A novel disposable electrochemical sensor based on modifying graphite pencil lead electrode surface with nanoacetylene black for simultaneous determination of antiandrogens flutamide and cyproterone acetate

Journal Pre-proof A novel disposable electrochemical sensor based on modifying graphite pencil lead electrode surface with nanoacetylene black for simultaneous determination of antiandrogens flutamide and cyproterone acetate

Hossieny Ibrahim, Yassien Temerk PII:

S1572-6657(20)30019-9

DOI:

https://doi.org/10.1016/j.jelechem.2020.113836

Reference:

JEAC 113836

To appear in:

Journal of Electroanalytical Chemistry

Received date:

11 December 2019

Revised date:

4 January 2020

Accepted date:

7 January 2020

Please cite this article as: H. Ibrahim and Y. Temerk, A novel disposable electrochemical sensor based on modifying graphite pencil lead electrode surface with nanoacetylene black for simultaneous determination of antiandrogens flutamide and cyproterone acetate, Journal of Electroanalytical Chemistry(2020), https://doi.org/10.1016/ j.jelechem.2020.113836

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© 2020 Published by Elsevier.

Journal Pre-proof A novel disposable electrochemical sensor based on modifying graphite pencil lead electrode surface with nanoacetylene black for simultaneous determination of antiandrogens flutamide and cyproterone acetate Hossieny Ibrahim* and Yassien Temerk* Chemistry Department, Faculty of Science, Assiut University, Assiut, Egypt

Abstract

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A novel nanoacetylene black coated the surface of graphite pencil lead electrode (nano-AB/GPLE) is successfully constructed. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV)

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indicated that the new designed nano-AB/GPLE possessed a fast electron transfer compared with the bare GPLE and enhanced electrochemical response toward electroreduction of antiandrogens flutamide (FLU)

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and cyproterone acetate (CPA) simultaneously. From the nano-AB/GPLE, two well separated reduction signals of coexistence FLU and CPA at nano-AB/GPLE were obtained. Electrochemical sensing of FLU

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and CPA in the presence of the physiological common interferents at nano-AB/GPLE was examined and confirmed the selectivity for the detection analysis of the two antiandrogens without cross interference of

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biological compounds. The limits of detection for coexistence of FLU and CPA are 1.39 nM and 4.74 nM, respectively. Finally, the applicability of the designed sensor is performed for detection of the two

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accuracy and precision.

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antiandrogens FLU and CPA simultaneously in pharmaceutical tablets and biological fluids with good

Keywords: Flutamide, Cyproterone acetate, Simultaneous detection, Nanoacetylene black, Electrochemical sensor, Pharmaceuticals

*Corresponding Authors E-mails: [email protected]; [email protected] (H. Ibrahim), [email protected], [email protected] (Y. Temerk)

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Journal Pre-proof 1. Introduction Antiandrogens are a class of cytostatic drugs used therapeutically in prostate cancer patients [1-7]. Among them, flutamide (FLU, Scheme 1), represents a nonsteroidal and androgen receptor antagonist [8]. As FLU has a structure similar to testosterone, which is a natural hormone, it works by blocking the effect of testosterone and prevents the attachment of the male of hormone [9]. Prostatic cancer patients treated also with the

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steroidal antiandrogen cyproterone acetate (CPA, Scheme 1) [10,11]. In prostatic cells,

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CPA inhibits the binding dihydrotestosterone to androgen receptors and it also suppresses

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the corticotropic axis and thereby reducing effective plasma testosterone levels. It is

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noteworthy that steroidal CPA as well as non-steroidal FLU antiandrogens can be used to inhibit the tumor growth after gonadoreline analog therapy. FLU and CPA are also

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recently approved for use in patients with metastatic prostate cancer refractory to

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gonadorelin analog therapy. The overdosage of FLU and CPA in humans may cause severe side effects such as blood in urine, inflamed prostate, rectal bleeding, liver

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malfunction and methemoglobinemia [12]. Hence, the restricted quality control of these

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anticancer drugs is demanded in order to control potency in vivo. Consequently, the electroquantification of each cytostatic drug FLU and CPA in clinical samples without the effect of interferences is highly recommended. By today, several analytical techniques for the detection of FLU and CPA were reported including chromatography [13-20], spectrophotometry [21-28], spectrofluorimetry [29] and photochemistry [30]. Most of these methods are powerful and accurate, and even some of them are standard techniques for determination procedure of a single drug molecule, but on the other hand, they are very expensive, time consuming and require very complicated steps. In recent

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Journal Pre-proof years, voltammetric sensors have gained greater attention for drug analysis owing to their greater selectivity and sensitivity [31, 32]. In this respect, the detection of each FLU and CPA has been reported by using some electrochemical methods [33-44]. There is still an unmet need for a new sensor to determine the two antiandrogen drugs FLU and CPA simultaneously. In the last decade, graphite pencil lead electrodes (GPLEs) have acquired great attention in electrochemistry due to its low technology, low cost, disposability,

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renewability and ease of modification [45-50]. In order to improve the redox capacity and

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electrocatalytic effect of GPLE we present a novel concept of electrode composed of

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nano-AB coated GPLE. Nanoacetylene black has recently found comprehensive and

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impressive study applications in electroanalytical chemistry, particularly in electrode modification towards the electrochemical reduction of organic molecules, due to their

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excellent electrical conductivity, large surface area and powerful adsorptive capacity [51-

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54]. Herein, the electrocatalytic activity of the designed electrode enhanced which was applied for simultaneous detection of prostate anticancer drugs FLU and CPA in

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pharmaceutical tablets and human biological fluids. Surveying the literature, the

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advanced electrochemical sensor of nano-AB/GPLE as a new modified electrode has been not reported.

In the present work, nano-AB modified GPLE as a novel electrochemical sensor is designed for simultaneous analysis of cytostatic drugs FLU and CPA via square wave adsorptive cathodic stripping voltammetry (SWAdCSV) for the first time. The newly fabricated electrode achieved the detection of the two anticancer drugs FLU and CPA in a single voltammetric run. In view of this fact, the novel sensor (nano-AB/GPLE) was

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Journal Pre-proof used successfully for the determination of anticancer drugs FLU and CPA in human urine and serum blood samples and pharmaceutical tablets. 2. Experimental 2.1. Reagents and solutions Flutamide (FLU, ≥99%) and cyproterone acetate (CPA, ≥98%) were purchased from Sigma–Aldrich chemicals. Nanoacetylene black (nano-AB) was obtained from Alfa

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Aesar. Stock solutions of FLU (1×10-3 M) and CPA (1×10-3 M) were freshly prepared by

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dissolving an equivalent amount of drugs in ethanol and methanol, respectively.

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Phosphate buffer solution (PBS) as the supporting electrolyte was prepared using 0.1 M

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Na2HPO4 and NaH2PO4 solutions. The pH of buffer solution was adjusted using 0.1 M

2.2. Apparatus and procedures

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H3PO4 or NaOH solution. Deionized water was used in all experiments.

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Measurements of CV and SWAdCSV were performed using a polarographic analyzer EG&G PAR 384 B. An Interface 1000E Potentiostat / Galvanostat / ZRA model

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was used to measure EIS. The morphology of GPLE and nano-AB/GPLE was

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characterized by scanning electron microscopy (SEM). A three electrode system was used consisting of a reference electrode (Ag/AgCl, KClsat), the auxiliary electrode (Pt wire) and graphite pencil lead electrode modified with nano-AB (nano-AB/GPLE) as a working electrode. Graphite pencil leads (GPLs) 0.5 mm in diameter and 60 mm in total length (2B, Rotring, Germany) were employed. A new design of a mechanical pencil, which was used as the holder for the pencil lead, was constructed for the first time (Scheme 2). Electrical contact to the GPL was obtained by wrapping a copper wire to the metallic front part of the holder. The graphite pencil lead was retained vertically with 10

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Journal Pre-proof mm immersed in the solution. For voltammetric experiments, 5.0 ml of the electrolyte solution was placed in a voltammetric cell and saturated with nitrogen (15 min) to eliminate any oxygen. 2.3. Fabrication of nano-AB/GPLE The electrochemical sensor was constructed according to the following steps.

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Firstly, GPLEs were vertically put in a Teflon holder and were treated with 6 M HNO3 solution in ultra-sonication bath for 15 min. Next, they were washed in Millipore water

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for 30 min followed by sonication for 15 min in the acetone solution. To evaporate the

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solvent, the treated GPLEs samples were kept for drying for 2 h at 60 °C. A cellulose

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acetate (CA) solution (0.5%, w/v) was prepared in a mixture of solvents containing

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acetone and cyclohexanone (1:1). The solution was stirred for 3 h to completely dissolve CA. Thereafter, 4.5 mg nano-AB was dispersed in 1 mL CA solution and sonicated for 30

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min to form a black homogeneous suspension. The pretreated GPLEs were dipped in

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nano-AB dispersion. This led to the formation of a film of nano-AB on the surface of GPLE (nano-AB/GPLE). To accommodate the maximum amount of nano-AB on the

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GPLE surface, this complete process of dip coating was repeated three times. Finally, the fabricated nano-AB/GPLEs were heated in an oven for 2h at 90 °C to evaporate the solvents. The procedure of the fabricated nano-AB/GPLE was shown in Scheme 3. 2.4. Analysis of FLU and CPA in real samples Following approval by the Assiut University Hospital Ethical Clearance Committee, samples of human serum and urine were collected. Samples of serum and urine were diluted with PBS properly to decrease the complexity of the matrix. The analyzed

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Journal Pre-proof solution was transferred without further pretreatments into the voltammetric cell. To prepare the solution of FLU and CPA commercial samples, two dosage forms were used: Andoxin contained 250 mg of FLU and Androcur contained 50 mg CPA. Ten tablets of each examined pharmaceutical formulation were weighed accurately and finely powdered in a mortar. An equivalent amount of each Andoxin and Androcur was weighed then transferred to a 50 ml calibrated flask, which was completed to volume with ethanol and

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methanol, respectively. For the detection of FLU and CPA in real samples, the standard

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addition method was used.

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3. Results and discussion

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3.1. Surface morphology

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The studies of surface morphologies GPLE and nano-AB/GPLE were done by SEM analysis at different magnifications (Fig. 1). As shown in Fig. 1A, the bare GPLE has a

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regularly lined surface. The topography of the nano-AB/GPLE surface shown in Fig. 1D

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is completely different from that of Fig. 1A that confirms a successful coating of GPLE with nano-AB. The sphere-like structure morphology of nano-AB with its rough surface

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increases the active surface area and making surface of GPLE more electrically conductive (Fig. 1F). Moreover, the presence of nano-AB at the electrode surface leads to an increase in the surface coverage for adsorption of FLU and CPA. 3.2. Electrochemical characterization of nano-AB/GPLE The electrochemical characterization of the nano-AB/GPLE was examined and compared to unmodified GPLE using CV. In this context, CV of ferri/ferrocyanide is an effective tool to monitor the surface status. Fig. 2A shows the cyclic voltammograms of 5 mM [Fe(CN)6]3–/4– containing 0.1M KCl using unmodified GPLE and the modified nano6

Journal Pre-proof AB/GPLE. It is found that at bare GPLE, a pair of redox peaks of [Fe(CN)6]3–/4– was obtained with peak-to-peak separation (∆EP) as 368 mV (curve 1), whereas at nanoAB/GPLE both anodic and cathodic currents enhanced clearly with the ∆EP value decreased to 237 mV (curve 2), showing the superiority of nano-AB/GPLE than GPLE. This confirms that nano-AB is a specific carbon nanomaterial with a large surface area and high conductivity that can accelerate the transfer of electrons.

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For further characterization of the surface of nano-AB/GPLE, electrochemical

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impedance spectroscopy (EIS) was measured in 0.1M KCl using [Fe(CN)6]3–/4– as

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electroactive probe. Fig. 2B showed the Nyquist plots at the GPLE (curve 1) and nano-

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AB/GPLE (curve 2). As shown in Table S1, the charge transfer resistance (Rct) was significantly decreased confirming an effective nano-AB mediated charge transfer

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process at GPLE surface. In this context, the internal resistance of nano-AB/GPLE was

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230 Ω, while Rct of the bare GPLE was 1200 Ω, which could be attributed to the good conductivity of nano-AB/GPLE. Hence, nano-AB enhanced the electrochemical

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performance and facilitated the electron transfer process. This phenomenon also

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confirmed that nano-AB was successfully coated on the GPLE surface. The results obtained of CV and EIS measurements demonstrated that nano-AB/GPLE could promote the electron transfer rate of [Fe(CN)6]3–/4– effectively. The influence of different scan rates on the electrochemical behavior of [Fe(CN)6]3–/4– at GPLE and nano-AB/GPLE was studied using CV (Fig. S1). A linear relationship was found between the CV peak currents (Ip) and the square root of scan rate (ν1/2), indicating a diffusion-controlled process.

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Journal Pre-proof In order to evaluate the active surface area (A) for GPLE and nano-AB/GPLE, Randles – Sevcik Eq.(1) was used [55]: Ipa = 2.69 × 105 n3/2 A D1/2 ν1/2 C

(1)

From the slope of Ip ‒ ν1/2, the values of A are calculated to be 0.21 cm2 and 0.33 cm2 for GPLE and nano-AB/GPLE, respectively (Table S1). The result further showed that the

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existence of nano-AB increased the active surface area of the electrode, which led to greater enhancement of the electrochemical response of the sensor.

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3.3. Electrochemical reduction responses of FLU and CPA at nano-AB/GPLE

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The electrochemical reduction responses of coexistence of the anticancer drugs FLU

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and CPA were studied first by CV at GPLE and nano-AB/GPLE and the results were

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presented in Fig. 3A. In the case of the bare GPLE, the reduction of each FLU and CPA exhibited poor responses due to a slow kinetic electron transfer. However, after GPLE

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coated with nano-AB, two well-defined and sensitive cathodic peaks were obtained. The first reduction peak located at ‒ 0.81 V corresponding to the direct reduction of –NO2

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group of FLU molecule [34]. The second cathodic peak occurred at ‒ 1.21 V and is

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attributed to the reduction of the carbonyl group present in CPA [38]. On the reverse scan of the CV no anodic peaks are observed, which confirms that the electroreduction of both FLU and CPA is an irreversible process. The obtained results show that the reduction peak potentials derived for FLU and CPA in the binary system are well separated at the nano-AB/GPLE. Moreover, the significant improvement of the reduction peak currents at the nano-AB/GPLE reveals that the modified electrode has a good electrocatalytic sensing of FLU and CPA compared with GPLE. Therefore, the nano-AB/GPLE is highly

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Journal Pre-proof effective for rapid transfer of analysis, providing sensitive quantification of the two anticancer drugs FLU and CPA in biological fluids. Furthermore, the electroreduction responses of coexistence of FLU and CPA at the nano-AB/GPLE were studied by SWAdCSV as shown in Fig. 3B. SW voltammograms of the coexistence of FLU and CPA at the surface of nano-AB/GPLE demonstrated that the reduction peak currents of the two drugs significantly increased about 7-fold higher than

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the bare GPLE. Moreover, the difference of the reduction peak potentials for FLU-CPA

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was 425 mV which was sufficiently enough for simultaneous detection of FLU and CPA

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in a binary system.

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The electrochemical cathodic responses of FLU and CPA separately in pH 7.0 PBS were also investigated by SWAdCSV at the nano-AB/GPLE (Fig. 4). Individual SW

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voltammograms of FLU (curve 1) and CPA (curve 2) are identical to the voltammetric

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responses (curve 3) obtained in the mixture for both drugs. Hence, the determination of

detections.

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FLU and CPA simultaneously can be considered as efficient as their individual

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The effect of scan rate on the electroreduction of FLU and CPA at the nanoAB/GPLE was investigated by CV. Fig. S2 shows CVs of 5.66 μM FLU and 7.48 μM CPA at different scan rates. The cathodic peak intensity for each the reduction of FLU and CPA increased continuously with increasing the scan rate, indicating an adsorption controlled process. The plot of log Ip against log ν, straight lines were obtained (Fig. S2) that can be presented as: log IPc (μA) = 0.83 log ν (mVs-1) – 0.72 (R2 = 0.9993) for FLU log IPc (μA) = 0.77 log ν (mVs-1) – 0.59 (R2 = 0.9974) for CPA

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Journal Pre-proof The slopes of log IP ‒ log ν plots were found to be 0.83 and 0.77 for FLU and CPA, respectively, confirming the adsorption controlled process. Therefore, the adsorption of coexistence of FLU and CPA at the nano-AB/GPLE surface can be considered as a high sensitive possibility for their detection due to the successful accumulation step previous to the voltammetric analysis. The surface coverage of the two anticancer drugs FLU and CPA (ГFLU & ГCPA, mol

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cm−2) on the nano-AB/GPLE and GPLE can be calculated by using: Γ = Q/nFA, where Q

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is the electroreduction charge. The values of ГFLU and ГCPA on the nano-AB/GPLE are

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estimated to be 2.76×10–11 mol cm−2 and 3.19×10–11 mol cm−2, respectively. Whereas the

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corresponding values of ГFLU and ГCPA on GPLE are to be 3.74×10–12 mol cm−2 and

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5.59×10–12 mol cm−2. These values confirmed that nano-AB coated GPLE has distinctive

adsorptive capacity.

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characteristics, such as excellent electrocatalytic activity, large surface area and powerful

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3.4. Effect of pH on the electrochemical reduction of FU and CPA

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In this work, SWAdCSV was performed to characterize the influence of solution pH on the electrochemical behavior of FLU and CPA in the binary system at the nanoAB/GPLE (Fig. 5). The obtained results showed that a gradual increase in the cathodic peak current (Ipc) of the two drugs on the modified electrode when pH was increased from to 3 to 7 and decreased upon further increase of pH 8. Hence, pH 7.0 was selected as the optimum value for simultaneous determination of FLU and CPA. Furthermore, it is observed that the reduction peak potential values change linearly with pH (Fig. S3) and the regression equations for both FLU and CPA were defined as: Epc (V) = –0.31 – 0.062 pH (R2 = 0.997) for FLU 10

Journal Pre-proof Epc (V) = –0.71 – 0.063 pH (R2 = 0.981) for CPA The slopes 62 mV/pH for FLU and 63 mV/pH for CPA were obtained, which described the Nernst equation. Thus, it can be logically thought that the proton number equals the number of transfered electrons in the electroreduction of FLU and CPA. 3.5. Optimization of parameters 3.5.1. Effect of square wave parameters

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In order to evaluate the optimum electrochemical measurements for simultaneous

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determination of FLU and CPA, their SW voltammograms in PBS of pH 7.0 at the nano-

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AB/GPLE were studied at different parameters. The variable parameters of interest were

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examined over the range 5‒35 mV of pulse amplitude (ΔEa), 20‒120 Hz of square wave frequency (ƒ) and 2‒10 mV of scan increment (ΔEs). The following parameters were

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used to achieve the SW voltammograms of high sensitivity and best peak morphology: ƒ

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= 80 Hz, ΔEa = 25 mV and ΔEs = 6 mV using the nano-AB/GPLE. 3.5.2. Influence of accumulation time and potential

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The CVs of FLU and CPA recorded at the nano-AB/GPLE at different potential scan

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rates revealed that the reduction process of the two drugs is indicative of their adsorption on the nano-AB/GPLE surface. Hence, the influence of accumulation time (tacc) on the reduction responses of FLU and CPA was also investigated by SWAdCSV (Fig. S4). As expected the SW peak current of the two drugs increased greatly with first 90s and then it becam constant suggesting that the accumulation of FLU and CPA rapidly reached saturation [56]. Taking account, the sensitivity and the working efficiency an tacc = 90 s was chosen as the optimal accumulation for stripping analysis of coexistence FLU and CPA at the nano-AB/GPLE. The reduction peak current of both FLU and CPA was also

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Journal Pre-proof measured after 90 s at different accumulation potentials (Eacc) from ‒0.3 V to ‒0.55 V. The reduction peak currents of FLU and CPA increased notably as the Eacc changed from ‒0.3 V to ‒0.55 V. When Eacc was exceeded ‒0.55 V, the reduction response conversely showed gradual decrease. Hence, ‒0.55 V was applied as accumulation potential in the following experiments. 3.5.3. Effect of nano-AB

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Our results revealed that the modification GPLE surface using nano-AB

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significantly enhanced the redox capacity of the coexistence FLU and CPA. Hence, it

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was expected that the amount of nano-AB for modifying GPLE surface exerts a

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significant on the cathodic adsorptive stripping analysis of both FLU and CPA. In order to optimize the responses of FLU and CPA, six modified electrodes were performed

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containing different concentrations of nano-AB. Fig. 6 clearly revealed that the cathodic

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peak currents of FLU and CPA reached the highest values at 4.5 mg/mL of the modifier, indicating that the effective surface area of the nano-AB/GPLE is improved and then the

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reduction peak responses are enhanced. However, the reduction peak currents of FLU and

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CPA decrease when the amount of nano-AB modifying the GPLE increases from 6 to 8 mg/mL, suggesting too much nano-AB is not beneficial for FLU and CPA sensing. Therefore, the obtained result indicated that 4.5 mg/mL nano-AB is the saturation point to facilitate the charge transport between nano-AB/GPLE and both FLU and CPA. Hence, we have used 4.5 mg/mL nano-AB as the optimum concentration in the modification of GPLE surface. 3.6. Simultaneous electrochemical detection of FLU and CPA

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Journal Pre-proof In order to investigate the simultaneous electroanalysis of FLU and CPA, SWAdCSV was performed to examine the sensitivity of nano-AB/GPLE sensor towards the detection of the two anticancer drugs. In this context, two separate experiments were performed at pH 7.0. Fig. 7A shows the consecutive additions of FLU in the range of 2.03×10-8 M ‒ 3.37×10-7 M in the presence of 7.33×10-7 M CPA. Interestingly, the cathodic peak intensity of FLU increases with its concentration while the response of

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CPA remains constant. No obvious potential mutual interference of FLU and CPA was

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observed, confirming that their reduction at the nano-AB/GPLE sensor take place

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independently. The variation of peak current with concentration of FLU was linear at a fixed concentration of CPA (Fig. 7B). The linear function can presented as: IPc (μA) = ‒

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1.33 + 7.45 × 10-7 CFLU (M), (R2 = 0.9992) and the detection limit of FLU was 1.49 nM.

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Similarly, sensing of CPA in the presence of FLU at nano-AB/GPLE was achieved. In

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this context, the independent determination of CPA in the concentration range of 3.44×10-8 M to1.07×10-6 M CPA was accomplished in solution, containing 2.55×10-7 M

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FLU (Fig. 8A). At a fixed concentration of FLU, the reduction peak current of CPA

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increases regularly as its concentration is increased (Fig. 8B). The corresponding linear function can be presented as: IPc (μA) = ‒0.11 + 1.37 × 10-7 CCPA (M), (R2 = 0.9989) and the detection limit of CPA was 4.82 nM. The aforementioned results indicated that nano-AB/GPLE has a strongly electrocatalytic capability to simultaneously discriminate the two anticancer drugs FLU and CPA. Hence, we tried for recording reduction signals of these anticancer drugs, with different concentrations of them at the optimum conditions. As can be seen in Fig. 9A, the FLU and CPA appeared two separated reduction signals with ΔEP = 419 mV that is

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Journal Pre-proof sufficient for their simultaneous analysis. Their SWAdCSV reduction peak currents linearly increased with the increase in FLU and CPA concentrations (Fig. 9B). The sensitivities of the fabricated sensor toward the reduction of FLU and CPA were found to be 894.89 and 329.58 μA μM-1 cm-2, respectively (Table 1). The calibration plots for FLU and CPA present good linear responses in the concentration range of 0.026 – 0.477 µM FLU and 0.066 – 1.10 µM CPA. The corresponding calibration equations are IPc (μA)

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= ‒1.87 + 8.59 × 10-7 CFLU (M), (R2 = 0.9989) and IPc (μA) = ‒1.08 + 3.16 × 10-7 CCPA

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(M), (R2 = 0.9997). The calculated limits of detection (LOD) for FLU and CPA in the

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mixture are 1.39 and 4.74 nM, respectively which revealed that the nano-AB/GPLE

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shows promising for simultaneous determination of the two anticancer drugs in clinical samples. Moreover, Table 2 indicates that the detection limits are quite lower than those

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of many other methods for the determination of FLU and CPA separately. Therefore, the

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electrochemical responses of FLU and CPA on nano-AB/GPLE surface were greatly enhanced which provided a platform to simultaneously determine both drugs in

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commercial pharmaceutical tablets and biological fluids.

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The precision and accuracy of the proposed SWAdCSV using the fabricated sensor nano-AB/GPLE for simultaneous analysis of FLU and CPA were examined (Table S2). The obtained results indicated that the RSD% values were not higher than 1.9 and 2.1% for the intra-day and inter-day determinations, respectively. Also, the value of recovery varies from 97.33% to 102.5%. Hence, the precision and accuracy of the proposed SWAdCSV method were fairly good, confirming an excellent repeatability of response of nano-AB/GPLE. 3.7. Electrochemical sensing of FLU and CPA in the presence of interferents

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Journal Pre-proof Sensing the two drugs in the presence of interferents which commonly existed in clinical samples was investigated using SWAdCSV. The experimental results showed that the electrochemical responses of FLU and CPA were monitored in the presence of high concentrations of biological co-active species. The nano-AB modified GPLE displayed well-defined response in each addition of coexistence of FLU and CPA whereas no noteworthy responses for 500 fold excess concentrations of biologically co-

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active species were observed (Table S3). Moreover, the measurement results of analysis

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of FLU and CPA in the recovery range 98.98‒101.34% confirm the high performance

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ability of nano-AB/GPLE for analysis of these anticancer drugs. This indicates that

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sensing of coexistence of FLU and CPA could be achieved without cross interference of several pharmaceutical and biological substances. It may be concluded that the selectivity

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analysis in clinical samples.

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of the fabricated sensor toward FLU and CPA shows promising properties of their

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3.8. Stability and reproducibility of the nano-AB/GPLE

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To demonstrate the stability of nano-AB/GPLE, the SWAdCSV was conducted in solution containing FLU (1.03×10-7 M) and CPA (2.56×10-7 M). The observed voltammograms show no significant change in the reduction peak currents even after five consecutive SWAdCSVs. Furthermore, the storage stability was varied by keeping the fabricated sensor at room temperature for 30 days (Fig. S5). During this period there was only a slight decrease in the peak current, where the electrode retained 96.1% of its initial response and consequently there is no surface fouling of the modified electrode. To characterize the reproducibility of the fabricated electrode, eight nano-AB/GPLEs were fabricated simultaneously in the same conditions. The values of RSD% for FLU and CPA 15

Journal Pre-proof were estimated to be 2.27% and 2.3%, respectively. These results confirm that the nanoAB/GPLE possessed high reproducibility and stability for simultaneous determination of FLU and CPA in real samples. 3.9. Applications of nano-AB/GPLE sensor 3.9.1. Assay of FLU and CPA in commercial tablets

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The applicability of the proposed nano-AB/GPLE was examined by simultaneous

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determination of the content of FLU and CPA in Androxin and Androcur tablets as real

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pharmaceutical samples using SWAdCSV. Typical standard additions voltammograms for coexistence of FLU and CPA are shown in Fig. 10. The increase in peak currents after

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addition of the binary system of FLU and CPA to the pharmaceutical sample indicated

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that the well-defined peaks located at ‒0.79 V and ‒1.21 V corresponding to the reduction of FLU and CPA, respectively. The amount of the investigated drugs in

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pharmaceutical tablets was calculated by reference to the appropriate calibration plots.

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The accuracy and precision of the proposed SWAdCSV using nano-AB/GPLE for

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simultaneous analysis of FLU and CPA were investigated and the results are summarized in Table 3. The recovery values are in the range from 99.42% to 102.24%. Thus, the presented results indicate that the modified GPLE with nano-AB could be applied successfully for simultaneous determination the content of FLU and CPA in pharmaceutical tablets with high sensitivity and excellent selectivity. 3.9.2. Assay of FLU and CPA in biological fluids Simultaneous analysis of anticancer drugs FLU and CPA in biological fluids such as human blood serum and urine samples demonstrated the practical application of the 16

Journal Pre-proof developed sensor. Fig. 11A shows representative SWAdCS voltammograms for simultaneous determination of coexistence FLU and CPA spiked in human serum samples. As shown in Fig. 11A the two peaks at ‒0.8 V and ‒1.21 V correspond to the reduction of FLU and CPA, respectively, and are used for electroquantification of these anticancer drugs in serum samples. Potentially interfering compounds that may be present in serum samples do not indicate any electrochemical reduction where the analytical

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peaks appear. As can be seen in Fig. 11B, the reduction peak currents of FLU and CPA

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are linearly proportional to their concentrations. The corresponding calibration equations

CCPA (M), (R2 = 0.997). The LOD values for FLU and CPA in the spiked serum samples

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7

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are IPc (μA) = ‒2.1 + 5.43 × 10-7 CFLU (M), (R2 = 0.9998) and IPc (μA) = ‒1.55 + 1.94 × 10-

are 1.55 nM and 4.8 nM, respectively. The obtained results show that the modified

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electrode gives recoveries in the range of 97.86% to 103.75% with acceptable RSD%,

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indicating good accuracy of nano-AB/GPLE in the simultaneous analysis of anticancer

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drugs in human serum sample (Table S4). The fabricated nano-AB/GPLE was also applied successfully for simultaneous

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analysis of the two anticancer drugs FLU and CPA in spiked human urine samples. Fig. 12A illustrates the response of successive standard additions of FLU and CPA. The peak currents increased after a binary mixture of FLU and CPA was added to the urine sample. The linear relationships were obtained between the peak currents and the concentrations of FLU and CPA in the urine sample (Fig. 12B). The linear regression equations are IPc (μA) = ‒0.7 + 5.1 × 10-7 CFLU (M), (R2 = 0.9996) and IPc (μA) = ‒0.5 + 1.85 × 10-7 CCPA (M), (R2 = 0.9998). The LOD values for FLU and CPA are 1.6 nM and 4.7 nM, respectively indicating the sensitive determination of the two drugs in human urine 17

Journal Pre-proof sample at the disposable nano-AB/GPLE. Moreover, the recovery values from 97.5% to 102.28% with RSD% less than 2.5%, confirmed that nano-AB/GPLE has a high reproducibility for assay of FLU and CPA (Table S4). In view of the above results, the advanced electrochemical sensor nano-AB/GPLE is promising for biologically relevant determinations of coexistence of FLU and CPA in-vivo and in-vitro.

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4. Conclusions

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In the present work, we report the fabrication of a novel and advanced

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electrochemical sensor based on disposable graphite pencil lead electrode coated with nanoacetylene black for the first time. The electrochemical sensing of antiandrogens FLU

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and CPA at nano-AB/GPLE in PBS of pH 7.0 exhibits two separated reduction signals

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for simultaneous analysis of the two drugs with ΔEP = 425 mV. The detection limits for coexistence of FLU and CPA sensing at the developed nano-AB/GPLE sensor were 1.39

na

nM and 4.74 nM, respectively. The electrochemical sensor nano-AB/GPLE showed good

ur

ability for simultaneous analysis of the antiandrogens FLU and CPA in pharmaceutical

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tablets and blood serum and human urine samples. The recovery values ranged from 97.5% to 103.75% with an acceptable RSD% less than 2.5%. Moreover, the developed sensor has some dominant advantages including the cost effectiveness of the sensor, the short fabrication time, disposability, high stability, good reproducibility, environmental friendly and broad detection which provided a new avenue to simultaneously determine at least two anticancer drugs in clinical patient samples.

18

Journal Pre-proof References [1] R.O. Neri, N.Y. Kassem, Biological and clinical properties of antiandrogens, Prog. Cancer Res. Ther. 31 (1984) 507–518. [2] G.H. Jacobi, U. Tunn, T.H. Senge, Clinical experience with cyproterone acetate for palliation of inoperable prostate cancer, In Prostate Cancer 3 (1982) 305–319. [3] F. Labrie, A. Dupont, A. Belanger, C. Labrie, Y. Lacouriere, J.P. Raynaud, J. M.

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[31] H. Ibrahim, Y. Temerk, A novel electrochemical sensor based on B doped CeO2 nanocubes modified glassy carbon microspheres paste electrode for individual and simultaneous determination of xanthine and hypoxanthine, Sensors and Actuators B 232 (2016) 125–137. [32] H. Ibrahim, Y. Temerk, Sensitive electrochemical sensor for simultaneous determination of uric acid and xanthine in human biological fluids based on the nano-boron doped ceria modified glassy carbon paste electrode, J.Electroanal.Chem. 780 (2016) 176–186. [33] A. Snycerski, Polarographic determination of flutamide, J.Pharm.Biomed.Anal. 9 (1989) 1513–1518. 22

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serum, Biosensors and Bioelectronics 25 (2010) 2497–2502. [48] Y.M. Temerk, M.S. Ibrahim, M. Kotb, W. Schuhmann, Renewable pencil electrodes for highly sensitive anodic stripping voltammetric determination of 3hydroxyflavone and morin in bulk form and in biological fluids, Electroanalysis 25 (2013) 1381–1387. [49] Y. Temerk, H. Ibrahim, W. Schuhmann, Simultaneous anodic adsorptive stripping voltammetric determination of luteolin and 3-hydroxyflavone in biological fluids using renewable pencil graphite electrodes, Electroanalysis 31 (2019) 1095–1103. [50] Y.M. Temerk, H.S.M. Ibrahim, Individual and simultaneous square wave 24

Journal Pre-proof voltammetric determination of the anticancer drugs emodin and irinotecan at renewable pencil graphite electrodes, J. Braz. Chem. Soc. 24 (2013) 1669–1678. [51] P.H. Deng, Z. F. Xu, J. H. Li, Y. F. Kuang, Acetylene black paste electrode modified with a molecularly imprinted chitosan film for the detection of bisphenol A, Microchim. Acta 180 (2013) 861–869. [52] H. Ibrahim, M. Ibrahim, Y.Temerk, A novel megestrol acetate electrochemical sensor based on conducting functionalized acetylene black–CeO2NPs nanohybrids

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CRediT author statement

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Hossieny Ibrahim: Conceptualization, Methodology, Formal analysis, Data curation, Writing - Review & Editing, Submission of paper. Yassien Temerk: Writing - Original Draft, Reviewing, Visualization, Supervision.Both authors read and approved the final version of the manuscript.

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Declaration of interests

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☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Fig. 1 The SEM photographs of GPLE (A, B&C) and nano-AB/GPLE (D, E&F) at different magnifications Fig. 2 (A) CVs and (B) Nyquist plots of EIS of (1) bare GPLE and (2) nano-AB/GPLE in

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5 mM [Fe(CN)6]3–/4– containing 0.1M KCl at a scan rate of 100 mVs-1.

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Fig. 3 (A) CVs at bare GPLE (1) and nano-AB/GPLE (2) PBS (pH 7.0) containing 5.66 μM FLU and 7.48 μM CPA. (B) SWVs at bare GPLE (1) and nano-AB/GPLE (2) in PBS (pH 7.0) containing

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2.55×10-7 M FLU and 4.56×10-7 M CPA; accumulation potential, -0.55 V; accumulation time, 90 s;

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scan increment, 6 mV; frequency, 80 Hz and pulse height, 25 mV.

Fig. 4 SW voltammograms of (1) 2.12 ×10-7 M FLU, (2) 7.33 ×10-7 M CPA and (3) 2.12

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×10-7 M FLU + 7.33 ×10-7 M CPA at nano-AB/GPLE in PBS at pH 7.0.

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Fig. 5 SW voltammograms for a mixture 2.55×10-7 M FLU and 4.56×10-7 M CPA on the surface of nano-AB/GPLE at different pH values (PBS): (1) pH 3, (2) pH 4, (3) pH 5, (4) pH 6; (5) pH 7 and (6)

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pH 8; accumulation time, 90s; scan increment, 6 mV; frequency, 80 Hz and pulse height, 25 mV.

Fig. 6 Effect of the amount of nano-AB on the cathodic adsorptive stripping peak

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currents of a mixture of 2.55×10-7 M FLU and 4.56×10-7 M CPA at nano-AB/GPLE in PBS of pH 7.0.

Fig. 7 (A) SWVs of different concentrations of FLU in presence of 7.33×10-7 M CPA (pH 7) at nano-AB/GPLE (1) 0.0, (2) 2.03×10-8, (3) 4.47×10-8, (4) 7.10×10-8 , (5) 9.67×10-8, (6) 1.34×10-7 , (7) 1.62×10-7 , (8) 2.12×10-7, (9) 2.67×10-7 and (10) 3.37×10-7 M FLU. (B) Plot of IP (µA) versus [FLU]. Error bar represents the standard deviation of triple measurements. Fig. 8 (A) SWVs of different concentrations of CPA in presence of 2.55×10-7 M FLU (pH 7) at nano-AB/GPLE (1) 0.0, (2) 3.44×10-8, (3) 9.95×10-8, (4) 1.99×10-7, (5) 2.98×10-7, (6) 3.96×10-7 , (7) 4.95×10-7, (8) 5.90×10-7, (9) 6.90×10-7, (10) 7.85×10-7, (11)

27

Journal Pre-proof 8.85×10-7 and (12) 1.07×10-6 M CPA. (B) Plot of IP (µA) versus [CPA]. Error bar represents the standard deviation of triple measurements. Fig. 9 (A) SWVs for nano-AB/GPLE in PBS (pH 7.0) containing different concentrations of FLU + CPA in bulk solution, (1) to (10): (1) Blank, (2) 0.026 + 0.066, (3) 0.052 + 0.131, (4) 0.078 + 0.194, (5) 0.102 + 0.256, (6) 0.156 + 0.353, (7) 0.212 + 0.456, (8) 0.274 + 0.567, (9) 0.361 + 0.774 and (10) 0.477 + 1.10 μM, respectively. (B) Calibration plots of IP (μA) vs. [FLU] and [CPA]. Error bar represents the standard deviation of triple

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measurements. Fig. 10. (A) SW voltammograms for determination of FLU in Androxin tablet solution

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and CPA in Androcur tablet solution at nano-AB/GPLE. 1) tablet sample, 2) (1) + 0.06 + 0.18, 3) (2) + 0.13 + 0.38, 4) (2) + 0.22 + 0.62 , 5) (2) + 0.33 + 0.88 and 6) (2) + 0.44

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+1.14 μM FLU and CPA, respectively. (B) The corresponding FLU standard addition

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plot. (C) The corresponding CPA standard addition plot.

Fig. 11 (A) SWVs for nano-AB/GPLE in PBS (pH 7.0) containing different

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concentrations of FLU + CPA spiked in human serum samples, (1) to (9): (1) serum sample, (2) 0.052 + 0.131, (3) 0.084 + 0.202, (4) 0.11 + 0.282, (5) 0.14 + 0.379, (6) 0.181

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+ 0.568, (7) 0.253 + 0.779, (8) 0.357 + 0.976 and (9) 0.524 + 1.25 μM, respectively. (B) Calibration plots of IP (μA) vs. [FLU] and [CPA]. Error bar represents the standard

ur

deviation of triple measurements.

Fig. 12 (A) SWVs for nano-AB/GPLE in PBS (pH 7.0) containing different

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concentrations of FLU + CPA spiked in human urine samples, (1) to (9): (1) urine sample, (2) 0.052 + 0.066, (3) 0.078 + 0.133, (4) 0.103 + 0.199, (5) 0.153 + 0.264, (6) 0.191 + 0.363, (7) 0.277 + 0.552, (8) 0.355 + 0.767 and (9) 0.492 + 1.10 μM, respectively. (B) Calibration plots of IP (μA) vs. [FLU] and [CPA]. Error bar represents the standard deviation of triple measurements. Scheme 1 Chemical structures of Flutamide (FLU) and Cyproterone acetate (CPA)

Scheme 2 New design of graphite pencil lead holder Scheme 3 Schematic diagram showing the fabrication of the nano-AB/GPLE

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Table 1: Regression data of the calibration lines for simultaneous quantitative determination of FLU and CPA in PBS (pH 7.0) at nano-AB/GPLE using SWCASV. FLU

CPA

Linearity range (μM)

0.026 – 0.477

0.066 – 1.10

Slope (μAμM )

85.91

31.64

SE of slope

0.25

0.10

Intercept (μA)

–1.87

–1.08

SE of intercept

0.86 2

0.9989

LOD (nM)

1.39 -1

ro 0.9997

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Determination coefficient (R )

0.19

-p

-1

-2

894.89

Repeatability of peak current (RSD%)

4.74

329.58

2.13

2.35

2.27

2.30

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Reproducibility of peak current (RSD%)

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Sensitivity (μA μM cm )

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Parameters

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Journal Pre-proof Table 2: Comparison of limits of detection for determination of FLU and CPA using some analytical methods. Technique

Method

LOD (µM)

Ref.

0.19 0.18 9.33 0.026 0.42 0.21 3.62×10-3 1.12 0.041 1.39×10-3

[39] [40] [42] [41] [44]

0.31 0.26, 0.33 2.39 13.3 4.74×10-3

[38] [25] [27] [28] This work

ESI-MS Spectrophotometric HPLC Voltammetry

CASV DPV CV DPV DPV SWV -

SWAdCSV

[24] [22] [13] This work

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Voltammetry Voltammetry Voltammetry Voltammetry Voltammetry

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lP

DPP SWAdCSV

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CPA Polarography Spectrophotometry Spectrophotometry Spectrophotometry Voltammetry

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FLU

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CASV: cathodic adsorptive stripping voltammetry, DPV: differential pulse voltammetry, CV: cyclic voltammetry, ESI-MS: electrospray ionization mass spectrometry, DPP: differential pulse polarography

30

Journal Pre-proof Table 3:

Recovery %

Bias %

248.56

99.42

0.58

249.11

99.64

0.36

251.42

100.57

-0.57

248.98

99.59

0.41

51.05

102.10

-2.1

Androcur tablets

50.44

100.88

-0.88

50 mg

51.12

102.24

-2.24

50.87

101.74

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na

lP

-1.74

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250 mg

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Androxin tablets

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Found (mg)

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Labeled content

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Analysis of FLU and CPA in its commercial tablets (n=4)

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Highlights



A new design of a mechanical pencil holder was constructed, which subsequently



A novel disposable electrochemical sensor based on nano-AB coated graphite pencil

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lead electrode (nano-AB/GPLE) was fabricated.

The sensitive sensor was applied for the determination of antiandrogens flutamide

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

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reduced the voltammetric analysis time usage.

(FLU) and (cyproterone acetate) CPA in real samples.

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The new electrode showed good stability, sensitivity and selectivity and low detection

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limits.

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

32

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12