Electrochemical determination of vitamin B6 in pharmaceutical and energy drink samples http://dx.doi.org/10.5599/jese.1574 297 J. Electrochem. Sci. Eng. 13(2) (2023) 297-319; http://dx.doi.org/10.5599/jese.1674 Open Access : : ISSN 1847-9286 www.jESE-online.org Original scientific paper Electrochemical determination of vitamin B6 in pharmaceutical and energy drink samples Gizaw Tesfaye, Negussie Negash and Merid Tessema Department of Chemistry, Addis Ababa University, P. O. Box 1176, Addis Ababa, Ethiopia Corresponding author: gizawtes@gmail.com Received: January 24, 2023; Accepted: February 25, 2023; Published: March 1, 2023 Abstract A simple and low-cost electrochemical sensor based on poly(phenylalanine) and function- nalized multi-walled carbon nanotubes (F-MWCNTs) modified glassy carbon electrode (GCE) was developed for the determination of vitamin B6 (VB6). The surface morphology of modified glassy carbon electrodes was investigated with scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). The electrocatalytic activities of the bare and modified electrodes were investigated in the presence of ferri-ferrocyanide redox couple using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The exchange current density (jo = 2462 µA cm-2) and electron transfer rate constant (ko = 0.002 cm s−1) were calculated using 5 mM K3[Fe(CN)6]. The electrochemical activity of poly(phenylalanine)/F-MWCNT/GCE towards VB6 oxidation was investigated using CV. Parameters including the number of electrons transferred (n = 2), number of protons transferred (H+ = 2), electron transfer coefficient (α = 0.51) and surface concentration of VB6 ( = 0.24 nmol cm−2) were calculated. At the optimal experimental conditions, the oxidation peak current of VB6 measured by square wave voltammetry (SWV) was found proportional to its concentration in two linear ranges of 0.5 to 20 µM and 20 to 200 µM with a low detection limit (LOD) of 0.038 µM and limit of quantification (LOQ) of 0.125 µM. Finally, the sensor was successfully used to determine VB6 in soft drink and pharmaceutical formulation samples. Keywords Glassy carbon electrode; electro-polymerization; poly(phenylalanine); F-MWCNTs; pyridoxine; electrochemical sensor; real samples Introduction Vitamins are nutrients that are essential for normal cell development, energy generation and red blood cell production [1–3]. Furthermore, they are important cofactors of many enzymes in different metabolic processes occurring in the body [3]. Based on their solubility, vitamins are classified as water-soluble vitamins (vitamin B and vitamin C) and fat-soluble vitamins (vitamin A, vitamin D, http://dx.doi.org/10.5599/jese.1574 http://dx.doi.org/10.5599/jese.1674 http://www.jese-online.org/ mailto:gizawtes@gmail.com J. Electrochem. Sci. Eng. 13(2) (2023) 297-319 DETERMINATION OF VITAMIN B6 298 vitamin E, and vitamin K) [1]. Water-soluble vitamins are not stored in the body due to easy discharge through urine [2,4]. Furthermore, they cannot be synthesized in the human body [3,5]. Therefore, they must be obtained from dietary sources for normal cell growth. Among the water-soluble vitamins, VB6 (pyridoxine) (Figure 1A) plays an important role in body metabolism. VB6 is involved in amino acid metabolism, red cell production, hemoglobin synthesis, gene expression, enzyme- catalyzed reactions and nervous and immune systems [1,5]. VB6 is found in many foods such as chickpeas, turkey, fish, starchy vegetables, potatoes, bananas, meats, organ meats, fortified cereals and whole grains [6,7]. The recommended dietary allowance (RDA) for VB6 is 1.3 mg/day for adult males and females and even higher amounts are advised for males and females over the age of fifty years [6]. The normal range of VB6 in human blood is 5–50 μg L-1 [1]. The deficiency of VB6 causes colorectal cancer, skin problems, weakness, depression, convulsions, neurological diseases, anemia, hyperlipidemia, hypertension, obesity, cardiovascular diseases, mucous membranes and circulatory system problems [1,3]. However, excessive intake of VB6 for a long time leads to different allergic reactions such as rash, itching, severe dizziness and breathing [2]. It is vital to develop sensitive and selective methods for the quick determination and monitoring of VB6 in various matrices, such as food and pharmaceuticals, in light of its significance and potential negative effects. For the purpose of determining VB6, a number of analytical techniques, including liquid chromatography [8], gas chromatography-mass spectrometry [9], chemiluminescence [10], capillary zone electrophoresis [11] and spectrophotometry [12] have been described in the literature. Many of these analytical techniques require tedious and time-consuming procedures and involve highly sophisticated instrumentation and toxic organic reagents [5,13,14]. Due to great selectivity, sensitivity, quick response time, ease of use and low cost, electrochemical methods have received a lot of interest in electroanalysis [4,15]. Furthermore, combined with portable detection equipment, electrochemical methods offer great promise for on-site environmental monitoring [16]. Glassy carbon electrode (GCE) has been widely used as a working electrode for sensor fabrication due to its resistance to chemical attack, easy modification, good reproducible surface, low porosity, high electrical conductivity, wide potential window and good mechanical stability. However, at bare electrode surfaces, VB6 shows weak or no electrochemical response. In order to increase the rate of electron transfer and lower the overpotential, it is required to change the electrode surface with appropriate modifiers [17]. Various modified electrodes, such as GCE modified with a hybrid of polydopamine and reduced graphene [18], multi-walled carbon nanotube and cobalt phthalocyanine (CoPc) modified pyrolytic graphite electrode [19], NiO-carbon nanotubes (CNTs) nanocomposite and 1-methyl-3-octylimidazolium hexafluorophosphate modified carbon paste electrodes [20], poly(arginine) modified carbon nanotube paste electrode [21], cobalt hexacyanoferrate modified carbon paste electrode [22] and gold nanostructures modified carbon paste electrode [23] have been fabricated for the electrochemical determination of VB6. However, all these electrodes suffer from tedious modification processes. Therefore, it is important to develop low-cost and simple electrochemical method for the VB6 determination. Recently, polymers have received much attention for electrode modification due to their biocom- patibility, chemical stability, ease of synthesis, more active sites and low-cost processability [24,25]. The advantages of electrochemical polymerization over chemical polymerization for polymer preparation include homogeneous and stable polymer film formation on the electrode surface, ease of preparation, cost-effectiveness, strong adhesion of the polymer film to the electrode surface and simple control of film thickness by adjusting electrochemical parameters only [25]. Because of their ease of processing, high electrocatalytic capacity, easy interaction with the target analyte via G. Tesfaye et al. J. Electrochem. Sci. Eng. 13(2) (2023) 297-319 http://dx.doi.org/10.5599/jese.1574 299 hydrogen bonding or electrostatic interaction caused by the presence of amine and carboxyl groups, stable film forming ability, high biocompatibility and low cost, poly(amino acids) were used extensively as electrode modifiers [26]. Different amino acids have been used to modify electrode surfaces for the electrochemical analysis of various analytes [27-31]. Among the major amino acids, phenylalanine (Figure 1B) is easily electropolymerized on the electrode surface to form poly(phenylalanine) [32]. A number of analytes have been studied electrochemically using electrodes modified with poly(phenylalanine) [33-35]. With respect to the intended analytes, the modified electrode demonstrated high electrocatalytic activity and stability. Figure 1. Chemical structures of (A) VB6 and (B) phenylalanine On the other hand, CNTs modified electrodes have been widely used in electrochemical sensors due to their high electrical conductivity, biocompatibility, ease of functionalization, mechanical strength, high adsorption capacity, chemical stability and high surface area [26,36]. Furthermore, carbon nanotubes are easily incorporated into other materials to produce synergistic effects that increase sensitivity to a target analyte. However, a significant obstacle to the current development of CNT-based devices is the limited solubility of CNT in most solvents. Because of the strong - interactions between the aromatic rings, CNTs tend to aggregate in aqueous solutions, making challenging to disperse them and use for development of electrochemical sensors. Therefore, it is important to prepare hydrophilic surface CNTs to overcome the dispersion problem. Functionalization of CNT enhances solubility, processibility and interaction with other materials [37]. CNTs can be covalently functionalized by oxidation with strong acids such as HNO3, H2SO4 or their mixtures [38]. Oxidation of CNTs results in open-ended nanotubes containing oxygenated functional groups such as carboxylic acid, ketone, alcohol and ester groups [38]. The presence of these oxygen-containing functional groups on CNT increases its interaction with a polymer during composite formation [38]. As described in many literature studies, the combination of carbon nanotube with polymer significantly improves the electrocatalytic activity of the modified surfaces compared to individual CNTs or polymer-modified electrodes due to the synergistic effect of CNT and polymer [25,26]. In this study, taking into account the synergistic effect of F-MWCNT and polymer, we constructed an effective and low-cost electrochemical sensor for the determination of VB6 in food and pharma- ceutical samples. The electro-polymerization of phenylalanine on carbon nanotubes is carried out using potentiodynamic polarization. The prepared electrode, poly(phenylalanine)/F-MWCNT/GCE is characterized by Fourier-transform IR (FTIR), scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. The electrochemical behavior of VB6 at the modified electrode is investigated using CV, while its analytical determination is performed by square wave voltammetry (SWV). The preparation procedures for the F-MWCNT and poly(phenylalanine) modified GCE and sensing mechanism are illustrated in the Graphical abstract. http://dx.doi.org/10.5599/jese.1574 J. Electrochem. Sci. Eng. 13(2) (2023) 297-319 DETERMINATION OF VITAMIN B6 300 Experimental Reagents and chemicals All chemicals used in this study were analytical grade and were used without any further purification: pyridoxine hydrochloride (VB6) (≥ 98 %), vitamin B1 (≥ 99 %), potassium nitrate (99 %), sodium sulfate (≥ 99 %) and ascorbic acid (≥ 99.7 %) were obtained from (BDH, England). Vitamin B2 (≥ 98 %), L-phenylalanine (99 %), caffeine (99 %), tartaric acid (98 %), acetic acid (99.8 %), glucose (≥ 99.5 %), starch (AR grade), multi-walled carbon nanotube (MWCNT) (> 90 % carbon basis) synthesized by chemical vapor deposition, potassium ferricyanide (99 %), sucrose (≥ 99.5 %), lactose (≥ 99%), vitamin B12 (≥ 98 %), vitamin B9 (≥ 97 %), sodium hydroxide (98 %) and hydrochloric acid (37 %) were obtained from Sigma-Aldrich (USA). Citric acid (99 %), sodium citrate (98 %) and dipotassium hydrogen phosphate (98 %) were obtained from Research–Lab. Chem. Industries (Mumbai, India). Potassium dihydrogen phosphate (98 %), magnesium chloride (99.5%), copper nitrate (98 %), iron(II) nitrate (98 %), sodium carbonate (99.5 %) and sodium bicarbonate (99.7 %) were obtained from Hopkin and Williams LTD (England). Potassium chloride (98 %) was purchased from Riedel-de Haën (France). Boric acid (99.5 %) was obtained from Carlo Erba reagent SPA (Italy). 5 mM stock solution of VB6 was prepared by dissolving accurately weighed amounts of VB6 in distilled water. Until use, the stock solution was kept in a refrigerator. A suitable quantity of phenylalanine was dissolved in phosphate buffer solution pH 8.0 to prepare a 2mM phenylalanine solution. Acetate buffer solutions (ABS) were prepared by mixing 0.1 M acetic acid and 0.1 M sodium acetate. 0.1 M NaOH and 0.1 M HCl solution were used to adjust the pH of the solution. The stock solutions were diluted with ABS pH 4.5 to prepare the working solutions. Apparatus and instruments Electrochemical experiments, including CV, SWV and EIS were carried out using CHI 760D Electrochemical Workstation (CH Instruments, USA). A three-electrode system consisting of bare GCE or modified GCE (geometric surface area of 0.07 cm2) as a working electrode, platinum wire as a counter electrode and Ag/AgCl (3 M KCl) as a reference electrode were used. The pH of the solution was measured using a pH meter (sensION, SHA Snilu Instruments Co. Ltd., China). The surface morphology and chemical structure of the modified electrodes were studied using SEM (Cx-200 Coxem, Korea) and FTIR (Spectrum 65 FT-IR (Perkin Elmer, USA) using KBr disk, respectively. Real sample preparation Beverage sample Energy drink samples (predatory energy drink, Ambo mineral water S.C. Ethiopia) were obtained from a nearby supermarket in Addis Ababa, Ethiopia, and kept in a refrigerator until analysis. 100 mL of the liquid sample was transferred into a beaker and degassed in an ultrasonic bath before voltammetric analysis. Then 2.0 mL of the sample was diluted to 10 mL with 0.1 M ABS pH 4.5 solution. The determination of VB6 in the sample was performed using the standard addition method. The samples were spiked with a stock solution of VB6 for the recovery test. Pharmaceutical sample A commercial sample of the vitamin B complex (N-VIT, Ningbo Shuangwei Pharm.Co., Ltd., China, containing vitamins B1, B6 and VB12) was obtained from a local drugstore in Addis Ababa. Five tablets containing 100 mg of VB6 were precisely weighed and ground into a fine powder. An amount equivalent to one tablet was taken in a 50 mL volumetric flask and dissolved in distilled water by G. Tesfaye et al. J. Electrochem. Sci. Eng. 13(2) (2023) 297-319 http://dx.doi.org/10.5599/jese.1574 301 sonicating it for 10 min. Then, the solution was filtered using Whatman filter paper to obtain a clear filtrate and transferred to a 100 mL volumetric flask and the volume was made up to the mark with distilled water. To achieve a final concentration in the range of calibration curve, the sample solution was further diluted with ABS pH 4.5. SWV quantification of VB6 in the pharmaceutical formulation was carried out by the standard addition method. For the recovery test, the samples were spiked with various concentrations of the standard solution of VB6. Functionalization of multi-walled carbon nanotubes Functionalization of MWCNTs was performed utilizing the acid oxidation method according to the procedure described in the literature with a minor modification [39]. Briefly, 500 mg of pristine MWCNTs was added to 100 mL of a concentrated solution of HNO3 and H2SO4 at a ratio 1:3 (v/v) in a 200 mL conical flask and heated at 80 °C with continuous starring for 5 h. The resulting mixture was diluted with 200 mL of distilled water and filtered. Then, the mixture was thoroughly rinsed with distilled water until the pH of the filtrate reached 7.0. Finally, the functionalized F-MWCNTs were gathered and dried in an oven at 40 °C for 12 h. Preparation of modified electrodes The GCE was properly rinsed with double-distilled water after being carefully polished with 0.05 µm alumina slurry on the polishing pad. The electrode was then successively sonicated in distilled water and ethanol for five minutes to remove any remaining polishing agent from the electrode surface. F-MWCNT suspension was made by dispersing 5 mg of F-MWCNT in 5 mL of double-distilled water and sonicating the mixture for 30 minutes. To prepare F-MWCNT modified GCE, 8 µL of an optimal amount of F-MWCNT suspension was drop cast onto the cleaned GCE and allowed to dry for five minutes in the open air. Then, electro-polymerization was carried out by cyclic voltammetry in the potential range −1.5 to 2.5 V at a scan rate of 0.05 V s−1 for 5 cycles in 0.1 M phosphate buffer solution pH 8.0 containing 2 mM phenylalanine. To remove unreacted monomers, the fabricated poly(phenylalanine)/F-MWCNT/GCE was rinsed with double distilled water and dried for five minutes at room temperature. For comparison purposes, poly(phenylalanine)/GCE and F-MWCNT were also prepared with the same procedures described above. Results and discussion Construction of poly(phenylalanine)/F-MWCNT/GCE Poly(phenylalanine) film was deposited on the surface of bare GCE and F-MWCNT/GCE by electrochemical polymerization of phenylalanine using 5 cycles of CV in the potential range −1.5 to 2.5 V at the scan rate of 0.05 V s−1 in 0.1 M phosphate buffer solution (pH 8) containing 2 mM phenylalanine monomer. The mechanism of electro-polymerization of phenylalanine was- already reported [40]. The cyclic voltammograms (CVs) for the electro-polymerization of phenylalanine are displayed in Figure 2. In the forward scan, the phenylalanine monomer oxidation peak was observed at 1.6 V, corresponding to the formation of monomer radical due to the oxidation of the amino functional group of phenylalanine. During the reverse scan, the cathodic peak at −0.50 V was observed, corresponding to phenylalanine reduction. The monomer cation radicals can form carbon-nitrogen linkages on the surface of the carbon electrode [28,30]. The deposition of the polymer film on the electrode surface is demonstrated by an increase in the anodic and cathodic peak currents with increasing cycle numbers [28]. After a few cycles, the anodic and cathodic peak currents remain almost stable, indicating that the formation of a polymer film has reached the level http://dx.doi.org/10.5599/jese.1574 J. Electrochem. Sci. Eng. 13(2) (2023) 297-319 DETERMINATION OF VITAMIN B6 302 of saturation. A blue coating was noticed on the electrode surface after washing with distilled water, indicating the formation of poly(phenylalanine) film [30]. Scheme 1 describes the electro- polymerization mechanism of phenylalanine. E / V vs. Ag/AgCl Figure 2. CVs of electro-polymerization of 2mM phenylalanine in 0.1 M phosphate buffer solution (pH 8.0) on F-MWCNT/GCE surface in the potential range −1.5 and 2.5 V for 5 cycles at a scan rate of 0.05 V s−1. Inset: peak current of 200 µM VB6 versus the number of polymerization cycles Scheme 1. Mechanism of electro-polymerization of phenylalanine -2 -1 0 1 2 3 -500 0 500 1000 1500 2 3 4 5 6 7 8 9 10 0 5 10 15 20 25 30 I (m A ) Number of polymerization cycle I / m A E / V ns. Ag/AgCl I / m A I / m A G. Tesfaye et al. J. Electrochem. Sci. Eng. 13(2) (2023) 297-319 http://dx.doi.org/10.5599/jese.1574 303 The thickness of the polymer film has an impact on the electrocatalytic capability of the prepared electrode [41]. Therefore, the effect of the number of cycles of electro-polymerization on the electrocatalytic activity of the prepared electrode was examined from 3 to 15 cycles. Due to an increase in active sites at the electrode surface, the peak current of VB6 increased along with the number of cycles up to 5 cycles (inset of Figure 2). After 5 cycles, the thickness of the polymer film increased and blocked the electron transfer between the electrode and VB6. Thereby, 5 potential scans were chosen to electropolymerize phenylalanine on the electrode surface. Electrochemical properties of bare and modified GCE The electrochemical behavior of the bare GCE and modified electrodes were examined by CV and EIS in 0.1 M KCl with 5 mM [Fe(CN)6]3–/4– taken as a redox probe. The CV responses of bare GCE (curve a), F-MWCNT (curve b), poly(phenylalanine)/GCE (curve c) and poly(phenylalanine)/F- MWCNT/GCE (curve d) are shown in Figure 3. Weak redox peaks and a broad voltammogram were observed for bare GCE (curve a) in comparison to the modified electrodes. Due to the high surface area and conductivity of F-MWCNT, the modification of bare GCE with F-MWCNT enhanced the current response of [Fe(CN)6]3–/4– and lowered the peak-to-peak potential separation. After electro- depositing poly(phenylalanine) on the GCE, the redox peak current increased and peak-to-peak potential separation reduced because the high conductivity of polymer and more active sites made it easier for [Fe(CN)6]3–/4– to reach the electrode surface. Similar to this, after F-MWCNT/GCE was modified with poly(phenylalanine), the redox peak currents increased further, and the peak-to-peak separation decreased because of the synergistic effect of F-MWCNT and poly(phenylalanine) for the electron transfer between [Fe(CN)6]3–/4– and poly (phenylalanine)/F-MWCNT/GCE. Using Randles- Ševčik equation, the electroactive surface areas of the unmodified GCE and modified GCE were estimated [27,42]: Ip = (2.69×105) AD1/2z3/2n1/2C (1) where Ip / A is peak current, z is a number of electrons involved in the reaction (z = 1), A / cm2 is electrode active surface area, D (7.6×10-6 cm2s−1) is diffusion coefficient of ferricyanide ions in 0.1 M KCl solution, C is the concentration of K3[Fe(CN)6] (mmol cm−3) and n / V s−1 is scan rate. E / V vs. Ag/AgCl Figure 3. CVs (n = 0.1 V s-1) of 5.0 mM [Fe(CN)6]3−/4− in 0.1 M KCl at: bare GCE (a), F-MWCNT/GCE (b), poly (phenylalanine)/GCE (c) and poly(phenylalanine)/F-MWCNT/GCE (d) According to the scan rate analysis, the anodic and cathodic peak currents of all electrodes were found to be proportional to the square root of the scan rate in the range 0.025 to 0.4 V s−1. From the -0.25 0.00 0.25 0.50 0.75 -150 -75 0 75 150 I / m A E/ V ns. Ag/AgCl a b c d A I / m A http://dx.doi.org/10.5599/jese.1574 J. Electrochem. Sci. Eng. 13(2) (2023) 297-319 DETERMINATION OF VITAMIN B6 304 slopes of Ip versus n 1/2, plots, the electroactive surface area was calculated for each electrode. The calculated values were 0.05, 0.08, 0.09 and 0.12 cm2 for bare GCE, F-MWCNT/GCE, poly(phenyl- alanine)/GCE and poly(phenylalanine)/F-MWCNT/GCE, respectively. Electrochemical impedance spectroscopy (EIS) was used to further examine electrochemical characteristics of unmodified and modified electrodes. Important information about the kinetics of electron transfer at the electrodes can be provided by EIS [43]. In Nyquist plots, the semicircle diameter refers to the electron transfer resistance (Rct) at the electrode surface [44]. Figure 4A shows Nyquist plots of EIS for all electrodes in 0.1M KCl containing 5.0 mM [Fe(CN)6] 3−/4− redox probe. In comparison to other modified electrodes, bare GCE exhibited the highest charge transfer resistance (Rct) and the lowest double-layer capacitance (Cdl) value, as shown in Table 1. It was evident that the F-MWCNT modified surface has higher conductivity and surface area than bare GCE since the Rct value of the F-MWCNT/GCE was lower than that of bare GCE. Electrodeposition of poly(phenylalanine) on the surface of bare GCE can also reduce the Rct value by accelerating the electron transfer rate due to its high electrocatalytic ability. Poly(phenylalanine)/F-MWCNT/GCE exhibits the lowest Rct and the highest Cdl compared to all other electrodes. The high value of Cdl at poly(phenylalanine)/F-MWCNT/GCE indicates the increase in the surface area of the electrode after modification. Thus, more ions transfer from the solution to the electrical double layer, increasing the electrical double layer capacitance. Similarly, Bode plots (Figure 4B, C) revealed that MWCNT or poly(phenylalanine) modified electrode has lower charge transfer resistance (lower impedance at all frequencies) than bare GCE. Poly(phenylalanine)/F-MWCNT modified electrode exhibited the lowest charge transfer resistance. At very high frequencies where solution resistance (Rs) dominates, the impedance values are almost similar for all electrodes. This suggests that modifiers did not have any significant impact on the solution resistance value. Therefore, the combination of MWCNT and poly(phenylalanine) increases exclusively the electron transport between the electrode and [Fe(CN)6]3−/4−. In light of this, the EIS results confirm the CV results. Furthermore, the exchange current density (jo) and electron transfer rate constant (ko) for the electrodes were computed from the EIS data using the following equations [45]: 0 ct RT j zR AF = (2) 0 2 ct RT k R ACF = (3) where j0 / A cm-2 is the exchange current density, ko / cm s−1 is the electron transfer rate constant, R is the universal gas constant (8.314 J K−1 mol−1), T is the temperature (298 K), F is Faraday constant (96485 C mol−1), Rct / Ω is the electron transfer resistance, A / cm2 is the electrode surface area and z is the number of electrons involved in the reaction (z = 1). The j0 and ko values obtained for the bare GCE, F-MWCNT/GCE, poly(phenylalanine)/GCE and poly (phenylalanine)/F-MWCNT/GCE are listed in Table 1. Table 1. Electron transfer rate constant, exchange current density, charge transfer resistance and double layer capacitance for bare and modified GCE Electrode ko / cm s−1 J0 / µA cm-2 Rct / Ω Cdl / µF GCE 0.0034 1633 314.4 1.22 MWCNT/GCE 0.0043 2100 152.8 1.62 poly(phenylalanine)/ GCE .0046 2246 127 2.45 poly(phenylalanine) /F-MWCNT/GCE 0.0051 2462 86.91 16.0 G. Tesfaye et al. J. Electrochem. Sci. Eng. 13(2) (2023) 297-319 http://dx.doi.org/10.5599/jese.1574 305 Z’ /  log (f / Hz) log (f / Hz) Figure 4. (A) Nyquist plots of 5.0 mM [Fe(CN)6] 3−/4− in 0.1 M KCl at: bare GCE (a), F-MWCNT/GCE (b), poly (phenylalanine)/GCE (c) and poly(phenylalanine)/F-MWCNT/GCE (d). Inset: Randles equivalent electrical circuit used for fitting Nyquist plots; Rs is solution resistance, Rct is charge transfer resistance, Cdl is double layer capacitance, and Zw is diffusion (Warburg) impedance. (B) Bode plots, log Z vs. log frequency and (C) phase angle vs. log frequency at bare GCE (a), F-MWCNT/GCE (b), poly(phenylalanine)/GCE (c) and poly(phenylalanine)/F-MWCNT/GCE (d). Frequency range of 100 kHz−0.1 Hz, applied potential of 0.15 V and amplitude of 0.005 V The findings demonstrated that greater j0 and ko values were achieved for the composite modified electrode, poly(phenylalanine)/F-MWCNT/GCE, due to its large surface area and presence of additional functional groups that were active and facilitated the transport of electrons between the electrode and [Fe(CN)6]3–/4– . It seems that the electron transfer process is simpler and quicker at the composite-modified electrode. Surface characterization FTIR was used to investigate the surface functional groups of functionalized MWCNT and the deposition of polymer film on the electrode surface. Figure 5 shows FTIR spectra of pristine MWCNT, F-MWCNT and poly (phenylalanine)/F-MWCNT. It could be seen that the F-MWCNT exhibited new bands compared to pristine MWCNT due to the attachment of new functional groups on their surface. The FTIR spectrum of MWCNT showed weak peaks at 3430 and 1646 cm−1 corresponding to O–H stretching vibration of the C–OH, which can be introduced during their synthesis and conjugated C=C stretching vibration of pristine MWCNT, respectively. Absorption bands at 2918 and 0 200 400 600 800 0 150 300 450 600 -Z " ( O h m ) Z' (Ohm) c d a b A -3 -2 -1 0 1 2 3 4 5 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 lo g ( Z /O h m ) log (Frequency/Hz) a b cd B -3 -2 -1 0 1 2 3 4 5 0 10 20 30 40 50 -P h a s e a n g le / d e g re e log (frequency / Hz) a b c d C -Z “ /  lo g ( Z /  ) Rs Rct Cdl ZW -  /  http://dx.doi.org/10.5599/jese.1574 J. Electrochem. Sci. Eng. 13(2) (2023) 297-319 DETERMINATION OF VITAMIN B6 306 2853 cm−1 are assigned to asymmetric and symmetric stretching vibrations of the C–H [46]. For F- MWCNT, a strong absorption band observed at 3445 cm−1 is attributed to OH stretching vibration of the ─COOH groups [39,46]. The absorption bands at 2927 and 2862 cm–1 are assigned to asym- metric and symmetric stretching vibrations of the C–H bond in CH2 of F-MWCNT, respectively [46]. A weak band at 1749 cm−1 is assigned to the stretching vibration of C=O of the carboxyl group [39]. The absorption peak at about 1639 cm–1 is assigned to the C = C skeletal stretching vibration of F- MWCNT. The band at 1447 cm−1 is assigned to C-H bending vibration of –CH2 [46]. The peak at 1380 cm−1 corresponds to O–H bending vibration [39]. The absorption band at about 1083 cm–1 can be ascribed to the C–O stretching vibrations of epoxy groups [46]. The increase in the intensity of O–H stretching vibration and formation of C=O and C–O functional groups after oxidation of pristine MWCNT indicate successful functionalization of MWCNT [39]. The structural properties of poly(phenylalanine) /F-MWCNT have also been investigated using FTIR spectroscopy. The absorption band at 3443 cm−1 corresponds to O–H stretching of MWCNT and the N–H stretching of poly (phenylalanine) [47]. The peaks at 2922 and 2852 cm−1 are attributed to asymmetric and symmetric stretching vibrations of the C–H in CH2 [47]. The characteristic band at 1738 cm−1 is assigned to the stretching vibration of C=O of the carboxylic group. The band at 1634 cm−1 is attributed to the C=O stretching vibration of the amide group on the poly (phenylalanine) [47]. The band at 1465 cm−1 is attributed to C–H bending vibration of the CH2 groups and phenyl ring stretching. The band observed at 1383 cm−1 is assigned to O–H bending vibration and phenyl ring stretching [39]. The band at 1226 cm−1 is due to C–N stretching of secondary amine. The peak observed at 1081 cm−1 is assigned to C–O stretching of the alkoxy group and C–H bending in the phenyl ring [46]. The results obtained from the FTIR spectrum indicated the successful functionalization of MWCNT and deposition of the polymer film. Wavenumber, cm-1 Figure 5. FTIR spectra of MWCNT, F-MWCNT and poly(phenylalanine) /F-MWCNT The morphologies of bare GCE, F-MWCNT/GCE, poly(phenylalanine)/GCE and poly(phenylala- nine)/F-MWCNT/GCE were investigated by SEM. As shown in Figure 6A, bare GCE has a smooth surface structure. After the deposition of F-MWCNT on GCE, porous and thread-like structures of F- MWCNT were observed (Figure 6B). Figure 6C shows a loose and rough surface of poly (phenylalanine) film on GCE. After the deposition of poly(phenylalanine) on F-MWCNT (Figure 6D), a dense and tight surface structure of poly(phenylalanine) film is observed on F-MWCNT, providing 0 1000 2000 3000 4000 5 10 15 20 25 30 1210 1220 1230 1240 1250 1260 1226 T ra n s m it tn c e / % Wavenumber / cm-1 MWCNT F-MWCNT poly (phenylalanine) /F-MWCNT 3443 2922 2852 1738 1634 1465 1383 1081 T ra n sm it ta n ce , % G. Tesfaye et al. J. Electrochem. Sci. Eng. 13(2) (2023) 297-319 http://dx.doi.org/10.5599/jese.1574 307 supplementary surface area for adsorption of VB6. The π-π interaction and hydrogen bonding between F-MWCNT and poly(phenylalanine) were strong enough to make the polymer film attached to F-MWCNT. This strong interaction between F-MWCNT and poly (phenylalanine) results in high stability of the composite material. The results obtained from the SEM study reveal the successful deposition of F-MWCNT on bare GCE and the uniform deposition of poly(phenylalanine) on F- MWCNT. Figure 6. SEM images of bare GCE (A), MWCNT/GCE (B), poly (phenylalanine/GCE (C) and poly(phenylalanine) /MWCNT/GCE (D) Cyclic voltammetric performance of VB6 at bare and modified GCE The electrochemical behavior of VB6 at bare GCE and modified GCE was studied by CV at the scan rate of 0.1 V s−1 in ABS pH 4.5. Figure 7 shows CVs of 200 µM VB6 at GCE (curve a), F-MWCNT/GCE (curve b), poly(phenylalanine)/GCE (curve c) and poly(phenylalanine)/F-MWCNT/GCE (curve d). At the bare GCE (curve a), VB6 shows a weak oxidation peak indicating slow electron transfer kinetics. Compared to bare GCE, an increase in peak current and decrease in peak potential are observed at F-MWCNT/GCE due to the presence of oxygen-containing functional groups and high conductivity of F-MWCNT which facilitate the accumulation of VB6 and electron transfer rate at the surface of the electrode. At poly (phenylalanine)/GCE, oxidation peaks higher than the bare GCE and F- MWCNT/GCE were observed. The enhanced peak current observed at poly(phenylalanine)/F- MWCNT/GCE indicates the enhanced kinetics of the electrochemical reaction due to the synergistic effect of MWCNT and poly(phenylalanine), such as high conductivity and presence of functional groups, which facilitate the accumulation of VB6 through hydrogen bonding and π-π interaction (Scheme 2). Furthermore, the deposition of F-MWCNT and poly(phenylalanine) onto the GCE provide more conducting pathways (more active sites) to ease the electron transfer between the surface of the electrode and the analyte. In the reverse scan, no cathodic peak was observed at all the electrodes, indicating that the electrochemical oxidation of VB6 is irreversible [22]. http://dx.doi.org/10.5599/jese.1574 J. Electrochem. Sci. Eng. 13(2) (2023) 297-319 DETERMINATION OF VITAMIN B6 308 E / V vs. Ag/AgCl Figure 7. CVs (0.1 Vs-1) of bare GCE (a), F-MWCNT/GCE (b), poly(phenylalanine)/GCE (c), poly(phenylalanine) /F-MWCNT/GCE (d) (background subtracted) in the presence of 200 µM VB6 in 0.1 M ABS pH 4.5 after accumulation at 0.0 V for 120 s Scheme 2. Possible interaction of VB6 with poly(phenylalanine)/F-MWCNT modified electrode and its mechanism of electrooxidation The performance of the sensor was examined to determine the impact of the order of deposition of F-MWCNT and polymer. The electrocatalytic activity for the oxidation of VB6 was found to be higher when F-MWCNT is deposited on the GCE before phenylalanine polymerization (poly(phenylalanine)/F-MWCNT/GCE), as opposed to the case when phenylalanine polymerization was followed by F-MWCNT deposition (F-MWCNT/poly(phenylalanine)/GCE). It is likely that the superior performance of poly(phenylalanine)/F-MWCNT/GCE is caused by the increased surface area when F-MWCNT was applied first. This makes it easier to apply poly(phenylalanine) and increases the number of active sites on the electrode surface [48]. Additionally, it was examined how well the poly(phenylalanine)/F-MWCNT/GCE worked during successive measurements. The oxidation peak current was not considerably altered after 10 SWV measurements of 200 µM VB6 solution, demonstrating high operational stability of the modified electrode. I / m A G. Tesfaye et al. J. Electrochem. Sci. Eng. 13(2) (2023) 297-319 http://dx.doi.org/10.5599/jese.1574 309 Optimization of experimental conditions Effect of amount of F-MWCNT SWV was used to examine how the amount of F-MWCNT affected the sensitivity of VB6. To get optimum amount of F-MWCNT, different amounts of F-MWCNT were dropped onto GCE. Increasing the amount of F-MWCNT up to 8 µL improved the current response of VB6. However, the sensitivity was reduced at volumes greater than 8 µL. This is explained by the thickening of the F- MWCNT layer, which interferes with electron transfer and decreases electrocatalytic activity. Therefore, 8 µL was selected as the optimum amount of F-MWCNT for the preparation of the sensor electrode. Impact of solution pH CV measurements were made in the pH range of 3.0 to 7.0 to determine how the pH of ABS affects the current response of 200 µM VB6 at poly(phenylalanine)/F-MWCNT/GCE (Figure 8A). The peak current of VB6 increased progressively from pH 3.0 to 4.5, as shown in Figure 9B, and then fell gradually with increasing pH. From the fact that pKa values of VB6 are pKa1 = 5 and pKa2 = 8.96 [4,49] while pKas of phenylalanine are pKa1= 2.47 and pKa2 = 9.13 [32], the increase in current response with pH from 3.0 to 4.5 might be due to the increase in electrostatic attraction between the positively charged VB6 (pH ˂ 5.0) and negatively charged polymer film (pH ˃ 2.47). The charge density on both VB6 and poly (phenylalanine) increases as the pH rises, which causes the electrostatic attraction between VB6 and poly (phenylalanine) to decrease. This reduces the accumulation of VB6 at the electrode surface. Thus, 0.1 M ABS pH 4.5 was chosen as the appropriate supporting electrolyte for subsequent investigations. Furthermore, the pH of the solution also affects the oxidation peak potential of VB6. The anodic peak potential shifted negatively with the increasing pH of the solution, demonstrating that peak potential is dependent on solution pH. The oxidation peak potential of VB6 changed linearly with the solution pH in the range 3.0 to 7.0. The regression equation for the dependence of the peak potential on the pH is given by the equation (Figure 8B): Ep = −0.063 pH +1.18 (R2 = 0.994). The slope is close to the Nernstian value of −59 mV, indicating the same number of protons and electrons are involved in the electrochemical oxidation of VB6, which is in agreement with previous works [4,5,20]. E / V vs. Ag/AgCl pH Figure 8. (A) CVs of 200 µM VB6 at poly (phenylalanine)/F-MWCNT/GCE in 0.1 M ABS of various pH values (3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6, 6.5 and 7) at scan rate of 0.1 V s−1. (B) Effect of pH on the peak current and peak potential values of VB6 0.2 0.4 0.6 0.8 1.0 1.2 1.4 -20 -10 0 10 20 30 40 50 60 I / m A E / V ns. Ag/AgCl A 37 3 4 5 6 7 4 6 8 10 12 14 pH I / m A B 0.75 0.80 0.85 0.90 0.95 1.00 1.05 E / V n s . A g /A g C l Ep (V) = -0.063 pH + 1.18 R2 = 0.994I / m A I / m A E / V v s. A g /A g C l Ep = -0-063 pH + 1.18 R2 = 0.994 http://dx.doi.org/10.5599/jese.1574 J. Electrochem. Sci. Eng. 13(2) (2023) 297-319 DETERMINATION OF VITAMIN B6 310 Scan rate study By using cyclic voltammetry, the influence of scan rate on the oxidation peak current and peak potential of 200 M VB6 in ABS pH 4.5 at the poly(phenylalanine)/F-MWCNT/GCE was investigated in order to determine the electrochemical reaction mechanism of VB6 at the modified electrode (Figure 9A). As shown in Figure 9B, the oxidation peak current is proportional to the scan rate in the range 0.025 to 0.3 V s−1. According to regression equations Ipa = 112.6 + 1.08 (R2 = 0.998), the electro- oxidation of VB6 at the surface of the modified electrode is adsorption controlled [15]. The relationship between the logarithm of peak current (log I) and the logarithm of scan rate (log υ) in the range 0.025 to 0.3 V s−1 was further investigated. As shown in Figure 9C, there is good linearity between log I and log υ. The corresponding regression equations is: log Ipa = 0.89 log n + 1.98 (R2 = 0.996) with a slope close to the theoretical value of 1, confirming the electrochemical oxidation of VB6 at poly (phenylala- nine)/F-MWCNT/GCE as a surface-controlled process. Moreover, the influence of scan rate on the peak potential of VB6 was studied. With increasing scan rate, the oxidation peak potential of VB6 shifted to a more positive value, indicating that the electrooxidation of VB6 at poly(phenylalanine)/ /F-MWCNT/GCE is irreversible [20]. The linear regression equations for the variation of the oxidation peak potential (Ep) with the logarithm of scan rate (log υ) in the range 0.025 to 0.3 V s−1 is given by the equation: Epa = 0.052 log n - 0.93, R2 = 0.996 (Figure 9D). According to Laviron’s equation [50,51], the relationship between Ep and log ν for an irreversible electrode reaction is given by: p 2.303 2.303 log log o RT RTk RT E E zF zF zF n    = + + (4) where Eo is the formal potential, T is the temperature (298 K), α is the transfer coefficient, z is the number of electrons transferred, n is scan rate, F is Faraday’s constant (96,480 C mol−1), R is the universal gas constant (8.314 J mol−1 K−1), and k is the heterogeneous electron-transfer rate constant. From the plot of Ep versus log n, the slope 2.303RT/ αzF equals 0.052. Using this value, αn was calculated to be 1.12. For an irreversible electrode reaction [51], α can be given as: pa pa/2 47.7 E E  = − (5) where Epa/2 is the potential where the current is half the peak value. Thus, from eq. (5) α was found to be 0.51. Additionally, it was determined that 2.2 (2) electrons were involved. Thus, the oxidation of VB6 requires two electrons [5,18]. From the relationship between Epa and pH, an equal number of electrons and protons are transferred during the electrooxidation of VB6. Therefore, the electrode reaction mechanism for VB6 oxidation at poly(phenylalanine)/F-MWCNT/GCE involves two electrons and two protons. Furthermore, it is possible to determine the surface concentration of VB6 at poly (phenylalanine)/F-MWCNT/GCE from the slope of the linear plot of oxidation peak current vs. v using the following equation [52]: 2 2 pa 4 z F A I RT  n = (6) where  / mol cm-2 is the surface concentration of electroactive species, A / cm2 is the electrode surface area and z, F, n, R, and T are as described in Eq. (4). The surface coverage of VB6 at the poly(phenylalanine)/F-MWCNT/GCE was calculated to be  = 0.24 nmol cm-2. G. Tesfaye et al. J. Electrochem. Sci. Eng. 13(2) (2023) 297-319 http://dx.doi.org/10.5599/jese.1574 311 I / m A I / m A E / V vs. Ag/AgCl n / V s-1 lo g ( I / m A ) E p / V log (n / V s-1) log (n / V s-1) Figure 9. (A) CVs of 200 µM VB6 at poly (phenylalanine)/F-MWCNT/GCE in ABS pH 4.5 at scan rates of 0.025, 0.05,0.075, 0.1, 0.125, 0.15, 0.175, 0.2, 0.25, 0.3, 0.350, 0.4 V s−1. (B) Plot of the oxidation peak current versus scan rate (n). (C) Variation of the logarithm of the oxidation peak current (log I) with the logarithm of scan rate (log n). (D) Variation of the oxidation peak potential (Ep) with the logarithm of scan rate (log n) Determination of VB6 at poly(phenylalanine)/F-MWCNT/GCE Square wave voltammetry (SWV) determination of VB6 was performed under the optimized expe- rimental conditions in ABS pH 4.5 in the potential range 0 to 1.2 V. According to Figure 10A, the peak currents increased linearly from 0.5 to 200 M of VB6 concentration. I / m A I / m A E / V vs. Ag/AgCl C / mM Figure 10. (A) SWVs for different concentrations of VB6: 0.5, 0.8, 1.5, 2, 4, 6, 10, 15, 20, 30, 40, 60, 80, 100, 140, 160, 180 and 200 µM in 0.1 M ABS pH 4.5. (B) Plot of the peak current versus concentration of VB6 at poly(phenylalanine)/F-MWCNT/GCE. Conditions: Eacc, 0.0 V; tacc, 120 s. SWV parameters: frequency = 35 Hz; step potential = 8 mV and pulse amplitude = 40 mV 0.0 0.2 0.4 0.6 0.8 1.0 1.2 -40 -20 0 20 40 60 80 I / m A E / V ns. Ag/AgCl 0.025 V s− 1 0.3 V s−1 A 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0 5 10 15 20 25 30 35 40 I / m A Scan rate, n / V s−1 Ipa = 112.6 n + 1.08 R2 = 0.998 B -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 0.4 0.6 0.8 1.0 1.2 1.4 1.6 lo g I / m A log n/ Vs−1 log Ipa = 0.89 log n + 1.98 R2 = 0.996 C -1.6 -1.4 -1.2 -1.0 -0.8 -0.6 -0.4 0.84 0.85 0.86 0.87 0.88 0.89 0.90 0.91 E P / V log n / V s−1 Epa = 0.052 log n + 0.93 R2 = 0.996 D 0.4 0.6 0.8 1.0 1.2 -10 0 10 20 30 I / m A E / V ns. Ag/AgCl 0.5 mM 200 mM A 0 50 100 150 200 0 5 10 15 20 25 30 I / m A Concentration / mM I = 0.093 C + 9.11 R2 = 0.995 I = 0.504 C + 0.259 R2 = 0.995 B Ipa =112.6 n + 1.08 R2 = 0.998 Ipa = 0.052 log n + 0.93 R2 = 0.996 Ipa = 0.89 log n + 1.98 R2 = 0.996 I = 0.093 C + 9.11 R2 = 0.995 I = 0.504 C + 0.259 R2 = 0.995 http://dx.doi.org/10.5599/jese.1574 J. Electrochem. Sci. Eng. 13(2) (2023) 297-319 DETERMINATION OF VITAMIN B6 312 Two linear ranges could be seen on the VB6 calibration curve. The first linear range is 0.5 to 20 µM and the second is 20 to 200 µM with regression equations: I = 0.504 C + 0.259, R2 = 0.995 and I = 0.093 C + 9.11, R2 = 0.995, respectively (Figure 10B). Error bars are added to the graph using the standard deviation of triplicate measurements of each data point. LOD and LOQ limits were calculated from the oxidation peak current of VB6 using the following equations: LOD = 3s/m and LOQ = 10s/m, where s is the standard deviation of the oxidation peak currents (n = 3) of the lowest concentration in the linear range and m is the slope of the calibration curve. The calculated LOD and LOQ values are 0.038 and 0.125 µM, respectively. Furthermore, the linear range and detection limit obtained in this study were compared with the results of previously reported electrochemical methods for the determination of VB6 (Table 2). The findings suggest that the proposed sensor exhibits a better linear range and detection limit than most of the reported sensors. The proposed sensor is also inexpensive and easy to make. Table 2. Comparison of reported analytical data for VB6 determination obtained by sensor electrodes Modified electrode Technique Concentration linear range, µM LOD, µM Ref. aP-doped/PGE lDPV 0.5–300 0.065 [4] b44-DABP/GCE DPV 15.7–2210 4.7 [5] cAuNps/POAP/PGE DPV 5–200 0.30 [1] dPMG/fCNT/CE DPV 100–800 9.4 [53] eAu-CuO/MWCNTs/GCE DPV 0.79–18.4 0.15 [54] fCrHCF/GCE CV 1.33–13.2 0.035 [55] gNiZCB-GCE DPV 0.3–5.9 0.09 [49] hMWCNT/CoPc/PGE DPV 10–400 0.5 [19] iCoHCF/MCPE SWV 5–26 0.17 [22] jN,S-GQD-CS/ GCE SWASV 0.1–18 0.03 [56] kePADs SWV 200–2000 29.5 [57] poly(phenylalanine)/F-MWCNT/GCE SWV 0.5–20 and 20–200 0.038 This work aPhosphorus-doped pencil graphite electrode; b4,4’-diamino benzophenone modified glassy carbon electrode; cgold nanoparticles and non-conducting polymeric film of o-aminophenol; dpoly(methylene green) (PMG)and functionalized carbon nanotubes (fCNT) modified graphite composite electrode (CE); ecopper oxide (shell) nanoparticles-MWCNT; fchromium(III) hexacyanoferrate(II); gNi-zeolite/carbon black modified glassy carbon electrode; hmultiwalled carbon nanotube (MWCNT and cobalt phthalocyanine (CoPc) modified pyrolytic graphite electrode; icobalthexacyanoferrate modified carbon paste electrode; ldifferential pulse voltammetry; jnitrogen and sulfur co-doped graphene quantum dot-modified glassy carbon electrode; kelectrochemical paper-based analytical devices Repeatability and stability of poly(phenylalanine)/F-MWCNT/GCE To evaluate the repeatability of the prepared sensor electrode for determining 20 µM VB6, 10 consecutive measurements of 20 µM VB6 were performed using the same electrode. The relative standard deviation (RSD) of the current response was 2.38 %, indicating that the prepared electrode can be used for several measurements to determine VB6. The electrode fabrication repeatability for VB6 determination was evaluated by measuring the peak current of 20 µM with four different electrodes prepared under identical conditions. With an RSD of 2.16 %, the outcome demonstrates that all electrodes have essentially identical responses, showing good repeatability of the electrode preparation process. In order to determine the poly (phenylalanine)/F-MWCNT/GCE long-term stability, the current responses to 20 M VB6 were monitored weekly over one month. The electrode used every week is kept at 4 °C in the refrigerator. The synthesized poly(phenylalanine)/F- MWCNT/GCE was found to have effective long-term stability, as evidenced by the observation that the oxidation current response dropped by 6.3 % at the end of the month. Robustness study The robustness of the developed method was examined by studying the effect of small variations of some experimental parameters, such as pH (4.5 ± 0.1), accumulation potential (0.0 ± 0.02 V) and accumulation time (120 ± 2 s) on the recovery of VB6. As shown in Table 3, the recovery for VB6 G. Tesfaye et al. J. Electrochem. Sci. Eng. 13(2) (2023) 297-319 http://dx.doi.org/10.5599/jese.1574 313 under all studied conditions is in the range of 96.1 – 103.4 %, indicating that small variation of the studied experimental parameters has an insignificant effect on the current response of VB6. Therefore, the proposed SWV method has good robustness and is reliable for the quantification of VB6. Table 3. Robustness study for the voltammetric method used for determination of 20 µM VB6 (n=3) Parameters Mean, µM ± RSD, % Recovery, % Optimum values (pH = 4.5, Eacc = 0.0 V and tacc = 120 s) 20.68 ± 1.97 103.4 pH (variation) 4.4 19.42 ± 3.6 97.1 4.6 19.35 ± 1.2 96.8 Accumulation potential (variation) 0.02 V 19.48 ± 2.63 97.4 -0.02 V 19.22 ± 2.9 96.1 Accumulation time (variation) 118 s 19.42 ± 1.58 97.1 122 s 19.55 ± 3.8 97.8 Effect of interferents The impact of potential interfering compounds on the determination of 20 µM VB6 was examined by SWV under optimal conditions. Except for folic acid (FA), the data demonstrated that none of these interferants at poly(phenylalanine)/F-MWCNT/GCE exhibited a voltammetric response. As can be seen from Figure 11, the response of VB6 was unaffected by 200− fold excess concentrations of Ca2+, K+, Mg2+, Cu2+, Fe2+, NO3−, CO32− and HCO32− , 100− fold excess concentrations of ascorbic acid (AA), citric acid (CA), sodium citrate (NaCA), tartaric acid (TA), glucose, fructose, starch, sucrose, caffeine, vitamin B1 (VB1), vitamin B2 (VB2) and vitamin B12 (VB12). Interferent Figure 11. Change in peak current response in the presence of different interferents for 20 µM VB6 However, FA exhibited two oxidation peaks close to that of VB6. In the presence of folic acid, even at a concentration ratio of 1:1, the peak current of VB6 was reduced and the potential shifted to a positive value. Therefore, FA must be separated before the voltammetric measurement of VB6. Here, however, the examined tablet and beverage samples did not have any folic acid. Additionally, the standard addition method was applied for the quantification of VB6 to minimize the interference S ig n a l ch a n g e , % http://dx.doi.org/10.5599/jese.1574 J. Electrochem. Sci. Eng. 13(2) (2023) 297-319 DETERMINATION OF VITAMIN B6 314 effect. As a result, the proposed electrode can be successfully utilized to measure VB6 in pharma- ceutical and beverage samples. Real sample analysis The developed method was applied for the determination of VB6 in commercially available pharmaceutical tablets (vitamin B-Complex containing VB1, VB6 and VB12) and an energy drink (predatory energy drink). The samples were prepared and diluted to the appropriate concentration with the supporting electrolyte. SWV was used to record signals at poly(phenylalanine)/F- MWCNT/GCE and concentrations of VB6 were determined by the standard addition method to minimize the matrix effect. As shown in Table 4, the result obtained for VB6 using the proposed method was found to be 95 mg VB6/tablet, which is in good agreement with the labelled value of 100 mg VB6/tablet (relative error 5 %). Similarly, the value of VB6 in the energy drink was found to be 0.29 mg/100mL, which is close to the labelled amount (0.3 mg/100 mL) (relative error of 3.3 %). Furthermore, recovery studies were carried out by the standard addition method to validate the accuracy and precision of the developed sensor and the results are shown in Table 5. The recovery values varied from 90.0 to 100.4 %, suggesting that the developed method is reliable and effective for the determination of VB6 in vitamin B-complex and energy drinks without the effect of other constituents of the samples. Furthermore, the low values of RSD (ranging from 0.4 to 4.0 %) indicate good precision of measurements. Table 4. Comparison of experimental results with labelled values Sample Labelled content Experimental content ± SD Relative error, % Predatory energy drink 0.3 mg/100 mL 0.29 mg/100 mL ± 0.08 3.3 N-VIT 100 mg/tablet 95 mg/tablet ± 3.1 5.0 Table 5. Recovery study Conclusions In the present study, poly(phenylalanine) and F-MWCNT modified glassy carbon electrode was successfully prepared for the electrochemical determination of VB6 in pharmaceutical tablet and energy drink samples. The developed sensor exhibits high electrocatalytic activity towards the oxidation of VB6 due to the synergistic effect between poly(phenylalanine) and F-MWCNT. CV study showed that the electrode reaction of VB6 is the irreversible and adsorption-controlled process. Under the optimized conditions, SWV experiments showed peak currents that increased linearly with VB6 concentration in two linear ranges (0.5 to 20 and 20 to 200 µM). The detection limit (LOD) was found to be 0.038 µM. The prepared electrode showed high selectivity, stability, repeatability and reproducibility. The acceptable recovery values obtained for VB6 during real samples analysis Sample C / µM RSD, % n = 3 Recovery, % Added Found N-VIT 0 4.98 3.10 - 3 7.68 4.00 90.0 5 9.58 1.30 92.0 8 12.6 0.37 95.3 12 17.0 1.80 100.4 Predatory energy drink 0 3.48 2.40 - 3 6.28 0.26 92.7 5 8.17 0.95 93.8 8 10.7 2.90 90.8 12 15.3 0.40 98.8 G. Tesfaye et al. J. Electrochem. Sci. Eng. 13(2) (2023) 297-319 http://dx.doi.org/10.5599/jese.1574 315 demonstrate that the proposed sensor can be effectively used for the determination of VB6 in pharmaceutical tablets and energy drinks with high accuracy. Acknowledgment: The authors gratefully acknowledge Oromia Education Bureau for the financial support. We are grateful to the Department of Chemistry, Addis Ababa University for providing laboratory facilities. Conflicts of Interest: The authors declare no conflict of interest. References [1] R. Rejithamol, S. 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