chitosan paste electrode for

A novel carbon/chitosan paste electrode for electrochemical detection of normetanephrine in the urine Redouan El Khamlichi, Dounia Bouchta, Mounia Ben Atia, Mohamed Choukairi, Riffi Temsamani Khalid, Ihssane Raissouni, et al. Journal of Solid State Electrochemistry Current Research and Development in Science and Technology ISSN 1432-8488 Volume 22 Number 7 J Solid State Electrochem (2018) 22:1983-1994 DOI 10.1007/s10008-018-3906-2

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Author's personal copy Journal of Solid State Electrochemistry (2018) 22:1983–1994 https://doi.org/10.1007/s10008-018-3906-2

ORIGINAL PAPER

A novel carbon/chitosan paste electrode for electrochemical detection of normetanephrine in the urine Redouan El Khamlichi 1 & Dounia Bouchta 1 & Mounia Ben Atia 1 & Mohamed Choukairi 1 & Riffi Temsamani Khalid 1 & Ihssane Raissouni 1 & Saloua Tazi 1 & Ahrouch Mohammadi 1 & Abdellatif Soussi 1 & Khalid Draoui 1 & Chaouket Faiza 1 & Mohammed Lamarti Sefian 2 Received: 8 November 2017 / Revised: 23 January 2018 / Accepted: 27 January 2018 / Published online: 11 February 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Normetanephrine is a marker for pheochromocytoma, a rare catecholamine-secreting and neuroendocrine tumor, that arises from sympathetic and parasympathetic paraganglia. In this work, a novel carbon/chitosan electrode paste was used for sensitive voltammetric determination of normetanephrine and dopamine in the presence of ascorbic acid and uric acid. The modified electrode has shown an increase in the effective area of up to 68%, well-separated oxidation peaks, and an excellent electrocatalytic activity. The electrochemical response characteristics were investigated by cyclic and differential pulse voltammetry. Interestingly, high sensitivity and selectivity in the linear range of normetanephrine, dopamine, ascorbic acid, and uric acid concentrations were observed. The present method was applied in the urine sample and satisfactory results were obtained showing that this electrode is very suitable in pharmaceutical and clinical preparations. Keywords Carbon/chitosan paste electrode . Normetanephrine . Dopamine . Ascorbic acid . Uric acid . Pheochromocytoma

Introduction The electroanalytical application of carbon paste electrodes for pharmaceutical analysis and clinical diagnosis are well reviewed in the last 10 years; the applications include measurements in alternate sample types such as the urine, serum, and saliva. The advantage of functional materials (carbon/chitosan) as an immobilization matrix for modified electrodes is due to high surface to volume ratio, the presence of reactive groups on the surface, and fast electron transfer kinetics [1–15]. Normetanephrine (NMN) is a metabolite of norepinephrine and is excreted in the urine. It is a marker for pheochromocytoma, a rare catecholamine-secreting and neuroendocrine

* Redouan El Khamlichi [email protected] 1

Electrochemistry and Interfacial Systems (ERESI) Team, Faculty of Sciences, University Abdelmalek Essaâdi, M’Hannech II, B.P. 2121, 93002 Tétouan, Morocco

2

High Normal School, University Abdelmalek Essaâdi, Tétouan, Morocco

tumor, that arises from sympathetic and parasympathetic paraganglia and can be malignant in 10% of cases. The measurement of NMN is the best tool in the diagnosis of pheochromocytoma, an adrenal medullary neoplasm [16–20]. Dopamine (DA) is found to be the most important catecholamine that belongs to the neurotransmitter family [21]. It is produced in the adrenal glands and is involved in the central nervous, hormonal, renal, and cardiovascular systems. The low levels of DA concentration may cause several neurological diseases, such as Parkinson’s and schizophrenia [22], attention-deficit hyperactivity disorder (ADHD) [23], drug addiction, and restless legs syndrome (RLS) [24]. A high urinary excretion of NMN and DA is considered as a symptom of pheochromocytoma [18]. Ascorbic acid (AA) is an essential vitamin known for its antioxidant properties. It also has been used for the prevention and treatment of common cold, mental illness, infertility, and cancer [25]. Uric acid (UA) is the end result of purine metabolism. Abnormal levels of UA can be considered as a diagnostic threshold of several perturbations and a symptom of several diseases, such as hyperuricemia, gout, and Lesch-Nyan disease. UA and AA often coexist in the human serum and urine. Thus, the monitoring and the measurement of NMN and DA in the presence of UA and AA are clinically very important [26, 27]. Normal levels of

Author's personal copy 1984

NMN, DA, AA, and UA in the urine are, respectively, 445 μg/ 24 h for NMN, 500 μg/24 h for DA, and between 17 and 52 mg/24 h for AA and 250–750 mg/24 h for UA [28]. In this work, a carbon/chitosan paste electrode (C/Chi) is proposed for a sensitive and selective pulse voltammetry determination of normetanephrine and dopamine in the presence of ascorbic acid and uric acid in real samples.

J Solid State Electrochem (2018) 22:1983–1994

electrode cell (20 mL) was used at room temperature (25 ± 1 °C). The counter electrode was a platinum wire, the reference electrode was an SCE (3 M KCl), and the working electrode was a carbon/chitosan paste electrode. The scanning electron microscope (SEM) image was obtained using a HITACHI X-650 SEM instrument.

Preparation of the C/Chi electrode

Experimental Reagents and materials Normetanephrine, dopamine, ascorbic acid, uric acid reagent grade ≥ 98% (HPLC) and chitosan were purchased from Sigma Aldrich (USA). KH2PO4, K2HPO4 used for phosphate buffer preparation and carbon powder (< 20 μm) were purchased from Fluka. Paraffin oil was purchased from a pharmacy. All other chemicals were of reagent grade and were used directly without further purification. Solutions were prepared using deionized double-distilled water with a measured resistance higher than 15 μS cm−1. Plastic capillary tubes, i.d. 2 mm, were used as the bodies for the composite electrodes.

Instrumentation The electrochemical impedance (EI), cyclic voltammetry (CV), and square wave voltammetry (SWV) electrochemical techniques were applied to study the behavior of C/Chi electrodes. They were all performed with a Voltalab®40, type PGZ301 from Radiometer (France). A conventional three-

Chitosan is a natural linear biopolymer cationic polysaccharide composed essentially of (1 to 4)-related β-D-glucosamine oneness in a body with a proportion of N-acetyl-β-D-glucosamine units [15]. It has drawn particular attention of many researchers in modified electrodes with nanomaterials including carbon nanotubes, graphene, and gold nanoparticles [29–31]. The C/Chi was prepared as follows: 0.1 g of chitosan was dispersed in 0.5 M acetic acid solution, then 1 g of carbon graphite powder was added and dispersed until obtaining a unique phase, and then the mixture was heated at 120 °C to evaporate the acetic acid and water. Subsequently, the carbon powder modified with chitosan is dried and mixed thoroughly in a mortar with 40% of paraffin oil until obtaining a homogenous paste. Finally, the plastic capillaries were filled, leaving a little extra mixture sticking out of the tube to facilitate the subsequent polishing. For establishing electrical contact, a copper wire was inserted into the capillary. Before usage, the electrodes were polished with emery paper No 1200 and were electrochemically cleaned by cyclic voltammetry until obtaining stable cyclic voltammograms between − 0.60 to 1.00 V in 0.05 mol L−1 of KCl. The basic strategy for the C/ Chi electrode preparation is given in Scheme 1. The

Scheme 1 The basic strategy for the graphite/chitosan-modified electrode in the simultaneous determination of normetanephrine, dopamine, ascorbic acid, and uric acid by differential pulse voltammetry

Author's personal copy J Solid State Electrochem (2018) 22:1983–1994

unmodified carbon paste electrodes were also prepared using the same procedure.

Results and discussion Surface and electrochemical characterization of modified electrodes The functionalization of carbon was evaluated. Carbon paste electrodes were prepared using functionalized and nonfunctionalized carbon. As expected, the functionalized carbon exhibits a higher analytical signal, which can be related to the introduction of polar hydrophilic surface groups, mainly carboxyl group. That can increase the selectivity and sensitivity towards NMN, DA, AA, and UA, as observed in a previous work. Initially, in order to evaluate the effect of the present modification, the electrochemical response of the electrode with and without chitosan was measured using cyclic voltammetry at 1 mM ferro-ferricyanide prepared in 1 M KCl in the potential range of − 400 to + 800 mV at a scan rate of 50 mV s−1. Figure 1a shows the electrochemical characteristics of the CE and C/Chi electrode. Both the oxidation and the reduction currents increase while using the C/Chi electrode (approximately two times higher). The redox potential shows clear anodic and cathodic current peaks, and the difference

Fig. 1 a Electrochemical characterization of C/Chi electrode and carbon electrode, mediated by the redox couple of ferro-ferricyanide prepared in 1 M KCl in the potential range of − 400 to + 800 mV. b Nyquist plots of EIS in 1 mM potassium ferricyanide prepared in 1 M KCl for C/Chi and

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ΔE passes from 500 mV (C) to 300 mV (C/Chi) indicates the change of character of the ferricyanide oxidation on the surface of the modified electrode. However, at present, the role of chitosan in the enhanced current is not sufficiently clear. Nevertheless, the hydrophobicity-hydrophilicity balance properties and the attractive feature of chitosan can improve the ion transportation by a mechanism involving the permselectivity of chitosan against anions, as recently described. This could be responsible for the enhanced analytical response of the electrode for metallic cations. The active surface area of the modified electrode was estimated according to the slope of the ip versus v1/2 plot, based on the Randles– Sevcik equation [32]: ip ¼ 2:69 105 n3=2 Aeff D1=2 C υ1=2 where Aeff is the effective surface area, n is the number of electrons transferred, D (= 7.6 × 10−6 cm2 s−1) is the diffusion coefficient of K3[Fe(CN)6], and C is the concentration of K3[Fe(CN)6]. The effective electrode area for (C/Chi)-modified electrode is approximately 0.047 cm2, whereas it is 0.032 cm2 for carbon electrode. Furthermore, the electrochemical impedance spectroscopy (EIS) is an effective method to characterize the interface features of a modified electrode surface. Figure 1b shows that the charge transfer resistance of (C/Chi) electrode is much smaller than that of carbon electrode, suggesting that it is easier to

carbon electrode, amplitude 5 mV, frequency range 100 kHz–10 mHz, potential 0 V. c SEM images obtained for C electrode. d SEM images obtained for C/Chi-modified electrode


transfer electrons at (C/Chi) electrode. Indeed, the incorporation of chitosan promotes electron transfer synergistically and accelerates the transportation of ferricyanide towards the modified electrode surface. External modality of CE and C/Chi electrode (Fig. 1c, d) was also investigated by SEM. The SEM images proved appearance of sidewall in functionalized carbon when compared with non-functionalized carbon. The surface morphology of electrode C/Chi shows a smooth surface of a composite carbon/chitosan material which presents fissures (see Fig. 1d).

Electrochemical behavior of NMN, DA, AA, and UA at the modified electrode Figure 2 shows the electrochemical behavior of the NMN, DA, AA, and UA at modified electrode in PBS (pH = 7.4) using CV. First, the NMN (pK a = 9.06) presents an electroactive character that appears with an oxidation peak in the studied potential ranges. For DA (pKa = 9.27), a redox couple shows the reversibility of DA. For AA (pKa = 4.1), an oxidation peak appears in potential range (from − 600 to + 400 mV). Finally, UA (pKa = 5.6) presents an electroactive character in potential ranges (from − 100 to + 500 mV). The relationship between the oxidation peaks currents (ip) and the square root of the scan rate (ν1/2) of NMN, DA, AA, and UA is linear with linear correlation coefficients RNMN = 0.9933, Ripa DA = 0.9906, Ripc DA = 0.9894, RAA = 0.9964, and RAU = 0.9978. Indeed, all spectrums indicating that the process of NMN, DA, AA, and UA is controlled by diffusion mechanism at the C/Chi electrode. Figure 3 depicts the SWVs of the electrooxidation of NMN, DA, AA, and UA molecules at the modified and nonmodified electrode in PBS (pH 7.4) with a scan rate of 20 mV s−1. As a result, the negative shift of the UA peak indicates that the C/Chi-modified electrode plays a strong electrocatalytic effect on the UA oxidation; contrarily, the positive shift of the AA peak indicates the malfunctioning electrocatalytic effect of the modified electrode on the AA oxidation. The peaks of NMN and DA have similar oxidation potentials at the modified and non-modified electrode. This might be the key factor to adjust the problem of interference and achieve simultaneous determination of NMN, DA, AA, and UA. However, all oxidation peaks of NMN, DA, AA, and UA were rather higher intensity at the modified electrode. As a result, it is obvious that NMN, DA, AA, and UA, which are characterized by different pKa and structure, have different interaction modes with the C/Chi electrode. The modified electrode should promote the sensitivity and the selectivity of NMN, DA, and AA detection in the presence of UA. The attractive feature and the hydrophobicityhydrophilicity of chitosan can accelerate the transportation of NMN, DA, AA, and UA according to their interaction modes, and this could be responsible for the enhanced


analytical response of the electrode towards this molecules. Indeed, the oxidation of DA, NMN, AA, and UA does not follow the known electron transfer mechanism. For our knowledge, the electrochemical oxidation mechanism of NMN has been reported. In our previous study, we proposed an electrooxidation mechanism for homovanillic acid an omethoxyphenols as the NMN [33]. Generally, the overall process of substituted methoxyphenols electrooxidation produces the corresponding o-quinones that intervene 2 e− and 2 H+ and release CH3OH (Scheme 2A). As reported on homovanillic acid, the dimerization of NMN implies two steps. In the first one, the oxidation of NMN leads to the formation of phenoxyl radical cations followed by an heterolytic bond dissociation of OH of catechol moiety yielding phenoxyl radicals. In the second step, the radical-radical or radical-neutral reactions generate diverse dimers (Scheme 2A). The rationalization with theoretical details of the different types of the obtained dimers has been reported by El Khamlichi et al. [33]. As demonstrated, the oxidation of NMN can formed different types of dimers (dimers, trimers, and tetramers) by a dimerization process between different radicals. NMN is the only methoxy-hydroxy compound included in this series and which follows therefore a different electrochemical mechanism due to the coupling of a fast chemical step involving the elimination of the methoxy group (EC mechanism). A potent parameter that needs to be considered is the electrocatalytic oxidation of AA by DA [34–38]. The electrooxidation of DA in the existence of even a small amount of AA leads to a homogeneous catalytic oxidation of AA through the following mechanism Scheme 2B. The dopamine-o-quinone (the result of electrochemical oxidation of DA) will react with AA to be chemically reduced and return to its original form. In consequence, the electrochemical oxidation of DA is affected by AA and reciprocally the AA oxidation is affected by dopamine-o-quinone. Thus, removing this homogeneous catalytic oxidation is required for the specific determination of DA and AA. For this purpose, we can assume the advantage of the difference in the ionization state and in the characteristic charge of this molecules, and considering the hydrophobicity-hydrophilicity balance properties and the attractive feature of chitosan that modified electrode could be considered simultaneously for dopamine and ascorbic acid in different ways, which makes possible the selective oxidation of DA (positively charged) from the oxidation of ascorbic acid (negatively charged). In view of the fact that, the diffusion part of the double layer separates the positives charges from the negatives charges. In addition, the shift of potential peak and the formation of the described wellresolved peaks are due to the different interaction of DA and AA with the modified electrode. Therefore, the modification by chitosan has eliminated the catalytic reaction between AA and DA and the separation between the two peak potentials of


Fig. 2 (A1) Cyclic voltammograms obtained at different scan rates from the modified electrode in a PBS at pH 7.4 containing 100 μM NMN. Scan rates 40, 60, 80,100, 120, 140,160, 180, and 200 mV s−1. (A2) Plots of anodic peak current as a function of square root of potential scan rate ν1/2. (B1) Cyclic voltammograms obtained at different scan rates from the modified electrode in a PBS at pH 7.4 containing 100 μM DA. Scan rates 20, 40, 60, 80,100, 150, 200,250, 300, 350, 400, and 450 mV s−1. (B2) Plots of anodic and cathodic peak currents as a function of square root of

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potential scan rate ν1/2. (C1) Cyclic voltammograms obtained at different scan rates from the modified electrode in a PBS at pH 7.4 containing 100 μM AA. Scan rates 50,100, 150, 200, and 250 mV s−1. (C2) Plots of anodic peak current as a function of square root of potential scan rate ν1/2. (D1) Cyclic voltammograms obtained at different scan rates from the modified electrode in a PBS at pH 7.4 containing 100 μM UA. Scan rates 50,100, 150, 200, 250, 300, and 350 mV s−1. (D2) Plots of anodic peak current as a function of square root of potential scan rate ν1/2



Fig. 3 SWVs of C/Chi-modified electrode and C electrode at PBS, pH 7.4 with concentration of AA (20 μM), NMN (5 μM), DA (5 μM), and UA (20 μM)

Scheme 2 A Suggested mechanism of NMN oxidation. B Homogeneous catalytic oxidation of DA and AA. C Suggested mechanism of UA oxidation


AA and DA is 200 mV which is large enough for the selective determination of DA in the presence of AA and also for the simultaneous determination of DA and AA in their mixtures. It is reported that allantoin is the most characteristic product of uric acid oxidation [27, 39]. A suggested mechanism of uric acid oxidation has been proposed (Scheme 2C), which consists of four main steps: In the first step, the formation of

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intermediate by the nucleophile attack of hydroxyl group at electrophilic site of carbonyl group CO and the opening of heterocycle. In a second step, the double oxidation of I1 leads to the formation of intermediate I2, which is converted to I3 in the presence of H2O. In the third step, decarboxylation of I3 leads to the formation of intermediate I4, which is automerized to give allantoin in a final step.

Fig. 4 Calibration plots and SWVof C/Chi-modified electrode at PBS, pH 7.4 with increasing concentration of a AA (from 1 to 120 μM), b NMN (from 1 to 80 μM), c DA (from 1 to 50 μM), and d UA (from 1 to 120 μM)


Analytical determination and calibration curves of NMN, DA, AA, and UA The SWV was used to obtain the calibration curve for NMN, DA, AA, and UA at the modified electrode in PBS (pH 7.4). For AA, the result in Fig. 4a shows the linear relationship Fig. 5 SWV at C/Chi-modified electrode in diluted urine 500% at pH 7.4 containing (A1) mixture of 1 μM DA and 140 μM NMN in the presence of different concentrations of AA (from 10 to 160 μM), (A2) calibration plots; (B1) mixture of 60 μM AA and 100 μM NMN in the presence of different concentrations of DA (from 1 to 22 μM), (B2) calibration plots; (C1) mixture of 80 μM AA and 2 μM DA in the presence of different concentrations of NMN (from 10 to 120 μM), (C2) calibration plots


between the oxidation peaks J and AA concentrations. The peak intensities are increased linearly in the range of 1– 120 μM, the slope is (0.0932 ± 0.0015) μA μM−1, the yintercept is (0.1822 ± 0.0804) μA, and the correlation coefficient (R2) is 0.9995. The detection limit (S/N = 3) is estimated to be 0.3 μM in terms of signal to noise ratio of 3:1. For NMN,

Author's personal copy J Solid State Electrochem (2018) 22:1983–1994 Table 1 Sample

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1991

Simultaneous determination of DA, AA, UA, and NMN in mixture synthesis samples (± SD; the standard deviation for n = 3) Added (μM)

Found (μM)

AA

DA

UA

NMN

AA

10 20

2 10

30 40

20 60

9.95 ± 0.3 19.4 ± 0.7

Recovery (%) DA 1.8 ± 0.16 9.4 ± 0.6

UA

NMN

AA

DA

UA

NMN

32 ± 0.6 40.1 ± 0.1

18.6 55.3

99.5 97

90 94

106.6 100.25

93 92.1

3

80

14

40

40

79.57 ± 1.4

13.8 ± 0.2

40.2 ± 0.2

38.2

99.4

98.5

100.5

95.5

4

100

16

20

100

98.2 ± 2.6

15.4 ± 0.3

20.6 ± 0.4

98.5

98.2

96.25

102.5

98.5

Fig. 4b shows the linear relationship between the oxidation peaks J and NMN concentrations, the peak intensities are increased linearly in the range of 1–80 μM, the slope is (0.2291 ± 0.0065) μA μM−1, the y-intercept is (0.2258 ± 0.042) μA, and the correlation coefficient (R2) is 0.9988. The detection limit is estimated to be 0.1 μM in terms of signal to noise ratio of 3:1. For DA, Fig. 4c shows the linear relationship between the oxidation peak J and DA concentration, the peak intensities are increased linearly in the range of 1– 50 μM, the slope is (1.053 ± 0.0313) μA μM −1, the yintercept is (0.6031 ± 0.665) μA, and the correlation coefficient (R2) is 0.9976. The detection limit (S/N = 3) is estimated to be 0.05 μM in terms of signal to noise ratio of 3:1. For UA, Fig. 4d shows the linear relationship between the oxidation peak J and UA concentration, the peak intensities are increased linearly in the range of 1–120 μM, the slope is (0.2097 ± 0.0055) μA μM−1, the y-intercept is (0.3205 ± 0.0602) μA, and the correlation coefficient (R2) is 0.9987. The detection limit (S/N = 3) is estimated to be 0.1 μM.

Simultaneous determination of NMN, DA, AA, and UA at C/Chi electrode in the urine UA is the principal organic constituent of urine. The phenomenon of interference on the electrochemical response of NMN, DA, and AA in the presence of the urinary UA is one of the

major problems that hinder electrochemical detection of these substances in biological media, since the unmodified carbon electrode could not separate AA and DA oxidation peaks. Also, it cannot separate the peak of UA from the peak of NMN. The development of a simple and inexpensive device for the simultaneous determination and separation of the electrochemical responses of these substances remains the challenge of this work. The C/Chi electrode possessed a higher active surface area and can separate NMN, AA, DA, and UA oxidation peaks. The catalytic oxidation of NMN, AA, and DA were studied by SWV in 500% diluted urine at pH = 7.4 for different concentrations (Fig. 5), in order to check the intermolecular effects between NMN, AA, DA, and urinary UA. The results (at Fig. 5a) were obtained by varying the concentration of AA from 10 to 160 μM in the presence of 1 μM of DA and 140 μM of NMN and urinary UA in diluted urine 500% at pH 7.4. This figure shows that the peak currents for AA increase linearly with an increase in their respective concentrations without considerable effects on the other peaks of DA, UA, and NMN, for the regression plot of ip vs AA concentration, the slope is (0.0625 ± 0.0045) μA μM−1, the yintercept is (0.7717 ± 0.4187) μA, and the correlation coefficient (R2) is 0.9949. In addition, various concentrations of DA from 1 to 22 μM in the presence of 60 μM AA and 100 μM NMN exhibit excellent SWV responses to AA, DA, UA, and NMN without any obvious intermolecular effects among

Table 2 Comparison of linear range and detection limit at the present modified electrode with the reported materials and chemically modified electrodes Linear range (μM)

-L-cys-sonogel–carbon MWCNT-CAN MWCNT-BZ PABS–MWNT-modified GCE MB-MWNTs/GCE MPCVD pt/BDDμE Nitrogen-doped graphene Pl-LEU/DNA Carbon graphite/chitosan

Detection limit (μM)

Ref

NMN

UA

DA

AA

NMN

UA

DA

AA

– – – – – 0.5–100 – – 1–100

10–100 – – 6–45 2.0–200 – 0.1–20 0.5–100 1–150

– 35–435 35–435 9–48 0.4–10 – 0.5–170 0.1–1 1–50

50–1000 – – – – – 5–1300 5–750 1–200

– – – – – 0.04 – – 0.1

10 – – 0.44 1 – 0.04 0.2 0.1

– 0.03 0.30 0.21 0.2 – 0.25 0.04 0.05

50 – – – – – 2.2 2 0.3

[40] [41] [42] [43] [44] [45] [46] This work


them, and the peak current of DA increased linearly with DA concentration, for the regression plot of ip vs DA concentration, the slope is (0.3638 ± 0.0317) μA μM−1, the y-intercept is (9.49 ± 0.43) μA, and the correlation coefficient (R2) is 0.9913 (Fig. 5b). Figure 5c indicates that the peak current of NMN increased linearly (in the presence of 80 μM AA and 2 μM DA) by increasing NMN concentrations (from 10 to 120 μM) without considerable effects on the other peak currents, for the regression plot of ip vs NMN concentration, the slope is (0.0651 ± 0.0057) μA μM−1, the y-intercept is (2.6924 ± 0.4154) μA, and the correlation coefficient (R2) is 0.9966. These results confirm that the oxidation processes of AA, DA, UA, and NMN at C/Chi electrode are independent from each other; the difference in the oxidation peak potentials for (AA–DA), (DA–UA), and (UA–NMN) were 200, 120, and 130 mV, respectively. This separation allows a simultaneous determination of DA, AA, UA, and NMN in a mixture. The objective of this study is the simultaneous detection of the AA, DA, UA, and NMN in the urine. In this context, and in order to evaluate the applicability of the proposed method for the determination of AA, DA, UA, and NMN in the urine, the measurements were conducted in urine samples diluted 500% (with 0.05 M PBS at pH 7.4), into which was added a deferred reports of AA, DA, UA, and NMN (Table 1). The feasibility of the C/Chi paste electrode is demonstrated for analytical application. The recovery test was performed by the standard addition method (Table 1), with four different additions of AA, DA, and NMN and three different additions of UA to the urine diluted samples. The obtained recoveries ranged from 97 to 99.5% for AA, 90 to 98.5% for DA, 100.5 to 106.6% for UA, and 92.1 to 98.5% for NMN. This high recovery and the perfect selectivity exerted by the proposed C/Chi electrochemical paste electrode are very promising for the simultaneous detection of DA, AA, UA, and NMN in urine sample. Table 2 presents the results of the proposed modified electrode as compared to other electrochemical methods reported in the literature.

Conclusions The use of electrochemical techniques for the sensitive and selective simultaneous determination of DA, AA, UA, and NMN in the urine by square wave voltammetry using graphite/chitosan-modified electrode showed that the C/Chi electrode presents a high selectivity for the detection of DA, AA, UA, and NMN in 0.05 M PBS at pH 7.4, with low limits of detection: 0.1 μM for NMN, 0.05 μM for the DA, 0.1 μM for the UA, and 0.3 μM for AA. This selectivity is also maintained when the investigation was conducted in biological fluids such as urine diluted 500% with 0.05 M PBS at pH 7.4. Indeed, our electrochemical paste electrode looks very promising and can be considered for early quantification of


normetanephrine, dopamine, ascorbic acid, and uric acid in pharmaceutical and clinical preparations. Acknowledgments The authors are grateful to Dr. Aisha Attar, University of California, Irvine, USA, and to Engr. Sara Elliazidi, University Abdelmalek Essaâdi, Tetouan, Morocco, for all their support.

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