Scholarly article on topic 'Simultaneous determination of caffeine and theophylline using square wave voltammetry at poly( l -aspartic acid)/functionalized multi-walled carbon nanotubes composite modified electrode'

Simultaneous determination of caffeine and theophylline using square wave voltammetry at poly( l -aspartic acid)/functionalized multi-walled carbon nanotubes composite modified electrode Academic research paper on "Chemical sciences"

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{"Poly(l-aspartic acid)/functionalized multi-walled carbon nanotubes composite modified electrode" / "Square wave voltammetry" / "Simultaneous determination caffeine and theophylline" / "Green tea" / "Blood serum" / "Panadol extra"}

Abstract of research paper on Chemical sciences, author of scientific article — Birhanu Mekassa, Merid Tessema, Bhagwan Singh Chandravanshi

Abstract A simple and reproducible poly(l-aspartic acid)/functionalized multi-walled carbon nanotubes composite modified glassy carbon electrode, P(LAsp)/f-MWCNTs/GCE, was constructed for simultaneous determination of caffeine and theophylline using square wave voltammetry. The electrode preserves and combines the properties of the individual modifiers synergistically. The electrochemical response of P(LAsp)/f-MWCNTs/GCE was characterized by cyclic voltammetry. A significant enhancement in the peak current response of CF and TP were observed accompanied with a negative shift in peak potential at the composite modified electrode compared to the bare electrode. The prepared P(LAsp)/f-MWCNTs/GCE exhibited excellent SWV response towards the simultaneous detection of CF and TP in the range of 1–150 and 0.1–50μM with limit of detection of 0.28 and 0.02μM (S/N = 3), respectively. Real sample analysis has been successfully carried out in green tea, blood serum and pharmaceutical formulation of Panadol extra samples, which revealed good recovery results, 92.0–106%. The proposed sensor also displayed good selectivity, repeatability and reproducibility with appreciable long-term stability, indicating the feasibility and reliability.

Academic research paper on topic "Simultaneous determination of caffeine and theophylline using square wave voltammetry at poly( l -aspartic acid)/functionalized multi-walled carbon nanotubes composite modified electrode"

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Simultaneous determination of caffeine and theophylline using square wave voltammetry at poly(l-aspartic acid)/functionalized multi-walled carbon nanotubes composite modified electrode

Birhanu Mekassa, Merid Tessema, Bhagwan Singh Chandravanshi

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S2214-1804(17)30163-0 doi:10.1016/j.sbsr.2017.11.002 SBSR 210

Sensing and Bio-Sensing Research

29 September 2017 1 November 2017

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Please cite this article as: Birhanu Mekassa, Merid Tessema, Bhagwan Singh Chandravanshi , Simultaneous determination of caffeine and theophylline using square wave voltammetry at poly(l-aspartic acid)/functionalized multi-walled carbon nanotubes composite modified electrode. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Sbsr(2017), doi:10.1016/ j.sbsr.2017.11.002

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Simultaneous determination of caffeine and theophylline using square wave voltammetry at poly( L-aspartic acid)/functionalized multi-walled carbon nanotubes composite modified electrode

Birhanu Mekassaa,b, Merid Tessemaa, Bhagwan Singh Chandravanshia,* bscv2006@yahoo.com

^Department of Chemistry, College of Natural Sciences, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia

bDepartment of Chemistry, College of Natural Sciences, Wachemo University, P.O. Box 667, Hossana, Ethiopia *Corresponding author.

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Abstract

A simple and reproducible poly(L-aspartic acid)/functionalized multi-walled carbon nanotubes composite modified glassy carbon electrode, P(L-Asp)/f-MWCNTs/GCE, was constructed for simultaneous determination of caffeine and theophylline using square wave voltammetry. The electrode preserves and combines the properties of the individual modifiers synergistically. The electrochemical response of P(L-Asp)/f-MWCNTs/GCE was characterized by cyclic voltammetry. A significant enhancement in the peak current response of CF and TP were observed accompanied with a negative shift in peak potential at the composite modified electrode compared to the bare electrode. The prepared P(L-Asp)/f-MWCNTs/GCE exhibited excellent SWV response towards the simultaneous detection of CF and TP in the range of 1-150 and 0.1-50 ^M with limit of detection of 0.28 and 0.02 ^M (S/N = 3), respectively. Real sample analysis has been successfully carried <out in green tea, blood serum and pharmaceutical formulation of Panadol extra samples, which revealed good recovery results, 92.0-106%. The proposed sensor also displayed good selectivity, repeatability and reproducibility with appreciable long-term stability, indicating the feasibility and reliability.

Keywords Poly(L-aspartic acid)/functionalized multi-walled carbon nanotubes composite modified electrode, Square wave voltammetry, Simultaneous determination caffeine and theophylline, Green tea, Blood serum, Panadol extra

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1. Introduction

Xanthines are purine bases which are known to act as anti-depressants, anti-therapeutic and anti-hyperuraemic agents [1]. Caffeine (1,3,7-trimethylxanthine), theophylline (1,3-dimethylxanthine) and theobromine (3,7dimethylxanthine), (Scheme 1), are xanthines that are widely found in the human diet. These compounds naturally occur in food products such as tea, coffee, and cocoa beans and, therefore, in the food and beverages made from them [2-5]. They have received an increased attention in the food and nutrition industry because they can cause various physiological effects. Very recently, xanthine derivatives such caffeine and theophylline have been identified as micromolar inhibitors of bacterial family, thereby opening up potential applications for such molecules as fungicides and nematocides, and renewing interest in their use as asthma therapeutics [6].

R1 = R2 = CH3: Caffeine R*= CH3; R2 = H: Theophylline R1 = H; R2 = CH3: Theobromine

Scheme 1. Chemical structure of methylated xanthenes.

Caffeine (CF) and theophylline (TP) are also found in a variety of pharmaceutical products and drugs because they possess the following properties: stimulate the central nervous system, induce gastric secretions, and act as a diuretic. Studies have also been done on these alkaloids to assess their antioxidant properties [3, 4]. CF and TP display multiple pharmacological effects, such as enhancing cognitive function, increasing endurance, and reliving anxiety. However, excessive intake of such compounds has been associated to several negative side effects ranging from simple tremor and tachycardia to cancer and even death [7].

Thus, the investigation of caffeine and theophylline give beneficial guidance to people's health and life in addition to clinical significances [7]. Since CF and TP usually coexist in real samples, the development of a selective and sensitive method for their simultaneous determination is highly desirable for analytical and diagnostic applications.

Various methods exist for the determination of caffeine and theophylline in different matrices such as food, drinks, clinical samples and pharmaceutical products. The most widely used analytical techniques are mainly chromatography, liquid chromatography and gas chromatography with various detectors [8-11], spectroscopy [1214], and electrochemical methods. Nevertheless, chromatographic and spectroscopic techniques are expensive and time-consuming; hence require extensive sample pretreatment and cleanup steps. Instead, electrochemical methods

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are characterized by practical advantages such as operational simplicity, high sensitivity, wide linear concentration range, good stability, low-cost instrumentation, possibility of miniaturization, suitability for real-time detection and less sensitive to matrix effects in comparison with separation and spectral methods [15, 16].

Several articles reported the electrochemical detection of CF and TP at different electrodes [17-21]. However, a key problem encountered is the overlap of the peak potentials of these species at conventional electrodes with pronounced fouling effect, resulting in poor selectivity and reproducibility. To overcome the above problems, various chemically modified electrodes were used. Recently, chemically modified electrodes (CM Es) have become important electrochemical methods for the determination of biologically important compounds because of their good sensitivity, selectivity and stability [22]. However, only few reports appeared for the simultaneous determination of CF and TP using CMEs [7, 23-25], such as, large mesoporous carbon/Nafion modified electrode [7], glassy carbon disk electrode modified with a composite consisting of poly(Alizarin Violet 3B), multiwalled carbon nanotubes and graphene [23], novel anisotropic gold nanoparticle-chitosan-ionic liquid/graphene [24], and poly(4-aminopyridine) modified electrode [25].

Since the discovery of carbon nanotubes (CNTs) by Iijima in 1991 [26], both single wall (SWCNTs) and multiwall carbon nanotubes (MWCNTs) have attracted increasing attention because of high electrical conductivity, extremely excellent mechanical strength and other extraordinary properties. In recent times, CNTs have been widely applied in electroanalytical chemistry based on the promotion ability of electron transfer reactions of target molecules when used as an electrode material [27-29]. Carbon nanotubes (CNTs) are the most widely used nonmaterial in electrochemical sensors due to their conductivity, chemical stability, flexibility and low cost [23]. Conducting polymer-modified electrodes have been fabricated as electrochemical sensory platforms for the detection of analytes because of their high selectivity, sensitivity, homogeneity, easy preparation, chemical stability and strong adherence of the poly mer film [30]. Polymers are also used as a support matrix for the immobilization of biomolecules and allow electrocatalysis [31]. In addition, CPs demonstrated anti-fouling ability, which is an important practical advantage over conventional electrode materials [32]. The selectivity of the polymer-modified electrodes as sensors can be attained by different mechanisms such as size exclusion, ion exchange, electrostatic interaction and hydrophobic interaction [33]. Electrochemical sensors modified with electropolymerized organic polymers are reproducible and possess more active sites than conventional electrodes.

Very recently, the use of composite materials based on CPs and CNTs, electropolymer/CNTs modified electrodes, with the aim of combining the properties of these materials have been intensively studied in a large number of publications. These composite materials have been shown to possess complementary properties of the individual components with a synergistic effect, which have demonstrated excellent electrocatalytic ability for some biological molecules. The desirable properties of CPs such as reproducibility, good stability, strong adherence, large number of active sites and homogeneity in electrochemical deposition, together with high surface area and nanoporosity of CNT films, leads to a superior performance of the resulting sensing devices [23, 34, 35].

In this work, conducting polymer/functionalized-MWCNTs composite modified electrode has been prepared and used for simultaneous determination of CF and TP. To the best of our knowledge, the simultaneous

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determination of CF and TP at poly(L-Asp)/f-MWCNTs composite film modified glassy carbon electrode has not been reported in the literature. 2. Experimental

2.1. Chemicals and reagents

All chemicals and reagents were of analytical grade and used as received without further purification. Double distilled water was used throughout the experiments. Caffeine and theophylline were purchased from Sigma -Aldrich, Germany. Multi-walled carbon nanotubes, (> 90% carbon basis), synthesized by chemical vapor deposition (CVD), was obtained from Sigma-Aldrich (USA). L-aspartic acid from Pharmacos Ltd (England) was used as received. Phosphate buffer solution of 0.1 M, prepared from Na2HPO4 (W agtech International Ltd, United Kingdom) and NaH2PO4 (BDH Chemicals Ltd, England) was used as supporting electrolyte; hydrochloric acid (0.1 M) and sodium hydroxide (0.1 M) were used to adjust the pH of the supporting electrolyte to the desired value.

2.2. Apparatus and instruments

The voltammetric experiments were performed using CHT760D electrochemical workstation (CHInstruments, USA) interfaced to a PC at room temperature. All experiments were carried out using a conventional three electrode system with a bare or modified glassy carbon electrode (GCE, 3 mm in diameter) as a working electrode, a platinum wire as an auxiliary electrode, and a silver/silver chloride (Ag/AgCl, KCl, saturated) as a reference electrode. The pH measurements were carried out using a pH meter (sensION, SHA Snilu Instruments CO. LTD, China). Electrode cleaning after each polishing of the GCE was carried out in an ultrasonic cleaner (YJ 5120-B, Shanghai, China). Centrifuge (model 800-1) was used during blood serum preparation and cleaning of the acid treated MWCNTs. To characterize pristine and functionalized MWCNTs, Fourier transform infrared spectrometer, FT-IR, (PerkinElmer, USA) was used.

2.3. Preparation of working standards and real samples

Stock solutions of caffeine and theophylline standards were freshly prepared immediately prior to the experiments in double distilled water. All working standard solutions were prepared in 0.1 M PBS. Appropriate quantity of L-aspartic acid was dissolved in distilled water followed by dilution in 0.1 M PBS of pH 6 to get a final concentration of 2.0 mM.

About 5 g green tea sample (Ethio Agri-CEFT PLC), purchased from a local super market, was weighed and transferred in to 50 mL of boiling water for 30 min to extract TP and CF. After filtration, the filtrate was collected in 100 mL volumetric flask and diluted to the mark with double distilled water. Before the measurements the filtrate was diluted with the supporting electrolyte by a factor 1:100 (v/v).

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Panadol extra (GSK Dungarvan, Ireland) containing caffeine (65 mg/tablet) was purchased from a local drug store from Addis Ababa, Ethiopia. Ten tablets were ground into fine powder, mixed and homogenized in agate mortar. The weight equivalent to one tablet was accurately weighed and dissolved in 25 mL double distilled water by ultrasonication for 30 min. Then, the solution was filtered using Whatman filter paper to obtain a clear filtrate and then quantitatively transferred into a 50 mL volumetric flask. To obtain final concentration in the range of the calibration curve, the sample solutions were suitably diluted with the supporting electrolyte.

Human blood sample was obtained from a healthy volunteer at the nearby hospital. About 5 mL fresh blood sample was taken and centrfuged at 1500 rpm for 20 min to remove all precipitating materials. The blank serum sample was prepared by diluting 0.5 mL of the serum to 25 mL with pH 4.5 phosphate buffer. To prepare the spiked samples, 0.5 mL of the serum was transferred to 25 mL volumetric flask containing 10 mL of PBS. After spiking with different concentrations of TP and CF, the mixture solution obtained was diluted to the mark using 0.1 M PBS of pH 4.5. Then, the blank and spiked serum samples were transferred to a voltammetric cell to detect TP and CF by the proposed SWV method without any further pretreatment. The standard addition method was used for the determination of TP and CF in all the real samples.

2.4. Functionalisation of MWCNTs

MWCNT is hydrophobic in nature and hence difficult to disperse in an aqueous solution to get a homogeneous mixture [36]. In order to obtain a uniform dispersion, strong interactions between the carbon nanotubes and the GC surface and achieve stable and uniform films of MWCNTs on the GCE surface, MWCNTs were functionalized by refluxing in a mixture of 3 M H2SO4 and 3 M HNO3 (3:1 by volume) at 100 °C for 6 h in a reflux condenser. The functionalisation also results in removing catalyst impurities present and generates more surface functional groups. The carbon atoms at the edge plane as well as at the defect sites on the basal plane undergo oxidation during acid treatment producing -COOH groups [37]. After cooling slowly to room temperature, the acid treated MWCNTs were washed several times by centrfugation at 3,500 rpm with double distilled water until the filtrate was neutral and then dried at 60 °C in an oven for 24 h. The functionalized MWCNTs were designated as f-MWCNTs. A uniform dispersion of the functionalized MWCNT in deionized water was prepared by dispersing 10 mg of f-MWCNTs in 5 mL of water by ultrasonication for 30 min.

2.5. Preparation of the modified electrode

Prior to modification the bare glassy carbon electrode was carefully polished using 1.0, 0.3 and 0.05 ^m alumina slurry sequentially on Buehler polishing cloth. The GCE was thoroughly rinsed several times with double distilled water and then ultrasonicated in double distilled water and ethanol for 5 min each to remove any adsorbed alumina particles on the electrode surface. Then, the electrode was treated by potential cycling between -0.80-0.80 V in 0.1 M H2SO4 until a stable cyclic voltammogram (CV) was obtained. After each step, the electrode was rinsed with water and ethanol, and dried at room temperature. To fabricate f-MWCNTs modified GCE, 20 ^L of f-

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MWCNTs suspension (following optimized procedure) was drop casted onto the well polished GCE surface and dried in open air.

To prepare the polymer/f-MWCNTs-composite modified electrode, poly(L-aspartic acid) was grown on the f-MWCNTs/GC electrode potentiodynamically by scanning the potential in the range of -1.0-1.7 V at a scan rate of 100 mV s-1 for 20 cycles in pH 6.0 PBS containing 2.0 mM L-aspartic acid. The modified electrode was rinsed repeatedly with distilled water and cycled in a monomer free PBS until a stable voltammogram was obtained. The poly(L-Asp)/f-MWCNTs composite film modified GCE was rinsed with distilled water, and then dried at room temperature. The prepared electrode denoted as P(L-Asp)/f-MWCNTs/GCE was kept in PBS (pH 6.0) solution at room temperature prior to use.

3. Results and discussion

3.1. FTIR spectroscopy characterization

The evidence of chemical functionalisation was confirmed by Fourier transform infrared (FTIR) spectral analyses of the pristine MWCNT and f-MWCNTs. FTIR spectra of MWCNTs and f-MWCNTs were recorded on Spectrum 65 FT-IR (PerkinElmer, USA) in the range 4000-400 cm-1 using KBr pellets. Fig. 1 shows the FTIR spectra of the pristine MWCNTs and f-MWCNTs. As shown in the figure, the increase in the intensity of all the absorption bands and the appearance of a new band at 1081 cm-1 for the functionalized MWCNTs compared to pristine MWCNTs is a clear evidence for the functionalisation of the carbon nano tubes. According to Canete-Rosales et al, CNTs present a few bands, indicating the presence of small fractions of functional groups most likely incorporated during their synthesis process. After chemical oxidation, an increase in the bands at about 3400 cm-1, 1700 cm-1 and 1100 cm-1 that corresponds to carboxylic acid groups was observed. All the bands that were associated with ether, alcohol and phenol groups increase the IR intensity [38]. This is in agreement with our results. The absorption band at 1732 cm-1 corresponds to C=O stretching of COOH, while the absorption bands at 1384 cm-1 and 1081 cm-1 are associated with O-H bending and C-O stretching, respectively. The absorption band at 1583 cm-1 is more likely from the C=C stretching mode of carbon nanotubes. Broad absorption band at 3400-3500 cm-1 is attributed to -OH stretching. The increase in intensity of these bands and the appearance of a new band suggests that the chemical functionalization of the MWCNTs have introduced more functional groups on the surface of MWCNTs. Similar observations of the FTIR spectra for f-MWCNTs were reported in the literature [39-41].

Fig. 1. FT-IR spectra of the pristine MWCNTs (bottom) and f-MWCNTs (top) powder samples.

3.2. Formation of poly(L-aspartic Acid) film on f-MWCNTs/GCE

The poly(L-aspartic acid)/f-MWNTs/GCE was prepared according to procedure described in the experimental section. Cyclic voltammetry (CV) was used for the electropolymerization of L-aspartic acid on bare

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GCE and f-MWCNTs/GCE. Fig. 2 shows the CV obtained during the electropolymerization of L-aspartic acid on f-MWCNTs coated GCE. As shown in the Fig., irreversible oxidation peaks were noticed in the positive potential direction in the first sweep, followed by continuous film growth in the higher positive potential region with successive potential scanning. The growth of the film tends to be stable after 18 scans, which confirmed the formation of the poly(L-aspartic acid) film. Hence, a total of twenty cycles of electropolymerization was selected to get a stable polymer film Moreover, a uniform adherent blue black film was also observed on the GCE surface after modification, which demonstrated that poly(L-aspartic acid) film was deposited on the surface of f-MWCNTs/GCE. However, for the electropolymerization of L-aspartic acid on bare GCE, no visible anodic peaks were observed in the first cycle, Inset of Fig. 2.

Fig. 2. CVs of the electropolymerization of 2.0 mM L-aspartic acid on f-MWCNTs/GCE in 0.1 M PBS (pH 6) at scan rate of 0.1 V s-1. Inset: Electropolymerization of L-aspartic acid on bare GCE.

3.3. Electrochemical behavior of TP and CF

The electrochemical behaviors of TP and CF were investigated individually by cyclic voltammetry (CV). Fig. 3 A and B show the CV responses of 50 ^M TP and 100 ^M CF in 0.1 M PBS (pH 4.5) at scan rate of 100 mV s-1 at bare GCE (a), P(L-Asp)/GCE (b), f-MWCNTs/GCE (c) and P(L-Asp)/f-MWCNTs/GCE (d), respectively. In all the voltammograms (Fig. 3 A and B), a single oxidation peak was observed for both TP and CF with no corresponding reduction peak in the reverse potential scan suggesting the irreversibility of the electrode reaction. In comparison to the bare glassy carbon electrode, the peak current response of TP and CF were increased at the modified electrodes with the maximum response observed for the composite modified electrode (curve d), indicating the catalytic activity of the modified electrodes. Obviously, there is a weak current response observed at about 1.20 and 1.45 V for TP and CF, respectively at the bare GCE (curve a, Fig. 3A and B), representing the slow rate of electron transfer. The oxidation peak potentials were slightly shifted negatively to 1.17 and 1.44 V for TP and CF, respectively at composite modified P(L-Asp)/f-MWCNTs/GCE.

When the bare GCE surface is coated with P(L-Asp) film, an increase in the anodic peak current of more than 2-fold of the bare electrode response was observed. Further improvement in oxidation peak current response of about 3-fold of the bare GCE was seen upon modification with functionalized MWCNTs. This indicates the electrocatalytic effect of the polymer and nanotube modifiers used in the electro -oxidation of theophylline and caffeine. The improved electrocatalytic performance of P(L-Asp)/GCE might be attributed to the electrostatic interaction between electrochemically polymerized membrane surface rich negative charge of poly(L-aspartic acid) film [42] and the positively charged TP and CF in the slightly acid pH of 4.5. The higher performance of the CNT modified electrode is due to the catalytic effect of f-MWCNTs which stems from the structure and unique properties they possess such as large specific surface area, strong adsorptive ability and the ability to promote electron transfer reaction, which provides enough effective reaction sites to increase the electron exchange rate when used as the electrode material in electrochemical reaction [29, 43-46]. At the composite of P(L-Asp)/f-MWCNTs/GCE the

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result showed a further enhancement in the peak current response of about 5 fold the current obtained at the bare electrode. This result is also higher than the responses obtained at the individual polymer and nanotubes modified electrodes. This clearly showed the synergic effect of the P(L-Asp) and f-MWCNTs, which effectively enhance the electrocatalytic activity of the composite modified electrode and promote the electron transfer rate between the analytes and the GCE. Moreover, the oxidation peak profiles of the composite modified electrode is better than both the peak resolution of the individual polymer and nanotube modified elec trode.

The electrochemical behavior of a mixture of 50 ^M TP and 100 ^M CF in 0.1 M phosphate buffer (pH 4.5) was investigated simultaneously by CV at scan rate of 100 mV s-1 (Fig. 3 C). The oxidation processes of TP and CF at the bare and modified electrodes are all irreversible. As can be seen in Fig. 3C, the voltammetric peak current responses of TP and CF are negligible at the bare GCE (curve a), indicating a slow electron transfer kinetics. So, it is impossible to use the bare electrode for simultaneous determination of the two species. However, the oxidation peak current responses of TP and CF were improved at the modified electrodes (Fig. 3C, curve b-d) without any interference effects on one another. The maximum current response was observed at the composite modified P(L-Asp)/f-MWCNTs/GCE for both TP and CF in the simultaneously detection of these drugs. In addition to the significant enhancement of peak currents, the oxidation peaks of TP and CF were clearly separated, with the peak potential of TP is about 250 mV lower than that of CF at the P(L-Asp)/f-MWCNT/GCE, Fig. 3C (curve d). These results demonstrate that the two analytes display distinguished peak potentials and can be simultaneously and sensitively determined at P(L-Asp)/f-MWCNTs/GCE.

Fig. 3. CVs of 50 ^M TP (A), 100 ^M CF (B) and a mixture of 50 ^M TP and 100 ^M CF (C) at bare GCE (a), P(L-Asp)/GCE (b), f-MWCNTs/GCE (c) and P(L-Asp)/f-MWCNTs/GCE (d) in pH 4.5 PBS at scan rate of 100 mV

3.4. Effect of amount ofMWCNTs

The effect of varying the amount of f-MWCNTs suspension on the oxidation peak current response of TP and CF was studied. As shown in Fig. S1 (supplementary information), the current response increased upon increasing the volume of the f-MWCNTs suspension up to 20 ^L for both TP and CF. After 20 ^L, the oxidation current for TP seems to decrease gradually and that of caffeine remains almost constant. Therefore, 20 ^L was selected as the optimum amount of f-MWCNTs suspension and was used to modify the glassy carbon electrode in this study.

3.5. Effect of scan rate

The effect of scan rate on the simultaneous oxidation of TP and CF at the P(L-Asp)/f-MWCNTs composite film modified electrode was investigated in 0.1 M PBS at different potential scan rates, Fig. 4. It can be seen that the oxidation peak currents for both TP and CF linearly increased with the square root of the scan rates (v1/2), Inset of

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Fig. 4, suggesting that the redox reaction at the electrode surface is predominantly diffusion-controlled process for both TP and CF [23, 46]. The linear regression equation relating Ipa with v1/2 in the range of 50 to 350 mV s-1, was found to be: Ip (^A) = -3.7(±0.3) + 49.4(±0.6)v1/2 (V1/2 s-1/2) (R2 = 0.998) for TP and Ip (^A) = -5.2(±0.5) + 82.0(±1)v1/2 (V1/2 s-1/2) (R2 = 0.998) for CF, respectively. Moreover, the oxidation peak potential (E) of both TP and CF shifted in the positive direction with increasing the scan rate. The analysis of these data showed that the plot of Epa vs. lnv gives a linear relation (R2 of 0.998 and 0.994 for TP and CF respectively), indicating that the oxidation of TP and CF at the composite modified electrode surface is irreversible [23, 48, 49].

Fig. 4. CVs of the mixture containing 50 ^M TP and 100 ^M CF at P(L-Asp)/f-MWCNTs /GCE in 0.1 M PBS at different scan rates (50-350 mV s-1). Inset: Plot of the dependence of peak current on the square root of scan rates.

3.6. Effects of pH

The effect of pH of supporting electrolytes on the redox behavior of electroactive molecules is an important factor. Hence, under the optimized experimental conditions, the effect of pH of common supporting electrolytes used in electrochemical analysis such as Britton-Robinson buffer (BRB), acetate buffer solution (ABS), phosphate buffer solution (PBS) and H2SO4 solution were investigated using CV for simultaneously determining TP and CF and the results were compared, Fig. S2(A). Owing to the high peak currents, good peak separation and peak shapes of TP and CF, PBS (pH 4.5) was chosen as the optimal supporting electrolyte for further work.

The influence of pH on the peak currents and potentials of TP and CF at the composite modified electrode were studied in 0.1 M PBS in the pH range from 3.5 to 8.0 by CV. The peak current of CF increases slowly with increasing the pH. However, the peak shape of CF is distorted at higher pH values, Fig. S2(B). On the contrary, the peak current of TP decreases as the pH value increases, Fig. S2(C). Therefore, the optimum pH value of 4.5 was selected for simultaneous determination TP and CF to obtain the best response in terms of peak current, peak shape and maximum peak separation.

The dependence of the peak potential (Ep) of TP and CF on the pH of buffer solution was also examined. The peak potential of TP shifts almost linearly towards negative potentials when the pH was increased (Fig. S2(C)), indicating that protons are directly involved in the rate determination step of the oxidation reaction of TP. A plot of peak potentials vs pH values was found to be linear in the range of 4.0 to 8.0 with a regression equation of: Epa (V) = -0.038(±0.0005)pH + 1.3(±0.003), R2 = 0.998, Inset of Fig. S2(C). The slope of -0.038/pH, which is about half of the theoretical Nernstian value of 0.059 V/pH, suggesting the redox process involve electrons and protons in 2:1 ratio [47, 50, 51]. However, the anodic peak potential (Epa) of CF did not significantly change with increasing pH, [23].

3.7. The effect ofSWV parameters for TP and CF determination

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Square wave voltammetry (SWV) was chosen as a sensitive voltammetric technique for the simultaneous determination of theophylline and caffeine in the present study. The dependence of peak currents of TP and CF on square wave parameters such as step potential, amplitude and frequency were investigated and optimized. The ranges studied were 1-16 mVfor step potential, 1-120 mV for pulse amplitude and 5-100 Hz for SW frequency and the optimum values are 5 mV, 25 mV and 25 Hz for step potential, amplitude and frequency, respectively.

3.8. Determination of TP and CF at P(L-Asp)/f-MWCNTs/GCE

Using the optimized experimental procedures described above, square wave voltammetry was used for determination of TP and CF individually and simultaneously at P(L-Asp)/f-MWCNTs/GCE in 0.1 M PBS pH 4.5. In the first case, individual detection of TP and CAF was performed. Fig. 5A illustrates the SWV curves for different concentrations of TP at constant concentration of caffeine. The peak currents of TP increased linearly with concentration in the range of 0.1-50 ^M and a regression equation of: Ip (^A) = 0.399(±0.006)C (^M) + 0.064(±0.1), R2 = 0.998, was obtained. The limit of detection was found to be 0.015 ^M (S/N = 3). Similarly, as shown in Fig. 5B, keeping the concentration of TP constant, the peak current of CF increases linearly with increasing concentration in the range of 1-120 ^M with a linear regression equation of: Ip (^A) = 0.110(±0.002)C (^M) +0.91 (±0.1), R2 = 0.995 and the LOD is 0.17 ^M (S/N = 3).

Fig. 5. SWVs obtained at P(L-Asp)/f-MWCNTs/GCE for different concentrations of (A) TP (0.1—50 pM) and (B) CF (1—>120 pM) in 0.1 M PBS for 10 pM constant concentration of CF and TP, respectively. Inset: Plot of peak current vs concentration.

One of the main objective of this study was to detect theophylline and caffeine simultaneously using P(L-Asp)/f-MWCNTs/GCE. Under the optimal experimental conditions, SWV of TP and CAF mixture with different concentrations are recorded. This was performed by simultaneously changing the concentrations of theophylline and caffeine, Fig. 6. It can be seen that the simultaneous determination of TP and CF at P(L-Asp)/f-MWCNTs/GCE provides two well-defined oxidation peaks and both peak current values are proportio nal to the corresponding concentrations in the mixture. The peak current response increases linearly with concentration in the range of 0.1 -50 pM for TP and 1-150 pM for CF, Inset of Fig. 6. The linear regression equations are: Ip (pA) = 0.299(±0.005)C (pM) + 0.20(±0.1), R2 = 0.997 and Ip (pA) = 0.0834(±0.002)C (pM) + 0.51(±0.1), R2 = 0.994, for TP and CF respectively. The LOD of TP is found to be 0.020 pM and that of CF is 0.28 pM (S/N = 3).

Fig. 6. Plot of the peak current of TP and CF vs concentration at P(L-Asp)/f-MWCNTs/GCE in 0.1 M PBS for the mixture of (TP+CF); (a—n): 0.1+1.0, 0.5+2.5, 1.0+5.0, 2.5+10, 5.0+20, 10+30, 15+40, 20+50, 25+60, 30+70, 35+80, 40+100, 45+120 and 50+150 pM. Inset: SWVs of a mixture of TP and CF TP concentrations.

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A comparison between the analytical performance of the present method and other similar literature reports for the simultaneous and individual determination of TP and CF is given in Table 1. The results suggest that the proposed method shows logical linear range and acceptable detection limit. Compared with the other electrochemical methods, the present method has many advantages for the simultaneous determination of TP and CAF, such as simplicity of the electrode preparation, good stability and reproducibility. These results reveal that the P(L-Asp)/f-MWCNTs modified glassy carbon electrode is highly suitable for the selective determination of one analyte in a wide concentration range in the presence of the other, and the proposed method is reliable for the simultaneous and quantitative determination of TP and CF.

Table 1 Comparison of the analytical parameters of the present method with other electrochemical methods reported in the literature.

Electrode type Method Analyte Linear-range ^ LOD Reference

(MM) (MM)

aED-GO/GCE LSV TP 0.8-60 0.10 [47]

bMWCNT/GCE CV TP 0.3-10 0.050 [52]

cMWCNT-PE DPV TP 2-150 0.020 [53]

dPoly(AHNSA)/GCE DPV TP 1-100 0.047 [54]

eMnO2 NPs/MWNT/GCE DPV TP 0.1-20 0.010 [15]

'CTAB-Gr/GCE DPV CF 0.3-100 0.091 [48]

gNafion-Gr/GCE DPV CF 0.4-600 0.12 [49]

hBDDE DPV CF 0.4-25 0.15 [18]

iNafion /SPG electrode DPV CF 3.1-247 1.0 [55]

J SWCNT/CCE DPV CF 0.25-100 0.12 [56]

kLMC/Nafion/GCE DPV TP 0.8-180 0.37 [7]

CF 1.3-230 0.47

lPoly (AV3B)/MWCNT- DPV TP 0.5-120 0.020 [23]

GR/GCE CF 1.0-120 0.10

mAnisotropic GNP/CHIT/IL/ DPV TP 0.025-2.1 0.0013 [24]

rGO/ GCE CF 0.025-2.49 0.0044

P (L-Asp)/f-MWCNT/ GCE SWV TP 0.1-50 0.020 [This work]

CF 1-150 0.28

aElectrodeposited graphene oxide onto a glassy carbon electrode; multi-wall carbon nanotube modified glassy carbon electrode; cmulti-wall carbon nanotube paste electrode; dpoly(4-amino-3-hydroxynaphthalene sulfonic acid) modified glassy carbon electrode; emanganese oxide nanoparticles/multiwalled carbon nanotube nanocomposite modified glassy carbon electrode; fcetyltrimethylammonium bromide-dispersed graphene; gnafion-graphen modified glassy carbon electrode; hboron-doped diamond electrode; inafion-modified disposable screen-printed graphite electrodes; Jsingle-walled carbon nanotubes on carbon-ceramic electrode; klarge mesoporous carbon and nafion composite modified electrode; lpoly(alizarin violet 3B), multiwalled carbon nanotubes and graphene modified electrode; "anisotropic gold nanoparticle-chitosan-ionic liquid modified electrode

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3.9. Interference study

The influence of various potentially interfering substances on the simultaneous determination of 50 pM TP and 100 pM CF were investigated. The tolerance limit was taken as the maximum concentration of the foreign substances that caused an approximately ±5% relative error in the determination. The results (Table S1) show that 50 fold of lactose, D-glucose, citric acid, oxalic acid, glycine, Ca2+, Mg2+, Na+, K+, NO3-, CO32- and 20 fold theobromine and ascorbic acid did not interfere in the simultaneous determination of TP and CF. However, uric acid begins to interfere when present at concentrations greater than two fold of TP and equimolar amounts with CF. From the interference experiments, it seems that the proposed method is selective enough and can be applicable to the simultaneous detection of TP and CF in real samples. Moreover, the interference study suggests the possibility of the simultaneous determination of theophylline, caffeine and theobromine at the composite modified electrode.

3.10. Analytical applications

To investigate matrix effects in the analysis of real samples and examine the analytical utility of the composite modified electrode, SWV was used under the optimized experimental conditions for the simultaneous determination of TP and CF. TP often coexists with CF in the same real samples, such as biological samples and tea products. Thus, green tea, blood serum and pharmaceutical formulation (Panadol extra) were used to see the practical applicability of the modified electrode in this study. The sample preparation is described in experimental section. The standard addition technique was used to determine TP and CF simultaneously by the proposed SWV method. The obtained results are summarized in Table 2. The accuracy and precision of the proposed method was checked by spiking the samples with known amounts of standard TP and CF. It can be seen that good recovery results (92.0 to 106%) were obtained, which indicates the practical applicability of the modified electrode for the simultaneous determination of TP and CF in the real samples.

Table 2 Simultaneous determination of TP and CF in green tea, human serum and Panadol extra samples (n = 3).

Sample Analyte Labeled Found Detected Detected Added Found Recovery

mg/tablet ±RSD (%) (MM) (MM) (MM) (%)

Green TP 10.5 15 25.2 98.0

Tea 40 47.4 92.3

CF 135 10 144 92.0

40 172 93.3

Blood TP - 20 21.1 106

Serum 30 28.6 95.3

CF - 40 38.2 95.5

60 60.4 101

Panado TP - - - - 30 29.7 99.0

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50 75 100

47.8 112 137

95.6 96.0 96.5

60.7±4

3.11. Repeatability, reproducibility and stability of the P(L-Asp)/f-MWCNTs/GCE

To test the repeatability of the modified electrode, the responses of a mixture of TP and CF were measured successively eight times at three different concentrations ratios of TP:CF, 10:40, 50:100 and 75:150 mM. The relative standard deviations (RSD) of the peak currents were found to be 1.4%, 0.96% and 2.8% for TP and 0.97%, 1.8% and 1.7% for CF, respectively. Similarly, the reproducibility of the P(L-Asp)/f-MWCNTs/GCEwas studied by measuring the peak current response of three different electrodes prepared under identical conditions for a mixture of 50 mM TP and 100 mM CF. The interelectrode relative standard deviations were determined to be 2.6% and 2.1% for TP and CF, respectively. These results demonstrate excellent precision and good reproducibility of the electrode.

The long-term stability was also evaluated by keeping the electrode in 0.1 M PBS for 4 weeks at room temperature. The peak current response of 50 mM TP and 100 mM CF was measured every week for one month using SWV in pH 4.5 PBS. The results show that the modified electrode retained 95.8% and 91.4% of the initial response with respect to TP and CF, respectively, which suggests that the proposed sensor has very good stability.

In this study, poly(L-Asp)/f-MWCNTs/GCE has been prepared and used successfully for the simultaneous determination of TP and CF. The modified electrode provides advantages such as ease of preparation, low cost, excellent peak-to-peak separation between theophylline and caffeine, good stability, reproducibility and repeatability. It also exhibits satisfactory sensitivity and high selectivity in voltammetric measurement of TP and CF simultaneously. Furthermore, the method was successfully used to detect TP and CF in green tea, human blood, and Panadol extra real samples with satisfactory recovery results, 92.0-106%. In general, the proposed method provides a useful tool for the simultaneous determination of TP and CF in food, biological, pharmaceutical analysis and clinical applications.

Acknowledgments

The authors acknowledge Department of Chemistry, Addis Ababa University, Addis Ababa, Ethiopia and Wachemo University, Hossana, Ethiopia for supporting this work.

4. Conclusion

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List of Figures

Fig. 1

1732 >-,041081 3401 15831384

f-MWCNTs

MWCNTs

4000 3500 3000 2500 2000 1500 1000 500

Wavenumber (cm-1)

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Fig. 2

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35 i 25 -

—I—

—i— 1.5

E (V) ra Ag/AgCl

Fig. 3

-10 H-1-1-1-1-1-1-1-1-1

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

E (V) ra Ag/AgCl

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Fig. 4

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Ip (^A) = 0.11 C (^M) + 0.914 R2 = 0.995

50 [CF] (^M) 00

0.2 0.4 0.6 0.8 1 1.2

E (V) ra Ag/AgCl

1.4 1.6

Fig. 5

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Fig. 6

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Highlights

P(L-Asp)/f-MWCNTs/GCE was proposed for simultaneous detection of CF and TP. P(L-Asp)/f-MWCNTs composite presents an enhanced electrocatalytic activity. CF and TP were quantified by square wave voltammetry.

The method was sensitive enough to reach a lower LOD of 0.28 (CF) and 0.02 |jM (TP). The method was successfully applied for real sample analysis.