The Nyquist plot was simulated with an equivalent circuit in order to determine EIS parameters i.e. Rct; charge transfer resistance, Rs; solution resistance, W; Warburg impedance and CPE; constant phase element as listed in Table 1. The Rct value was found to be the lowest for TiO2NPs/fMWCNTs/GCE (1036 Ω)
3.5. Impact of pH on the electrochemical signal The influence of pH of buffer medium on the anodic peak potential and peak current response of OMZ was investigated to get insights about its action mechanism. In order to determine complete oxidative mechanism of this drug, higher concentration of OMZ i.e. 200 μM and 100 μM was analyzed by DPV in a wide pH range (pH 2.0–10.0) at bare GCE and TiO2NPs/fMWCNTs/GCE, respectively as shown in Fig. S2. The DP voltammograms in Fig. S2A shows that OMZ produces two anodic peaks in solution of pH 4.7 to pH 10.0 while the second peak (Peak 3) could not appear in the pH range 2.0–3.7. Moreover, a shoulder on the peak 1 merged in the DP voltammograms recorded in solution of pH 4.7 to pH 10.0. This appearance of a slight shoulder in peak indicated the presence of another peak (Peak 2) for the oxidation of OMZ. The presence of peak 2 was confirmed by the DP voltammograms obtained at TiO2NPs/fMWCNTs/GCE (Fig. S2B). Here, one more prominent signal came to sight at the same potential of the shoulder of Peak 1 in the DPVs obtained at bare GCE. Hence, the oxidation of OMZ was found to take place in three steps as indicated by the appearance of three peaks. Moreover, the peak clipping experiment revealed that all these three peaks are independent as shown in Fig. S2C. The plot of Ip versus pH using DPVs data obtained at TiO2NPs/fMWCNTs/GCE shows the Ip values of all the three peaks in media of different pH (Fig. 3A).
Table 1 Parameters calculated at bare and modified GCE using EIS data. Modifiers Bare GCE TiO2NPs/GCE fMWCNTs/GCE TiO2NPs/fMWCNTs/GCE
Rct (Ω)
CPE (μF)
2805 2225 1827 1036
403.4 424 424.4 427.5
Rs (Ω)
N
1.3 × 10−7 1.2 × 10−7 1.5 × 10−7 1.4 × 10−7
0.84 0.91 0.93 0.89
60
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Fig. 2. SEM images of (A) bare GCE, (B) fMWCNTs/GCE, (C) TiO2NPs/GCE, and (D) TiO2NPs/fMWCNTs/GCE.
The maximum Ip value for peak 1 and peak 3 was found in solution of pH 7.0 while Ip value of peak 2 remained sufficiently constant after pH 4.7. So, pH 7.0 was selected as the optimum pH for conducting further studies. To calculate the number of electrons involved in the oxidation process, half peak width (W1/2) was calculated for all three peaks. The average W1/2 values for peak 1, 2 and 3 were 63, 110 and 89 mV, respectively. These values suggest involvement of two electrons in oxidation process of peak 1 and one electron oxidation mechanism corresponding to peak 2 and 3 as calculated according to the following equation [18–20].
16
W1/2 = 90 mV/n As the pH of the system changed from pH 2.0 to pH 10.0, a gradual shift in peak potential (Ep) towards less positive potential was noticed. The plot of Ep versus pH showed a linear response with slope values of −52.96 mVs−1, −57.39 mVs−1 and −73.09 mVs−1 for peak 1, peak 2 and peak 3, respectively as displayed in Fig. 3B. These slope values clearly depict that oxidation of OMZ involves equal number of electron and protons [21,22].
Peak 1 Peak 2 Peak 3
A
Peak 1 Peak 2 Peak 3
B
1000 y = -57.391 x + 989.74 2 R = 0.9964
Ep/ mV vs Ag/AgCl
12
Ip / A
1200
8 4
800
y = -73.097 x + 1578.2 2
R = 0.9978
600 400
0
y = -52.965 x + 784.42 2 R = 0.9979
200
2
4
6
8
2
10
4
6
8
10
pH
pH
Fig. 3. pH dependence on (A) Ip and (B) Ep of 10 μM OMZ in wide pH range (2.0, 3.0, 3.7, 4.7, 5.7, 6.0, 7.0, 8.0, 9.0, 10.0) and 20% methanol at TiO2NPs/fMWCNTs/ GCE using DPV. 61
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10
Ip / A
120
-4.0
A
2
8
R = 0.8981
6
-4.5 Peak 2
4
2
Ip / A
40
0.16
2
Peak 1 Peak 2
-5.0 -5.5
R = 0.9309
y = 0.9414 x - 3.7776 2
Peak 1
0 0.0
L og Ip
2 0.10 0.12 0.14 1/2 / V1/2s-1/2
y = 0.9376 x - 3.4976 R = 0.9997
80 0.06 0.08
B
0.2 1/2
0.4 1/2 -1/2 /V s
0.6
R = 0.9981
-6.0 0.8
0.5
1.0
1.5
2.0
2.5
3.0
Log
Fig. 4. (A) Effect of v on Ip of 100 μM OMZ in pH 7.0 PBS and 20% methanol at TiO2NPs/fMWCNTs/GCE by cyclic voltammetry, (B) plot of logv vs logIp.
3.6. Influence of scan rate
3.7. Oxidation mechanism of OMZ
The effect of scan rate on the redox signals of OMZ was also investigated to understand the type of redox process and to support the DPV results about the electrochemical fate of OMZ. In this regard, cyclic voltammograms of 100 μM OMZ were recorded at a scan rate of 5–500 mVs−1 on the surface of TiO2NPs/fMWCNTs/GCE in PBS at pH 7.0 within the potential range of 0–1.5 V. Unlike DPV, the CV signals were not sensitive enough to detect the weak signal of Peak 3. Hence, the potential range of 0–1.0 V was selected for recording CVs to obtain clear and distinct peaks as shown in Fig. S3A. The CVs displayed two peaks at lower scan rate (5 mVs−1 to 25 mVs−1) which is in accordance with the DPV results. While, at higher scan rates (50 mVs−1 to 500 mVs−1), these peaks were combined in the form a single peak. The plot of Ip versus scan rate (υ) showed linear behavior with correlation coefficient (R2) of 0.991 as represented in Fig. 4A. The R2 value of this plot was found greater than the R2 value of the plot of Ip versus square root of υ (0.9309 and 0.8981, respectively) (Fig. S3B) suggesting the adsorption controlled nature of the redox process of OMZ drug [23]. The plot of logarithm of Ip versus logarithm of υ showed linear response having slope values of 0.9414 and 0.9376 for both peaks, respectively as displayed in Fig. 4B. These slope values further also support the adsorption controlled nature of the oxidation of OMZ drug as their values are close to the theoretical value of 1 for an adsorption controlled redox process [24]. The Ep of the oxidation peak of OMZ was found to shift towards positive potentials on increasing the scan rate between 5 and 500 mVs−1 indicating irreversible redox process. According to Laviron, Ep of a completely irreversible electrode process varies according to the following equation [25].
The electrooxidation of OMZ was examined by both DPV and CV techniques. For this purpose, the electrochemical response of imidazoline derivatives i.e. xylometazoline (XMZ) and naphazoline (NZ) were studied at pH 4.7, pH 7.0 and pH 9.0 by CV and DPV. Both model compounds appeared to give only one anodic peak at 1.03 V and 0.96 V, respectively, nearly equal to 1.0 V shown by Peak 3 of OMZ as displayed in Fig. S4. Hence, this anodic peak was proposed to generate due to oxidation of their common imidazoline moiety. While, the appearance of peak 1 and peak 2 was assumed to be due to hydroxyl group present only in OMZ. Considering all the previous results, we suggest that the oxidation of OMZ (Peak 1) involves removal of two electrons and one proton from the free carbon atom in the benzene ring to form a benzoxy cation. This benzoxy cation further removes one proton and accepts one hydroxyl group [27] to form a stable compound i.e. 2-tertbutyl-5-((4,5-dihydro-1H-imidazol-2-yl)methyl)-4,6-dimethylbenzene1,3-diol. The peak 2 of OMZ is proposed to be due to the oxidation of hydroxyl group of the molecule. The oxidation of phenol takes place by removal of one electron and one proton to a phenoxy radical. The phenoxy radical is a thermodynamically unstable specie, which may exists in three isomeric forms [28–30]. The unstability of these radical species is expected to lead to the formation of dimer in the last step. The emergence of peak 3 can be attributed to the oxidation of imidazoline group. In the first step, one proton is removed from -NH site of the molecule followed by the removal of one electron to produce a radical [31,32]. This radical exists in three thermodynamically unstable resonance forms, which undergo dimerization in order to gain stability as depicted in Scheme 1.
Ep = E 0
3.8. Effect of deposition potential and deposition time
2.303RT RTk 0 2.303RT log + log v nF nF nF
The study of influence of deposition potential and deposition time on Ip is necessary for understanding adsorption process of OMZ. Therefore, the effect of deposition potential and deposition time on Ip of 2 μM OMZ was investigated by ASDPV as depicted in Fig. S5. The maximum Ip was attained at 0 V and 180 s (Fig. 5). Therefore, consequent studies were performed at this optimum deposition potential and deposition time.
where α, n, k0, E0, υ, F, R and T are transfer coefficient, number of transferred electrons, standard heterogeneous rate constant, formal potential, scan rate, Faraday's constant, general gas constant and temperature, respectively. The Ep versus logυ was plotted for peak 1 using current potential data obtained from 5 mVs−1 to 500 mVs−1. The slope value was observed to be 0.0405 V with correlation coefficient of 0.9775. Considering 0.5 as the α value for an irreversible redox process, the number of electrons (n) for the oxidation of OMZ was obtained to be 2.922 (∼3) within the studied potential range [26]. This n value complemented well with the combined n value calculated from W1/2 of peak 1 and peak 2 of OMZ by ASDPV. Therefore, the number of electrons for peak 1 (2) and peak 2 (1) were confirmed for proposing oxidation mechanism of OMZ drug.
3.9. Calibration curve and analytical parameters The calibration curve of OMZ was recorded in the range of concentration from 0.12 μM to 1.5 μM for sensitive detection of this drug by ASDPV at the surface of TiO2NPs/fMWCNTs modified GCE (Fig. 6A). The TiO2NPs/fMWCNTs/GCE displayed a linear behavior with R2 of 0.996 and 0.995 for both peaks, respectively. The limit of detection (LOD) and the limit of quantification (LOQ) were calculated according 62
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Scheme 1. Proposed oxidation mechanism of OMZ.
to the following equations:
days (three days) measurements of 0.75 μM OMZ were taken at TiO2NPs/fMWCNTs/GCE by ASDPV (Fig. S6). The relative standard deviation (% RSD) was calculated for Ip and Ep of both peaks of OMZ. This % RSD values came out to be in acceptable range i.e. less than 1% for peak 1 and less than 5% for peak 2 as tabulated in Table 2.
LOD = 3 s / m
LOQ = 10 s/ m where m is the slope of relevant calibration plot and s is the standard deviation of Ip of blank solution (five runs) [33]. The LOD value of OMZ was found to be 4.40 nM as calculated from the most sensitive peak (Peak 1). The summary of the regression data and parameters obtained from the calibration curve can be seen in Table 2.
3.11. Nasal spray analysis The reliability of the designed method was assessed from results obtained for the pharmaceutical commercial spray (Iliadin®). The ASDPV findings were found consistent with the amount of OMZ given in spray label as shown in Table 3. The recovery studies were also performed to check the accuracy of the developed method for practical applications. Recoveries of OMZ were determined by spiking a known amount of pure OMZ in pharmaceutical commercial spray using TiO2NPs/fMWCNTs modified GCE. Five repeated measurements were
3.10. Repeatability and reproducibility of fabricated GCE The repeatability studies of fabricated GCE were performed in order to get insights about the precision of our developed method [34,35]. For this purpose, five repeated within day and five repeated between
2.5
1.6 A
B
2.0 1.2
0.8
Ip / A
Ip / A
1.5 Peak 1 Peak 2
Peak 1 Peak 2
1.0 0.5
0.4
0.0 0.0
0.1
0.2
0.3
0
Deposition Potential / V
50
100
150
200
250
Deposition Time / s
Fig. 5. (A) The effect of deposition potential on peak current at constant deposition time i.e. 60 s (B) the effect of deposition time on peak current at constant deposition potential i.e. 0 V, of 2 μM OMZ in pH 7.0 PBS and 20% methanol at TiO2NPs/fMWCNTs/GCE by ASDPV. 63
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2.0
2.4 A
Ip / A
Peak 1
1.0 Peak 2
0.5
1.8
Ip / A
1.5
Peak 1 Peak 2
B
0.05 M 0.125 M 0.25 M 0.50 M 0.75 M 1.0 M 1.25 M 1.50 M
1.2 2
R = 0.9960
0.6 2
R = 0.9950
0.0
0.0 0.2
0.4
0.6
0.8
1.0
0.0
Ep/ V vs Ag/AgCl
0.4
0.8 Concentration
1.2 M
1.6
Fig. 6. (A) ASDP voltammograms and (B) calibration plot of OMZ within the range of 0.05–1.5 μM in pH 7.0 PBS and 20% methanol at TiO2NPs/fMWCNTs/GCE using 0 V deposition potential and 180 s deposition time.
i.e. xylometazoline and naphazoline, structurally related to OMZ, was also investigated to get clues about the involvement of imidazoline group. The influence of pH, scan rate, deposition potential, deposition time and concentration on the current signals of OMZ was examined. The LOD was calculated to be 4.40 nM within the linear concentration range of 0.12 μM to 1.5 μM. Moreover, the developed method exhibited good repeatability and stability, hence, suggesting its suitability for the analysis of OMZ in pharmaceutical dosage forms.
Table 2 Regression data of the calibration lines of OMZ peaks by ASDPV in standard solution at TiO2NPs/fMWCNTs/GCE.
Measured potential (mV) Linearity range (μM) Slope (μA μM−1) Correlation coefficient (R) SE of slope LOD (nM) LOQ (nM) Repeatability of peak current (RSD%)a Repeatability of peak potential (RSD%)a Reproducibility of peak current (RSD%)a Reproducibility of peak potential (RSD%)a a
Peak 1
Peak 2
413 0.05–1.5 1.111 0.996 4.26 × 10−2 4.40 14.68 0.465 0.516 1.009 0.632
572 0.05–1.5 0.497 0.995 1.57 × 10−2 9.84 32.79 2.432 0.317 4.029 0.517
Acknowledgements Azeema Munir gratefully acknowledges and thanks TUBITAK (The Scientific & Technological Research Council of Turkey) for providing the full scholarship during her visit to Ankara, Turkey.
Obtained from five experiments using 0.75 μM OMZ.
Appendix A. Supplementary data
Table 3 Results obtained for the determination and recovery experiments of OMZ at TiO2NPs/fMWCNTs/GCE using ASDPV.
Labeled claim (mg) Amount found (mg)a RSD% Bias% Added (μM) Found (μM)a Average recovered % RSD% of recovery Bias% a
Peak 1
Peak 2
5.00 5.05 0.89 −1.00 0.25 0.249 99.80 0.88 0.20
5.00 5.28 3.34 −5.60 0.25 0.283 113.20 3.90 −13.20
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Obtained from five experiments.
taken for each result and the average value of these repeated measurements were used to calculate the recovery data by regression equation from calibration plot. The results of spray analysis are summarized in Table 3. 4. Conclusion In the current work, a nasal decongestant drug OMZ was studied by GCE modified with nanomaterials i.e. TiO2NPs and fMWCNTs. The possible number of electrons and protons involved in reaction mechanism were calculated by CV and DPV. The electrochemical oxidation of OMZ was found to occur in an irreversible manner following adsorption controlled process. Based upon the experimentally obtained evidences, the electro-oxidation mechanism was proposed and discussed in detail. The voltammetric behavior of imidazoline derivatives 64
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A novel electrochemical method for the detection of oxymetazoline drug based on MWCNTs and TiO2 nanoparticles Azeema Munira,b, Burcin Bozal-Palabiyikb, Amjad Khanc, Afzal Shaha,d, Bengi Uslub,
T
⁎
a
Department of Chemistry, Quaid-i-Azam University, 45320 Islamabad, Pakistan Department of Analytical Chemistry, Faculty of Pharmacy, Ankara University, 06560, Ankara, Turkey c Barking, Havering and Redbridge University Hospitals NHS Trust, London, UK d Department of Chemistry, College of Science, University of Bahrain, Sakhir 32038, Bahrain b
ARTICLE INFO
ABSTRACT
Keywords: Oxymetazoline Carbon nanotubes TiO2 nanoparticles Electrochemistry Voltammetry
Oxymetazoline, a nasal decongestant drug was investigated by a sensitive, simple and reproducible electrochemical method using Titania (TiO2) nanoparticles and carboxyl group functionalized multi-walled carbon nanotubes fabricated on glassy carbon electrode using cyclic voltammetry and differential pulse voltammetry. The possible electro-oxidation mechanism of this drug was also studied in detail. The performance of designed method was determined by electrochemical impedance spectroscopy, cyclic voltammetry and anodic stripping differential pulse voltammetry. The obtained results revealed enhanced electrochemical behavior of oxymetazoline as compared to bare glassy carbon electrode. Under optimum conditions i.e. buffer pH; pH 7.0 phosphate buffer solution, accumulation potential; 0 V, and accumulation time; 180 s, the differential pulse voltammetric response of oxymetazoline was probed between the linear concentration range of 0.12 μM to 1.5 μM in aqueous medium containing pH 7.0 PBS and 20% methanol. The limit of detection of oxymetazoline was evaluated as 4.40 nM. Moreover, the developed sensing method was validated for its practical application in nasal spray with excellent recovery.
1. Introduction Topical nasal decongestants are drugs that react immediately to cure nasal congestion i.e. allergic rhinitis, common cold and sinusitis. Generally, they include inhalers, drops and sprays. They work by constriction of nasal arterioles leading to ease in drainage of secretions in nasal mucous membrane, thus resulting in elimination of nasal congestion. They can be classified into two major groups: sympathomimetic amines (cocaine, amphetamine, adrenaline, ephedrine) and imidazolines (oxymetazoline and xylometazoline) [1]. One member of imidazoline i.e. oxymetazoline (OMZ), (6-tert-butyl-3-(2-imidazolin-2ylmethyl)-2,4-dimethyl-phenol) has been used as nasal vasoconstrictor for more than forty years by influencing the α-adrenoceptor (an agonist material used as nasal mucosa decongestant) [2,3]. The use of this drug does not cause severe toxicity rather than mild side effects at cardiovascular or/and central nerve systems caused by systemic absorption of excess dose of unprescribed OMZ drug from nasal mucosa [2]. Literature survey of OMZ reveals that the determination of OMZ and its oxidation degradants has previously been performed by flow injection analysis with chemiluminescence detector [4], high performance
⁎
liquid chromatography [5] and liquid chromatography/mass spectrometry method [6]. While, no report is available about the electrochemical analysis of OMZ from pharmaceutical formulations. The electrochemical techniques are considered as efficient methods for the sensitive and selective determination of drugs as well as their redox properties. Moreover, their low cost and small size of the instrument makes them alternative classical analytical techniques for providing rapid and sensitive determination of desired organic compounds [7–10]. Therefore, in the present work electrochemical techniques were used for OMZ analysis. For the electrochemical sensing of redox components at the electrode surface, surface modifiers i.e. carbon structures, metallic nanoparticles and conductive polymers or combination of these materials are used to accelerate the transformation of irreversible oxidation processes to reversible processes due to rapid electron transfer rate. Among these nanoparticles are widely used for electrochemical analysis of various compounds owing to their distinguishing characteristics such as improved conductivity via mass transport, electro-catalytic effect, enhanced effective surface area and stability of electrode's chemical microenvironment [11]. The carbon nanomaterials are useful for
Corresponding author. E-mail address: [email protected] (B. Uslu).
https://doi.org/10.1016/j.jelechem.2019.05.017 Received 16 January 2019; Received in revised form 8 May 2019; Accepted 8 May 2019 Available online 10 May 2019 1572-6657/ © 2019 Elsevier B.V. All rights reserved.
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electrode modification owing to their large surface area, wide potential window and electrocatalytic effect [12]. Among carbon nanostructures, single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs) and graphene are extensively used to enhance the electrochemical response of analytes [13]. Similarly, metallic nanoparticles are used for the electroanalysis of electrochemical processes due to their unique physical and chemical properties. The electrodes modified with noble metallic nanoparticles show enhanced electrocatalytic effect as compared to bare electrodes. TiO2 nanoparticles find extensive use in the designing sensing devices due to their adsorption ability, chemical and thermal stability, cost affordability, bio-compatibility and environment-friendliness. Such nanomaterials can also be used as surface-charge regulators and adsorbents for modification of electrodes which allow the researcher to study at a wider potential range and improve the stability and reproducibility of the designed electrode [14,15]. Therefore, in the present study TiO2 nanoparticles were used in combination with -COOH functionalized MWCNTs for the modification of glassy carbon electrode to combine the sensing properties of both nanomaterials for the sensitive detection of OMZ from its pharmaceutical dosage form by anodic stripping differential pulse voltammetry (ASDPV).
(21 nm, Sigma Aldrich) was prepared in ultrapure water. 2.2. Preparation of modified TiO2NPs/fMWCNTs/GCE Homogenous and stable fMWCNTs suspension (0.5 mg mL−1) was prepared in DMF after sonication in ultrasonic bath for 2 h. The mechanical cleaning of GCE surface was performed with alumina slurry (0.05 μm) on a polishing pad followed by washing it with distilled water and drying in air. For the fabrication of electrode surface, 3 μL, 5 μL, 7 μL and 10 μL volume of TiO2NPs and fMWCNTs were loaded independently in order to check the distinct and the highest analytical response of OMZ. The optimum loading selected for TiO2NPs/ fMWCNTs/GCE preparation was 5 μL of both fMWCNTs and TiO2NPs. After each casting of drop, the electrode was dried at 40 °C in vacuum oven. 2.3. Nasal spray and recovery assay procedure In order to check the validity of the designed method for practical applications, 1 mM stock solution of OMZ was prepared in 20% methanol by using 7.5 μL of Iliadin® (containing 0.05% of OMZ) nasal spray. The working solutions were then prepared by diluting the stock solution with PBS (pH 7.0) and 20% methanol. This solution was subjected to electrochemical analysis. The nominal amount of OMZ in nasal spray was then calculated by corresponding regression equation of calibration curves. The recovery studies were also carried out to determine the accuracy of the developed method and to check the possible interferences from excipients present in the spray solution. For this purpose, a known amount of OMZ solution was added to pre-analyzed nasal spray solution. The recovery results were further investigated using calibration equation by DPV for five repeated measurements.
2. Experimental 2.1. Apparatus and reagents AUTOLAB-PGSTAT302 (Eco Chemie, Utrecht, The Netherlands) was used for electrochemical measurements with General Purpose Electrochemical Software (GPES) 4.9 using a conventional three electrode system in one compartment. In this system, glassy carbon electrode, Ag/AgCl electrode and platinum wire were used as working, reference and auxiliary electrodes, respectively. In differential pulse voltammetric (DPV) measurements: pulse amplitude, 0.0505 V; step potential, 0.00795 V; interval time, 0.500 s and modulation time, 0.050 s were used. The accumulation potential of 0 V and accumulation time of 180 s were further selected for adsorptive stripping differential pulse voltammetric analysis (ASDPV). While the pulse width of 0.010 V was adjusted for average baseline correction. The electrochemical impedance spectroscopy (EIS) was performed using AUTOLABPGSTAT100N (Eco Chemie, Utrecht, The Netherlands) electrochemical analyzer controlled by NOVA 2.1 software. For EIS measurements, the frequency range of 0.1 Hz to 100 kHz was used. While the amplitude and potential of AC voltage were selected as 10 mV and 0.0 V, respectively. Scanning electron microscopy (SEM) was performed by Zeiss Evo 40 instrument to characterize the fMWCNTs/GCE, TiO2NPs/GCE and TiO2NPs/fMWCNTs/GCE. For pH measurements 538 pH meter (WTW, Austria) was used. OMZ was kindly supplied from Santa Farma (Istanbul, Turkey) and its pharmaceutical dosage form (nasal spray) i.e. Iliadin® (0.05% of OMZ) was purchased from a local pharmacy. For the study of model compounds, naphazoline (NZ) and xylometazoline (XMZ) were also provided by Santa Farma (Istanbul, Turkey). Stock solutions of OMZ (2 × 10−3 M) were prepared in methanol and kept in refrigerator. For voltammetric experiments, working solutions of OMZ were prepared by dilution of a stock solution with the desired buffer solutions. The amount of methanol (20%) was kept constant in all working solutions. Acetate buffer solution (pH 3.7–5.7), phosphate buffer solution PBS (pH 2.0, 3.0 and 6.0–8.0) and borate buffer solution (pH 9.0–10.0) were used as supporting electrolytes. The reagents for preparation of buffers i.e. CH3COOH and H3PO4 were obtained from Sigma–Aldrich while NaH2PO4·2H2O and Na2HPO4 were purchased from Riedel-de Haen. The molarities of acids and bases used in the formation of these buffers are given in Table S1. For the modification of electrode, fMWCNTs were purchased from DropSens (DRP-MWCNTCOOH; 10 nm diameter approx. and 1–2 μm average length). The solution of fMWCNTs was suspended in dimethyl formamide (DMF, Merck) while that of TiO2NPs
3. Results and discussion 3.1. Voltammetric behavior of OMZ In this study, the electrochemical response of 10 μM OMZ was firstly analyzed with bare GCE in pH 7.0 PBS and 20% methanol by DPV in the potential range of 0–1.5 V. One defined oxidative peak of 0.22 μA intensity was observed at 401 mV. So, the potential range of 0–1.0 V was selected for further experimentation. The study of blank solution showed no distinct oxidative response with bare GCE in the absence of OMZ, thus, indicating the observed oxidative peak to be the result of oxidation of OMZ drug. Later, the oxidative signature of OMZ was studied by modification of GCE with different nanomaterials as shown in Fig. 1. The anodic peak current of OMZ was increased to 0.43 μA and 2.37 μA by modification of GCE with TiO2NPs and fMWCNTs, respectively. Moreover, on fabrication of GCE with fMWCNTs, the electrode surface became sensitive enough to detect one more anodic peak of 2.37 μA at 585 mV along with the previous peak. In order to get the best anodic response, the GCE was simultaneously modified with fMWCNTs and TiO2NPs. As expected, the intensity of the peak of OMZ enhanced about 11 times than that of the bare GCE. This enhancement of peak current can be attributed to the increase on the surface area of the electrode using fMWCNTs and TiO2NPs. Therefore, TiO2NPs and fMWCNTs were collectively selected as modifiers to study the oxidation mechanism of OMZ drug. 3.2. Effect of type and quantity of nanomaterials As the sensing properties of nanomaterials depend on the type and quantity of nanoparticles used, different types of metal oxide nanoparticles and CNTs were fabricated on the electrode surface in order to get the best oxidative response of OMZ. The metal oxide nanoparticles 59
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3
d Peak 2
Peak 1
c
Ip / A
2
as compared to bare GCE (2805 Ω) indicating increase in surface area of the electrode that could cause rapid electron transfer process. The increase in electronic transduction can be attributed to adsorption of fMWCNTs onto the GCE surface and sp2 hybridization of MWCNTs leading to π-π interaction between adjacent MWCNTs layers resulting in more surface roughness. These surface defects are expected to attract TiO2NPs producing even larger surface area for TiO2NPs/fMWCNTs/ GCE to detect OMZ [16]. The value of inhomogeneity factor/surface rough roughness (N) was found to vary in the range of 0.84–0.93 suggesting smoother surface for getting reproducible results. A prominent change in the EIS parameters of both unmodified and nanomaterials modified electrodes as the recognition layer suggests successful fabrication of the electrode surface with nanomaterials. The least resistive and capacitive values of TiO2NPs/fMWCNTs/GCE qualify this electrode as a preferred sensing platform for OMZ analysis. Secondly, CV was conducted to determine the effective surface area of bare, TiO2NPs, fMWCNTs and TiO2NPs/fMWCNTs modified GC electrodes in 0.1 M KCl and 5.0 mM K3Fe(CN)6 solution for finding the accuracy of surface fabrication procedure. K3Fe(CN)6 solution was taken as a redox probe. The surface areas of bare and modified electrodes were calculated by Randles-Sevcik equation. The effective surface areas of unmodified, TiO2NPs, fMWCNTs and TiO2NPs/fMWCNTs modified GC electrodes were evaluated as 0.0238 cm2, 0.0286 cm2, 0.0492 cm2 and 0.0647 cm2, respectively. The TiO2NPs/fMWCNTs/ GCE was found to have approximately three times greater surface area than that of bare GCE surface (Fig. S1B). Thus, these findings are consistent with EIS results indicating the efficiency of the designed electrode surface.
Bare GCE TiO2NPs/GCE fMWCNTs/GCE fMWCNTs/TiO2NPs/GCE
1 b a
0 0.0
0.3
0.6 0.9 Ep/ V vs cg/AgCl
1.2
Fig. 1. DPV of 10 μM OMZ in pH 7.0 PBS and 20% methanol at (a) bare GCE, (b) TiO2NPs/GCE, (c) fMWCNTs/GCE and (d) TiO2NPs/fMWCNTs/GCE.
tested for the oxidation of OMZ were ZnO, Fe2O3 and TiO2 nanoparticles (5% solution in water). TiO2NPs showed the highest current response, thus, TiO2 nanoparticles were selected as modifier among metal oxide nanoparticles. The concentration of these nanoparticles was then optimized by using 0.05%, 0.5% and 5% solution of TiO2NPs. 0.5% solution of TiO2NPs demonstrated the best results so further experiments were performed by using the same concentration of TiO2NPs. Moreover, a variety of CNTs i.e. SWCNTs, non-functionalized MWCNTs, -NH2 functionalized MWCNTs and -COOH functionalized MWCNTs were tested for improvement of the analytical response of OMZ after their fabrication at the surface of GCE. The -COOH functionalized MWCNTs/GCE registered the highest anodic current intensity so it was selected as modifier for further experimentation. The optimum concentration of 1 mM was used for -COOH functionalized MWCNTs in order to get distinct and the highest oxidative response of OMZ.
3.4. Analysis of surface morphology of modified electrodes The surface morphologies of modified electrodes were examined by SEM. The SEM image of bare GCE, fMWCNTs modified GCE and TiO2NPs modified GCE are displayed in Fig. 2A, B and C, respectively. Fig. 2D shows the SEM image of layer-by-layer modification of GCE surface with fMWCNTs and TiO2NPs simultaneously. The SEM images indicated the homogeneous modification of GCE surface which enabled the modified electrode to have much higher surface area hence resulting in the generation of clearer and repeatable electrochemical signals in the subsequent experiments [17]. The pictorial representation of electrode surface by SEM also supported the higher N value appeared in EIS.
3.3. Determination of charge transfer properties of modified electrodes The charge transfer properties of bare and modified electrodes were analyzed by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Firstly, in this study, EIS was performed using bare GCE and nanomaterials i.e. TiO2NPs, fMWCNTs and TiO2NPs/ fMWCNTs modified GCE in 0.1 M KCl and 5 mM K3[Fe(CN)6] solution. EIS data are presented in the form of Nyquist plot. A semicircle portion at high frequencies corresponds to electron transfer limited process and an oblique line like part at lower frequencies gives information about diffusion limited process. The diameter of semicircle points to charge transfer resistance (Rct). In the present study, the highest semicircular arc was obtained at bare GCE while it was appeared to decrease after each loading of TiO2NPs, fMWCNTs and TiO2NPs/fMWCNTs alternatively on GCE as shown in Fig. S1A. The Nyquist plot was simulated with an equivalent circuit in order to determine EIS parameters i.e. Rct; charge transfer resistance, Rs; solution resistance, W; Warburg impedance and CPE; constant phase element as listed in Table 1. The Rct value was found to be the lowest for TiO2NPs/fMWCNTs/GCE (1036 Ω)
3.5. Impact of pH on the electrochemical signal The influence of pH of buffer medium on the anodic peak potential and peak current response of OMZ was investigated to get insights about its action mechanism. In order to determine complete oxidative mechanism of this drug, higher concentration of OMZ i.e. 200 μM and 100 μM was analyzed by DPV in a wide pH range (pH 2.0–10.0) at bare GCE and TiO2NPs/fMWCNTs/GCE, respectively as shown in Fig. S2. The DP voltammograms in Fig. S2A shows that OMZ produces two anodic peaks in solution of pH 4.7 to pH 10.0 while the second peak (Peak 3) could not appear in the pH range 2.0–3.7. Moreover, a shoulder on the peak 1 merged in the DP voltammograms recorded in solution of pH 4.7 to pH 10.0. This appearance of a slight shoulder in peak indicated the presence of another peak (Peak 2) for the oxidation of OMZ. The presence of peak 2 was confirmed by the DP voltammograms obtained at TiO2NPs/fMWCNTs/GCE (Fig. S2B). Here, one more prominent signal came to sight at the same potential of the shoulder of Peak 1 in the DPVs obtained at bare GCE. Hence, the oxidation of OMZ was found to take place in three steps as indicated by the appearance of three peaks. Moreover, the peak clipping experiment revealed that all these three peaks are independent as shown in Fig. S2C. The plot of Ip versus pH using DPVs data obtained at TiO2NPs/fMWCNTs/GCE shows the Ip values of all the three peaks in media of different pH (Fig. 3A).
Table 1 Parameters calculated at bare and modified GCE using EIS data. Modifiers Bare GCE TiO2NPs/GCE fMWCNTs/GCE TiO2NPs/fMWCNTs/GCE
Rct (Ω)
CPE (μF)
2805 2225 1827 1036
403.4 424 424.4 427.5
Rs (Ω)
N
1.3 × 10−7 1.2 × 10−7 1.5 × 10−7 1.4 × 10−7
0.84 0.91 0.93 0.89
60
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Fig. 2. SEM images of (A) bare GCE, (B) fMWCNTs/GCE, (C) TiO2NPs/GCE, and (D) TiO2NPs/fMWCNTs/GCE.
The maximum Ip value for peak 1 and peak 3 was found in solution of pH 7.0 while Ip value of peak 2 remained sufficiently constant after pH 4.7. So, pH 7.0 was selected as the optimum pH for conducting further studies. To calculate the number of electrons involved in the oxidation process, half peak width (W1/2) was calculated for all three peaks. The average W1/2 values for peak 1, 2 and 3 were 63, 110 and 89 mV, respectively. These values suggest involvement of two electrons in oxidation process of peak 1 and one electron oxidation mechanism corresponding to peak 2 and 3 as calculated according to the following equation [18–20].
16
W1/2 = 90 mV/n As the pH of the system changed from pH 2.0 to pH 10.0, a gradual shift in peak potential (Ep) towards less positive potential was noticed. The plot of Ep versus pH showed a linear response with slope values of −52.96 mVs−1, −57.39 mVs−1 and −73.09 mVs−1 for peak 1, peak 2 and peak 3, respectively as displayed in Fig. 3B. These slope values clearly depict that oxidation of OMZ involves equal number of electron and protons [21,22].
Peak 1 Peak 2 Peak 3
A
Peak 1 Peak 2 Peak 3
B
1000 y = -57.391 x + 989.74 2 R = 0.9964
Ep/ mV vs Ag/AgCl
12
Ip / A
1200
8 4
800
y = -73.097 x + 1578.2 2
R = 0.9978
600 400
0
y = -52.965 x + 784.42 2 R = 0.9979
200
2
4
6
8
2
10
4
6
8
10
pH
pH
Fig. 3. pH dependence on (A) Ip and (B) Ep of 10 μM OMZ in wide pH range (2.0, 3.0, 3.7, 4.7, 5.7, 6.0, 7.0, 8.0, 9.0, 10.0) and 20% methanol at TiO2NPs/fMWCNTs/ GCE using DPV. 61
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10
Ip / A
120
-4.0
A
2
8
R = 0.8981
6
-4.5 Peak 2
4
2
Ip / A
40
0.16
2
Peak 1 Peak 2
-5.0 -5.5
R = 0.9309
y = 0.9414 x - 3.7776 2
Peak 1
0 0.0
L og Ip
2 0.10 0.12 0.14 1/2 / V1/2s-1/2
y = 0.9376 x - 3.4976 R = 0.9997
80 0.06 0.08
B
0.2 1/2
0.4 1/2 -1/2 /V s
0.6
R = 0.9981
-6.0 0.8
0.5
1.0
1.5
2.0
2.5
3.0
Log
Fig. 4. (A) Effect of v on Ip of 100 μM OMZ in pH 7.0 PBS and 20% methanol at TiO2NPs/fMWCNTs/GCE by cyclic voltammetry, (B) plot of logv vs logIp.
3.6. Influence of scan rate
3.7. Oxidation mechanism of OMZ
The effect of scan rate on the redox signals of OMZ was also investigated to understand the type of redox process and to support the DPV results about the electrochemical fate of OMZ. In this regard, cyclic voltammograms of 100 μM OMZ were recorded at a scan rate of 5–500 mVs−1 on the surface of TiO2NPs/fMWCNTs/GCE in PBS at pH 7.0 within the potential range of 0–1.5 V. Unlike DPV, the CV signals were not sensitive enough to detect the weak signal of Peak 3. Hence, the potential range of 0–1.0 V was selected for recording CVs to obtain clear and distinct peaks as shown in Fig. S3A. The CVs displayed two peaks at lower scan rate (5 mVs−1 to 25 mVs−1) which is in accordance with the DPV results. While, at higher scan rates (50 mVs−1 to 500 mVs−1), these peaks were combined in the form a single peak. The plot of Ip versus scan rate (υ) showed linear behavior with correlation coefficient (R2) of 0.991 as represented in Fig. 4A. The R2 value of this plot was found greater than the R2 value of the plot of Ip versus square root of υ (0.9309 and 0.8981, respectively) (Fig. S3B) suggesting the adsorption controlled nature of the redox process of OMZ drug [23]. The plot of logarithm of Ip versus logarithm of υ showed linear response having slope values of 0.9414 and 0.9376 for both peaks, respectively as displayed in Fig. 4B. These slope values further also support the adsorption controlled nature of the oxidation of OMZ drug as their values are close to the theoretical value of 1 for an adsorption controlled redox process [24]. The Ep of the oxidation peak of OMZ was found to shift towards positive potentials on increasing the scan rate between 5 and 500 mVs−1 indicating irreversible redox process. According to Laviron, Ep of a completely irreversible electrode process varies according to the following equation [25].
The electrooxidation of OMZ was examined by both DPV and CV techniques. For this purpose, the electrochemical response of imidazoline derivatives i.e. xylometazoline (XMZ) and naphazoline (NZ) were studied at pH 4.7, pH 7.0 and pH 9.0 by CV and DPV. Both model compounds appeared to give only one anodic peak at 1.03 V and 0.96 V, respectively, nearly equal to 1.0 V shown by Peak 3 of OMZ as displayed in Fig. S4. Hence, this anodic peak was proposed to generate due to oxidation of their common imidazoline moiety. While, the appearance of peak 1 and peak 2 was assumed to be due to hydroxyl group present only in OMZ. Considering all the previous results, we suggest that the oxidation of OMZ (Peak 1) involves removal of two electrons and one proton from the free carbon atom in the benzene ring to form a benzoxy cation. This benzoxy cation further removes one proton and accepts one hydroxyl group [27] to form a stable compound i.e. 2-tertbutyl-5-((4,5-dihydro-1H-imidazol-2-yl)methyl)-4,6-dimethylbenzene1,3-diol. The peak 2 of OMZ is proposed to be due to the oxidation of hydroxyl group of the molecule. The oxidation of phenol takes place by removal of one electron and one proton to a phenoxy radical. The phenoxy radical is a thermodynamically unstable specie, which may exists in three isomeric forms [28–30]. The unstability of these radical species is expected to lead to the formation of dimer in the last step. The emergence of peak 3 can be attributed to the oxidation of imidazoline group. In the first step, one proton is removed from -NH site of the molecule followed by the removal of one electron to produce a radical [31,32]. This radical exists in three thermodynamically unstable resonance forms, which undergo dimerization in order to gain stability as depicted in Scheme 1.
Ep = E 0
3.8. Effect of deposition potential and deposition time
2.303RT RTk 0 2.303RT log + log v nF nF nF
The study of influence of deposition potential and deposition time on Ip is necessary for understanding adsorption process of OMZ. Therefore, the effect of deposition potential and deposition time on Ip of 2 μM OMZ was investigated by ASDPV as depicted in Fig. S5. The maximum Ip was attained at 0 V and 180 s (Fig. 5). Therefore, consequent studies were performed at this optimum deposition potential and deposition time.
where α, n, k0, E0, υ, F, R and T are transfer coefficient, number of transferred electrons, standard heterogeneous rate constant, formal potential, scan rate, Faraday's constant, general gas constant and temperature, respectively. The Ep versus logυ was plotted for peak 1 using current potential data obtained from 5 mVs−1 to 500 mVs−1. The slope value was observed to be 0.0405 V with correlation coefficient of 0.9775. Considering 0.5 as the α value for an irreversible redox process, the number of electrons (n) for the oxidation of OMZ was obtained to be 2.922 (∼3) within the studied potential range [26]. This n value complemented well with the combined n value calculated from W1/2 of peak 1 and peak 2 of OMZ by ASDPV. Therefore, the number of electrons for peak 1 (2) and peak 2 (1) were confirmed for proposing oxidation mechanism of OMZ drug.
3.9. Calibration curve and analytical parameters The calibration curve of OMZ was recorded in the range of concentration from 0.12 μM to 1.5 μM for sensitive detection of this drug by ASDPV at the surface of TiO2NPs/fMWCNTs modified GCE (Fig. 6A). The TiO2NPs/fMWCNTs/GCE displayed a linear behavior with R2 of 0.996 and 0.995 for both peaks, respectively. The limit of detection (LOD) and the limit of quantification (LOQ) were calculated according 62
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Scheme 1. Proposed oxidation mechanism of OMZ.
to the following equations:
days (three days) measurements of 0.75 μM OMZ were taken at TiO2NPs/fMWCNTs/GCE by ASDPV (Fig. S6). The relative standard deviation (% RSD) was calculated for Ip and Ep of both peaks of OMZ. This % RSD values came out to be in acceptable range i.e. less than 1% for peak 1 and less than 5% for peak 2 as tabulated in Table 2.
LOD = 3 s / m
LOQ = 10 s/ m where m is the slope of relevant calibration plot and s is the standard deviation of Ip of blank solution (five runs) [33]. The LOD value of OMZ was found to be 4.40 nM as calculated from the most sensitive peak (Peak 1). The summary of the regression data and parameters obtained from the calibration curve can be seen in Table 2.
3.11. Nasal spray analysis The reliability of the designed method was assessed from results obtained for the pharmaceutical commercial spray (Iliadin®). The ASDPV findings were found consistent with the amount of OMZ given in spray label as shown in Table 3. The recovery studies were also performed to check the accuracy of the developed method for practical applications. Recoveries of OMZ were determined by spiking a known amount of pure OMZ in pharmaceutical commercial spray using TiO2NPs/fMWCNTs modified GCE. Five repeated measurements were
3.10. Repeatability and reproducibility of fabricated GCE The repeatability studies of fabricated GCE were performed in order to get insights about the precision of our developed method [34,35]. For this purpose, five repeated within day and five repeated between
2.5
1.6 A
B
2.0 1.2
0.8
Ip / A
Ip / A
1.5 Peak 1 Peak 2
Peak 1 Peak 2
1.0 0.5
0.4
0.0 0.0
0.1
0.2
0.3
0
Deposition Potential / V
50
100
150
200
250
Deposition Time / s
Fig. 5. (A) The effect of deposition potential on peak current at constant deposition time i.e. 60 s (B) the effect of deposition time on peak current at constant deposition potential i.e. 0 V, of 2 μM OMZ in pH 7.0 PBS and 20% methanol at TiO2NPs/fMWCNTs/GCE by ASDPV. 63
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2.0
2.4 A
Ip / A
Peak 1
1.0 Peak 2
0.5
1.8
Ip / A
1.5
Peak 1 Peak 2
B
0.05 M 0.125 M 0.25 M 0.50 M 0.75 M 1.0 M 1.25 M 1.50 M
1.2 2
R = 0.9960
0.6 2
R = 0.9950
0.0
0.0 0.2
0.4
0.6
0.8
1.0
0.0
Ep/ V vs Ag/AgCl
0.4
0.8 Concentration
1.2 M
1.6
Fig. 6. (A) ASDP voltammograms and (B) calibration plot of OMZ within the range of 0.05–1.5 μM in pH 7.0 PBS and 20% methanol at TiO2NPs/fMWCNTs/GCE using 0 V deposition potential and 180 s deposition time.
i.e. xylometazoline and naphazoline, structurally related to OMZ, was also investigated to get clues about the involvement of imidazoline group. The influence of pH, scan rate, deposition potential, deposition time and concentration on the current signals of OMZ was examined. The LOD was calculated to be 4.40 nM within the linear concentration range of 0.12 μM to 1.5 μM. Moreover, the developed method exhibited good repeatability and stability, hence, suggesting its suitability for the analysis of OMZ in pharmaceutical dosage forms.
Table 2 Regression data of the calibration lines of OMZ peaks by ASDPV in standard solution at TiO2NPs/fMWCNTs/GCE.
Measured potential (mV) Linearity range (μM) Slope (μA μM−1) Correlation coefficient (R) SE of slope LOD (nM) LOQ (nM) Repeatability of peak current (RSD%)a Repeatability of peak potential (RSD%)a Reproducibility of peak current (RSD%)a Reproducibility of peak potential (RSD%)a a
Peak 1
Peak 2
413 0.05–1.5 1.111 0.996 4.26 × 10−2 4.40 14.68 0.465 0.516 1.009 0.632
572 0.05–1.5 0.497 0.995 1.57 × 10−2 9.84 32.79 2.432 0.317 4.029 0.517
Acknowledgements Azeema Munir gratefully acknowledges and thanks TUBITAK (The Scientific & Technological Research Council of Turkey) for providing the full scholarship during her visit to Ankara, Turkey.
Obtained from five experiments using 0.75 μM OMZ.
Appendix A. Supplementary data
Table 3 Results obtained for the determination and recovery experiments of OMZ at TiO2NPs/fMWCNTs/GCE using ASDPV.
Labeled claim (mg) Amount found (mg)a RSD% Bias% Added (μM) Found (μM)a Average recovered % RSD% of recovery Bias% a
Peak 1
Peak 2
5.00 5.05 0.89 −1.00 0.25 0.249 99.80 0.88 0.20
5.00 5.28 3.34 −5.60 0.25 0.283 113.20 3.90 −13.20
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Obtained from five experiments.
taken for each result and the average value of these repeated measurements were used to calculate the recovery data by regression equation from calibration plot. The results of spray analysis are summarized in Table 3. 4. Conclusion In the current work, a nasal decongestant drug OMZ was studied by GCE modified with nanomaterials i.e. TiO2NPs and fMWCNTs. The possible number of electrons and protons involved in reaction mechanism were calculated by CV and DPV. The electrochemical oxidation of OMZ was found to occur in an irreversible manner following adsorption controlled process. Based upon the experimentally obtained evidences, the electro-oxidation mechanism was proposed and discussed in detail. The voltammetric behavior of imidazoline derivatives 64
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