Introduction

Dopamine (DA) is one of the significant catecholamines which belongs to the family of excitatory chemical neurotransmitters. DA is naturally produced and widely distributed in the brain of mammals where it acts as a neurotransmitter for message transfer in the central nervous system and for activating DA receptors. DA is also a neurohormone released by the hypothalamus; its main function as a hormone is to inhibit the release of prolactin from the anterior lobe of the pituitary. Abnormal levels of dopamine are symptomatic of some brain disorders and several diseases such as Parkinson’s disease, Alzheimer’s disease and Schizophrenia [1, 2].

Serotonin (5-hydroxytryptamine, 5-HT or ST) is a monoamine neurotransmitter synthesized in serotonergic neurons in the central nervous system and enterochromaffin cells in the gastrointestinal tract. In the central nervous system, ST is believed to play an important role in the regulation of mood, sleep, emesis (vomiting), sexuality and appetite. Low levels of ST have been associated with several disorders, notably depression, migraine, bipolar disorder and anxiety [3]. So investigation of chemical and neurological behaviour and also determination of DA and ST is of great importance because levels of DA and ST in biological fluids such as serum may also provide further information of diagnostic value in the aforementioned disorders. Furthermore, numerous reports have shown that DA and ST coexistent in biological systems and influence each other in their respective activities [4–6]. Therefore, the simultaneous determination of DA and ST is of great importance for the elucidation of their precise physiological functions and many chemists, biochemists and neuroscientists are actively researching this topic.

A range of techniques such as chromatographic methods [7–9], electrophoresis [10, 11], mass spectroscopy [12, 13], spectrophotometry [14], spectrofluorimetry [15] and chemiluminesence [16, 17] are reported in the literature for detection of DA and ST in real samples such as biological fluids, nervous tissues and pharmaceutical formulations. However these methods suffer from some disadvantages including long analysis times, high costs, the requirement for sample pretreatment, and in some cases low sensitivity and selectivity. These disadvantages make them unsuitable for routine analysis.

DA and ST are both electro-active compounds with very similar electrochemical properties and they can be oxidized electrochemically, so application and development of electrochemical methods and sensors for determination of neurotransmitters such as DA and ST has received considerable interest in the past few decades [18–20]. But voltammetric determinations of DA and ST suffer from high concentrations of coexisting interferences such as ascorbic acid (AA) in real samples which are typically present in the range 100–1,000 μM, and cannot be neglected because they can be oxidized at similar potentials at conventional electrodes [21]. To overcome this problem a wide variety of electrodes and different electrochemical techniques have been utilised for determination of DA in the presence of AA [22–25]. There are a few reports in the literature about the simultaneous determination of DA and ST in the presence of AA which used different electrochemical methods and electrodes for their simultaneous determination. For example, the simultaneous determination of DA and ST has been reported in the presence of AA using electrodeposited nanostructured platinum on Nafion-coated glassy carbon electrodes [26], poly(o-phenylenediamine)- and poly(phenosafranine)-modified glassy carbon electrodes [27, 28], DNA-immobilized carbon fibre microelectrodes [18, 20], choline- and acetylcholine-modified glassy carbon electrodes [19], carbon-nanotube-modified glassy carbon electrodes [29], carbon-nanotube-intercalated graphite electrodes [30], carbon paste electrodes modified with iron(II) phthalocyanine complexes [31] and graphite electrodes reinforced by carbon [32]. A proposed methodology for the discrimination between DA and ST in the presence of AA with the combination of a large amplitude/high frequency voltage excitation and signal processing techniques [33] is also reported in the literature.

To the best of our knowledge, almost all of the literature reported for determination of DA and ST in the presence of AA has used modified electrodes. However using an unmodified electrode is much more convenient and also often much more repeatable than using modified electrodes, there is no literature report of the successful simultaneous determination of DA and ST in the presence of AA using unmodified electrodes probably because unmodified conventional electrodes are unable to discriminate signals of DA and ST, especially in the presence of AA that usually has a huge overlap with DA. Unmodified electrodes also suffer from low sensitivity and fouling effects.

Recently we have advocated the use of edge plane pyrolytic graphite electrodes (EPPGEs) for broad use in electroanalysis [34]. Pyrolytic graphite is an electrode material which contains both basal plane and edge plane surfaces, with the graphite monocrystal size and basal/edge ratio depending on the quality of the used pyrolytic graphite [35]. In highly ordered pyrolytic graphite (HOPG) electrodes (see Fig. 1), the basal plane surface consists of layers of graphite parallel to the surface and with an interlayer spacing of 3.35 Å. Surface defects occur in the form of steps exposing the edges of the graphite layers. Due to the nature of the chemical bonding in graphite, the two planes, edge and basal (see Fig. 1), can exhibit completely different electrochemical properties [36]. For a large variety of redox couples, electron-transfer rate constants at edge plane graphite have been found to be over 103 times faster than for basal plane graphite [35]. The edge plane pyrolytic graphite electrode gives low background currents and improved electrocatalytic signals in comparison with those obtained by use of basal plane pyrolytic graphite, boron-doped diamond, glassy carbon or carbon-nanotube-modified basal plane pyrolytic graphite electrodes [36, 37]. We have recently used these electrodes for a variety of electroanalytical tasks, including the oxidation of thiols and ascorbic acid [36, 38], sensing of gases including chlorine [39] and NO2 [40], stripping voltammetry [38], detection of halides [41], NADH analysis [42] uric acid and ascorbic acid determination [43] and also the determination of cadmium and lead [44].

Fig. 1
figure 1

Schematic representation of a step edge on a highly ordered pyrolytic graphite electrode (HOPG) surface. The steps are multiples of 3.35 Å deep and typically range between 1 and 20 layers for HOGP [34]

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In this paper we investigate the direct electrochemical oxidations of DA, ST and AA using edge plane pyrolytic graphite electrodes, and compare them with other carbon-based electrodes, specifically glassy carbon electrode (GCE), boron-doped diamond (BDD) electrode, basal plane pyrolytic graphite electrode (BPPGE) and carbon nanotube/carbon composite electrode (CNT-CCE). It is shown that oxidation at edge plane pyrolytic graphite shows a highly improved analytical response in comparison to existing conventional electrodes. Well-defined and well-resolved voltammetric peaks are observed for the direct oxidation of DA, ST and AA in 0.1 M phosphate buffer (pH 7) using the edge plane pyrolytic graphite electrode and no fouling effect is observed during a set of experiments. A fresh surface can then be obtained by gentle polishing of the electrode with alumina powder on a polishing pad. No electrode modification is required in this determination method and because of its simplicity, high peak resolution, sensitivity and its resistance to fouling, this method has the potential to be used for routine analysis of DA and ST in the presence of AA.

Experimental

Chemicals and apparatus

The following chemicals were of analytical grade and were used as received without any further purification: dopamine hydrochloride (98.5%, Alfa Aesar), serotonin hydrochloride (99.0%, Alfa Aesar) and L-ascorbic acid (99+% Aldrich). All solutions were prepared with deionized water of resistivity not less than 18.2 MΩ cm (Vivendi water systems, UK). Cyclic voltammetric and differential pulse voltammetric measurements were carried out using a μ-Autolab II (ECO-Chemie, The Netherlands) potentiostat. Parameters for differential pulse voltammetry were step potential 5 mV and modulation amplitude 25 mV.

All measurements were conducted using a three-electrode configuration. The pyrolytic graphite disk was purchased from Le Carbone Ltd., Sussex, UK, and then used for the fabrication of EPPG electrode and BBPG electrode in our mechanical workshop. A 4.9-mm-diameter EPPGE, and also BPPGE, GCE (BASi Company ,USA), BDD (Windsor Scientific Company, Berkshire, UK) and CNT-CCE (CNT from NanoLab Company, USA and graphite powder from Aldrich) were used as the working electrodes. For the EPPGE, discs of pyrolytic graphite were machined to 4.9-mm diameter, which was oriented with the disc face parallel with the edge plane as required. The EPPGE electrode was polished before each experiment with alumina (Buehler, USA). For preparing a CNT-CCE, 20% (w/w) of bamboo-like carbon nanotube is mixed with 80% (w/w) carbon composite paste (a mixture of graphite powder, resin and hardener) and then placed and compressed in a glass tube with the internal diameter of 5 mm. Then the electrode is left in an oven at 40 °C for 48 h to be dried and hardened, and the electrode is then polished with polishing machine until a smooth surface is obtained.

The EPPGE and CNT-CCE were polished before each set of experiments with alumina (Buehler, USA) of decreasing particle size (5–0.1 μm). The BPPGE was prepared by renewing the electrode surface with Sellotape. This procedure involves polishing the BPPGE surface on carborundum paper (P100 grade) and then pressing Sellotape on to the cleaned BPPG surface before removing with attached graphite layers. This is repeated several times and the electrode is then cleaned in acetone to remove any adhesive. GCE and BDD are polished with diamond paste (Kemet, Kent, UK) of decreasing particle size (3–0.1 μm) sprayed on the polishing pad. A saturated calomel electrode (Radiometer, Copenhagen, Denmark) was used as the reference electrode. The counter electrode was a bright platinum wire.

Procedures

The EPPGE was polished by hand or using a polishing machine and rinsed with deionized water before each set of experiments. The EPPGE is normally placed in a 0.1 M phosphate buffer solution (pH = 7) containing target species such as DA, ST and AA and then simply followed by scanning the potential. The electrochemical techniques used for determination of DA, ST and AA in this report are cyclic voltammetry and differential pulse voltammetry.

Results and discussion

Comparison of EPPGE with other carbon-based electrodes

As mentioned before there is no report in the literature about the simultaneous determination of DA, ST and AA using unmodified electrodes. So in order to clarify the advantages of our proposed method, we have compared the signals for simultaneous detection of DA, ST and AA on different carbon-based electrodes. Figure 2 depicts the cyclic voltammetric responses for electrochemical oxidation of 40 μM DA, 40 μM ST and 40 μM AA on the EPPGE, BPPGE, GCE, BDD electrode and CNT-CCE in a pH 7 phosphate buffer solution, recorded at a scan rate of 10 mV s−1.

Fig. 2
figure 2

Cyclic voltammograms for 40 μM AA, DA and ST in a 0.1 M phosphate buffer solution pH 7 on different carbon-based electrodes as mentioned in the inset of this figure. Scan rate for all CVs are 10 mV s−1

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The dashed line shows the cyclic voltammetric signal of AA, DA and ST with aforementioned concentrations on a basal plane pyrolytic graphite electrode which resulted in three broad, undefined and partially overlapping peaks for AA, DA and ST at potentials of ca. 0.05 V(vs. SCE), 0.19 V (vs. SCE) and 0.35 V (vs. SCE), respectively. The thin solid line and the dotted line show the cyclic voltammograms for AA, DA and ST on GCE and BDD, respectively. No oxidation peak is obtained for AA and DA using BDD and the only observed broad peak is due to ST which exhibit an oxidation wave at ca. + 0.38 V.

In the case of the GCE, the voltammetric response is slightly improved and two peaks are observed at the potentials of ca. +0.16 V and 0.33 V (vs. SCE): the former is the signal of DA and the latter is the signal of ST, and no signal for AA is observed which is probably overlapped with DA. The solid thick line depicts the signal of AA, DA and ST on EPPGE, showing three well-defined, sharp and fully resolved peaks for AA, DA and ST. In comparison to the above electrode substrates, the EPPGE exhibits shifts in oxidation potentials of all three species to less positive values which is evident from the well-defined peaks at potentials of ca. −0.03 V, 0.16 V and 0.28 (vs. SCE) for AA, DA and ST, as shown in Fig. 2. The reason why the oxidation of AA, DA and ST occurs at the lower overpotentials is due to the edge plane sites of the EPPGE, which show much faster electron-transfer rate constants in comparison to other carbon-based electrodes and facilitate the electron transfer between species and electrode and increase the electrode sensitivity in comparison to other carbon-based electrodes. On EPPGE, ST and AA both show irreversible oxidation peaks but DA shows a characteristic anodic peak at E pa = 0.16 V and a complementary cathodic peak at E pc = 0.11 V.

Effect of pH

The effect of pH was studied in the range 3.0–8.0 for DA and 4.0–10.0 for ST in order to evaluate the number of protons involved in the voltammetric oxidation of DA and ST and also the optimum pH value for detection of DA and ST. Figures 3 and 4 show the cyclic voltammograms at different pH values for 100 μM DA and 100 μM ST, respectively. As can be seen in the insets of Figs. 3 and 4, the peak potential for DA (E p = 0.56 − 0.055 pH, R 2 = 0.99) and ST (E p = 0.7088 − 0.057 pH, R 2 = 0.99) oxidation varies linearly with pH and is shifted to less positive potentials with a slope of 0.055 V and 0.057 V per pH unit, which are very close to the theoretical value of −0.059 V per pH unit, suggesting the production of two electrons and two protons per molecule of DA and ST oxidized, which is in agreement with literature reports [19, 45]. The peak current of DA is increased from pH 3 to 4 and decreased slightly with increasing the pH value from 4 to 7 and then decreased significantly at pH values more than 7. Below pH 8.0 DA occurs in its protonated form and is, therefore, readily adsorbed on the charged electrode surface. But at pH values more than 7, DA loses protons and becomes a neutral species. The highest peak current for DA oxidation was obtained at pH 4 and the highest peak current for oxidation of ST was obtained at pH 7; hence, the pH value of 7.0 was chosen for the analytical development, because of the similarity to the conditions in physiological media.

Fig. 3
figure 3

Cyclic voltammograms for 100 μM DA in a Britton–Robinson buffer solution on an EPPGE at different pH values from 3 to 8. Inset shows the plot of peak potential of DA versus pH

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Fig. 4
figure 4

Cyclic voltammograms for 100 μM ST in a Britton–Robinson buffer solution on an EPPGE at different pH values from 4 to 10. Inset shows the plot of peak potential of ST versus pH

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Effect of scan rate

The effect of scan rate on the oxidative peak potential and peak current of DA (Fig. 5) and ST (Fig. 6) at the surface of EPPGE in a 0.1 M phosphate buffer solution was studied and the cyclic voltammetric curves of DA and ST obtained in the range 0.01–1 V s−1 in order to investigate the kinetics of electrode reactions and verify whether diffusion is the only controlling factor for the mass transport process or not.

Fig. 5
figure 5

Cyclic voltammograms for 50 μM DA in a 0.1 M phosphate buffer solution pH 7 on an EPPGE at different scan rates from 0.01 to 1 V s−1. Inset shows the plot of oxidative and reductive peak currents for DA versus different scan rates

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Fig. 6
figure 6

Cyclic voltammograms for 50 μM ST in a 0.1 M phosphate buffer solution pH 7 on an EPPGE at different scan rates from 0.01 to 1 V s−1. Inset shows the plot of oxidative peak currents for ST versus different scan rates

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A linear relation between oxidative peak current and scan rate from 0.01 to 1 V s−1 is observed for both DA (inset of Fig. 5) and ST (inset of Fig. 6). This linearity suggests that the electrochemical reaction of DA and ST at the surface of EPPGE is an adsorption-controlled process. Also the cyclic voltammetric results show that the oxidative and reductive peak potentials of DA are constant and do not shift with increasing scan rate from 0.01 to 1 V s−1, and a reductive peak related to DA is also observed in the back peak, which are all signs of reversibility for DA. But in the case of ST, no back peak is observed at low scan rates and only small back peaks are observed at high scan rates (1 V s−1 and higher), which suggests irreversible or quasi-reversible electrode process for ST.

Calibration data

The addition of DA in the presence of 50 μM ST and 100 μM AA in a phosphate buffer pH 7 using an edge plane pyrolytic graphite electrode was explored. Figure 7a shows the voltammetric responses to additions of DA over the range 0.2–25 μM in the presence of 50 μM ST and 100 μM AA: three defined and well-resolved peaks are obtained for DA, ST and AA. Using the plot of peak current (I p) versus added concentration of DA in the presence of 50 μM ST and 100 μM AA (Fig. 7b), one linear dynamic range between 0.2 and 25 μM \( {\left( {{I_{{\text{p}}} } \mathord{\left/ {\vphantom {{I_{{\text{p}}} } {{\text{ $ \mu $ A}}}}} \right. \kern-\nulldelimiterspace} {{\text{ $ \mu $ A}}} = 1.1 \times 10^{{ - 7}} {\left[ {{\left( {{{\text{DA}}} \mathord{\left/ {\vphantom {{{\text{DA}}} {{\text{ $ \mu $ M}}}}} \right. \kern-\nulldelimiterspace} {{\text{ $ \mu $ M}}}} \right)}} \right]} + 1.16 \times 10^{{ - 6}} {\text{ $ \mu $ A}};\;N = 8,\;R^{2} = 0.99} \right)} \) is obtained for the calibration plot of DA. Using this plot, a detection limit of 90 nM is obtained for DA. This detection limit is the same order of magnitude and even better than some of the modified electrodes which have been used for the detection of DA in the presence of ST and AA [19, 27, 28, 31].

Fig. 7
figure 7

a Differential pulse voltammograms for additions of 0.2, 1, 3, 6, 10, 15, 20 and 25 μM DA in the presence of 50 μM ST and 100 μM AA in a 0.1 M phosphate buffer solution pH 7 on an EPPGE. b Calibration plot for the additions of DA under the aforementioned conditions

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After the investigation of DA additions in the presence of ST and AA, we explored additions of ST in a phosphate buffer pH 7 using an EPPGE in the presence of 50 μM DA and 50 μM AA. Figure 8a depicts the differential pulse voltammetric responses from additions of ST in the range 0.1–100 μM in the presence of 50 μM DA and 50 μM AA. Analysis of peak current (I p) versus added concentration of ST in the presence of 50 μM DA and 50 μM AA was found to produce two linear ranges. The first linear range was from 0.1 to 20 μM \( {\left( {{I_{{\text{p}}} } \mathord{\left/ {\vphantom {{I_{{\text{p}}} } {{\text{ $ \mu $ A}}}}} \right. \kern-\nulldelimiterspace} {{\text{ $ \mu $ A}}} = 1.18 \times 10^{{ - 7}} {\left[ {{\left( {{{\text{ST}}} \mathord{\left/ {\vphantom {{{\text{ST}}} {{\text{ $ \mu $ M}}}}} \right. \kern-\nulldelimiterspace} {{\text{ $ \mu $ M}}}} \right)}} \right]} + 3.87 \times 10^{{ - 7}} {\text{ $ \mu $ A}};\;N = 6,\;R^{2} = 0.99} \right)} \) and the second linear range (not shown) from 20 to 100 μM \( {\left( {{I_{{\text{p}}} } \mathord{\left/ {\vphantom {{I_{{\text{p}}} } {{\text{ $ \mu $ A}}}}} \right. \kern-\nulldelimiterspace} {{\text{ $ \mu $ A}}} = 4.31 \times 10^{{ - 8}} {\left[ {{\left( {{{\text{ST}}} \mathord{\left/ {\vphantom {{{\text{ST}}} {{\text{ $ \mu $ M}}}}} \right. \kern-\nulldelimiterspace} {{\text{ $ \mu $ M}}}} \right)}} \right]} + 1.77 \times 10^{{ - 6}} {\text{ $ \mu $ A}};\;N = 5,\;R^{2} = 0.99} \right)} \). Using the calibration plot for the first linear ranges of ST additions in the presence of DA and AA, which is shown in Fig. 8b, a detection limit of 60 nM is obtained for ST in the presence of DA and AA which is also better than the detection limit of ST using some of the modified electrodes [19, 31, 32].

Fig. 8
figure 8

a Differential pulse voltammograms for additions of 0.1, 0.5, 1, 5, 10, 15, 20, 35, 50, 75 and 100 μM ST in the presence of 50 μM DA and 50 μM AA in a 0.1 M phosphate buffer solution pH 7 on an EPPGE. b Calibration plot for the first linear range of ST additions between 0.1 and 20 μM under the aforementioned conditions

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We then used an EPPGE for additions of AA in the presence of 50 μM DA and ST in a phosphate buffer pH 7. Figure 9a shows the differential pulse voltammetric signals from additions of AA in the range 0.5–60 μM in the presence of 50 μM DA and ST. A plot of peak current (I p) versus added concentration of AA in the presence of 50 μM DA and ST produced two linear ranges. The first linear range was from 0.5 to 10 μM, \( {\left( {{I_{{\text{p}}} } \mathord{\left/ {\vphantom {{I_{{\text{p}}} } {{\text{ $ \mu $ A}}}}} \right. \kern-\nulldelimiterspace} {{\text{ $ \mu $ A}}} = 7.23 \times 10^{{ - 8}} {\left[ {{\left( {{{\text{AA}}} \mathord{\left/ {\vphantom {{{\text{AA}}} {{\text{ $ \mu $ M}}}}} \right. \kern-\nulldelimiterspace} {{\text{ $ \mu $ M}}}} \right)}} \right]} + 3.17 \times 10^{{ - 6}} {\text{ $ \mu $ A}};\;N = 6,\;R^{2} = 0.99} \right)} \) and the second linear range was between 10 and 60 μM for AA additions \( {\left( {{I_{{\text{p}}} } \mathord{\left/ {\vphantom {{I_{{\text{p}}} } {{\text{ $ \mu $ A}}}}} \right. \kern-\nulldelimiterspace} {{\text{ $ \mu $ A}}} = 2.49 \times 10^{{ - 8}} {\left[ {{\left( {{{\text{AA}}} \mathord{\left/ {\vphantom {{{\text{AA}}} {{\text{ $ \mu $ M}}}}} \right. \kern-\nulldelimiterspace} {{\text{ $ \mu $ M}}}} \right)}} \right]} + 3.68 \times 10^{{ - 6}} {\text{ $ \mu $ A}};\;N = 7,\;R^{2} = 0.99} \right)} \). The calibration plot for the first linear ranges of AA additions in the presence of DA and ST is shown in Fig. 9b where a detection limit of 200 nM is obtained for AA in the presence of DA and ST.

Fig. 9
figure 9

a Differential pulse voltammograms for additions of 0.5, 1, 3, 6, 8, 10, 15, 20, 30, 40, 50 and 60 μM AA in the presence of 50 μM DA and 50 μM ST in a 0.1 M phosphate buffer solution pH 7 on an EPPGE. b Calibration plot for the first linear range of AA additions between 0.5 and 10 μM under the aforementioned conditions

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Analysis of a real sample

The EPPG electrode was next utilised for the simultaneous determination of DA and ST in laked horse blood (Oxoid Limited, Hampshire, UK) as a real sample. According to the literature, the concentration of neurotransmitters such as dopamine, serotonin and tryptophan in biological samples varies over a wide range between 10−7 and 10−3M from species to species [46]. For example the daily serotonin blood level in a horse varies in the range 180–200 μM [47]. The level of dopamine in biological samples is mostly of the same order of magnitude as serotonin, while it is believed that the concentration of ascorbic acid in biological samples is much more than dopamine and serotonin [45]. Their concentrations are all higher than or at least of the same order of magnitude as the achievable detection limit of our proposed method.

A solution of the laked horse blood diluted in a ratio of 1:250 with phosphate buffer solution pH = 7 which was spiked with 5 μM DA and 5 μM ST was used as a real sample test solution. The additions of DA and ST were then made to the real sample solution. The response of additions is shown in Fig. 10, where analysis of the peak current versus added concentration of DA and ST is shown in the inset a of Fig. 10. Based on this recovery experiment, recoveries of 95.5% and 101% are obtained for DA and ST, respectively. A typical differential pulse voltammogram of 20 μM DA and ST in the presence 40 μM AA spiked in the laked horse blood is also shown in the inset b of Fig. 10 which clearly shows the possibility of determination of DA, ST and AA in the real sample using the edge plane pyrolytic graphite electrode.

Fig. 10
figure 10

The dotted line shows the differential pulse signal for the blank of the buffered real sample (laked horse blood) on EPPGE before spiking DA and ST. The first solid line shows the differential pulse voltammogram of the real sample after spiking 5 μM DA and ST, and the next solid lines show simultaneous additions of 2, 4, 6, 8, 10, 12 μM of DA and ST on EPPGE in the spiked real sample. Inset a calibration plot for the additions of DA and ST on spiked real sample; b typical differential pulse voltammogram for 20 μM DA and ST in the presence 40 μM AA spiked in the laked horse blood

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Conclusions

An unmodified edge plane pyrolytic graphite electrode (EPPGE) has been successfully utilised for the simultaneous determination of DA, ST and AA in standard and real samples. DA, ST and AA have been detected in the presence of each other with detection limits of 90 nM, 60 nM and 200 nM, respectively. This method using an unmodified EPPGE has advantageous such as very easy handling, no need for modification, low detection limit, wide linear dynamic range, resistance against surface fouling and low cost and furthermore it can be used for the routine analysis of DA, ST and AA in different real samples. It worth mentioning that the use of edge plane pyrolytic graphite with its large number of edge plane sites facilitates and accelerates the electron-transfer rate between species and electrode and therefore results in sensitive, well-defined and resolved signals for DA, ST and AA without any modification, providing one of the easiest and best electroanalytical methods for determination of DA, ST and AA.