Electrochemical determination of melatonin hormone using a boron-doped diamond electrode

Diamond & Related Materials 21 (2012) 114–119

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Electrochemical determination of melatonin hormone using a boron-doped diamond electrode Abdulkadir Levent ⁎ Batman University, Faculty of Science and Art, Department of Analytical Chemistry, Batman, 72100, Turkey

a r t i c l e

i n f o

Article history: Received 16 July 2011 Received in revised form 9 September 2011 Accepted 18 October 2011 Available online 20 November 2011 Keywords: Melatonin Boron-doped diamond electrode Square-wave voltammetry Tablet Urine

a b s t r a c t In this study, a boron-doped diamond electrode was used for the electroanalytical determination of melatonin in the pharmaceutical tablet and urine samples by square-wave voltammetry. Melatonin yielded a welldefined voltammetric response in Britton-Robinson buffer, pH 3.0 at + 0.88 V (vs. Ag/AgCl). Using the optimal square-wave voltammetry conditions, the oxidation peak was used to determine melatonin in the concentration range of 5.0 × 10 7 M to 4.0 × 10 6 M (r =0.998, n = 8), a detection limit of 1.1 × 10 7 M (0.025 μg/mL) and relative standard deviation was 2.06% at the 2.0 × 10 6 M level (n = 10). Recoveries of melatonin were in the range of 97.67–105%, for both tablet and spiked human urine samples. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Melatonin, N-acetyl-5-methoxytryptamine, is a hormone mainly synthesised in the pineal gland, whose concentration depends on the circadian rhythms. Most of melatonin is produced at night [1]. This hormone has great influence on a variety of physiological and behavioural processes including neurological, psychiatric [2], reproductive [3] and as neuroprotective agent in Alzheimer and Parkinson's disease models [4]. Disorders such as anxiety and seasonal depression are related to it [5]. Melatonin was also recently reported to be an effective free radical scavenger, an antioxidant and immunomodulator in cancer therapy [6,7]. Exogenous melatonin has an estimated oral bioavailability of 40–70% for doses of 2.5–100 mg. Following an oral 80 mg dose, the time to peak plasma concentration ranges are 60–150 min. and the elimination half life is 20–70 min [8]. Several analytical techniques including high-performance liquid chromatography with different detectors [9–14], gas chromatography [15–17], liquid chromatography coupled with electrospray ionisation mass spectrometric detection [18], capillary electrophoresis [19,20], spectrofluorimetric–spectrophotometric [21–23]] and chemiluminescence [24] have been reported for the analysis of melatonin and its metabolites in pharmaceutical formulations, biological fluids and tissues. Some of the above mentioned methods require not only preliminary extraction and purification of melatonin from the sample matrix but also skilled personnel manipulating sophisticated instrumentation. Electrochemical methods are of great interest because of

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practical advantages such as low expense of instrument, operation simplicity, suitability for real-time detection and less sensitive to matrix effects compared to other analytical techniques [25,26]. To date, on the other hand, some electroanalytical methods have been reported for quantitative determination of melatonin [8,27–33]. These electroanalytical methods used in determination of melatonin amount were carried out with modified electrodes. However, in this study, boron-doped diamond electrode which does not require modification was used. Diamond electrodes have attracted considerable interest in recent years due to some superb electrochemical properties [34]. Moreover, highly boron-doped diamond (BDD) exhibits several technologically important properties that distinguish it from conventional electrodes, such as: an extremely wide potential window in aqueous and nonaqueous electrolytes, corrosion stability in very aggressive media, an inert surface with low adsorption properties and a strong tendency to resist deactivation, very low double-layer capacitance and background current [35]. These properties make the BDD electrode a promising material for electroanalytical applications. Recent works reported in the literature [34,36–41] have shown that several biomolecules can be satisfactorily determined using BDD electrodes. To our knowledge, a study related to the determination of continuous amperometric detection of co-released serotonin and melatonin from the mucosa in the ileum using a BDD microelectrode has appeared in the literature[42]. The oxidation peak potential for serotonin is at 500 mV, whilst the peak potential for melatonin is at 740 mV for the BDD microelectrode in the Krebs buffer pH 7.4 (vs. Ag/AgCl). The objective of the present paper is to develop a simple, accurate, selective and reliable method to detect melatonin in the tablet and human urine using a BDD electrode in 0.1 mol/L Britton-

A. Levent / Diamond & Related Materials 21 (2012) 114–119

Robinson (BR) buffer solution (pH 3.0) by square-wave voltammetry (SWV). 2. Experimental 2.1. Chemicals Standard melatonin was purchased from Sigma-Aldrich. Its stock solution 1.0 × 10 − 2 M for CV and 5.0 × 10 − 4 M for SWV studies were prepared in methanol due to its low solubility in water. On the day of the experiment, solutions worked on were prepared by diluting the stock solution with a selected supporting electrolyte. Six different supporting electrolytes were used in this paper, such as: sulfuric acid 0.1 M, perchloric acid (0.1 M, pH 1.0), acetate buffer (0.1 M, pH 4.7), BR buffer (0.1 M, pH 2–11), phosphate buffer (0.1 M, pH 2.5 and 7.4) and Tris buffer (0.1 M, pH 7.0). All stock solutions were preserved at 4 °C when not in use, and protected from daylight during use in the laboratory. All other chemicals were of analytical-reagent grade and used as received. Aqueous solutions were prepared with deionised water further purified via a Milli-Q unit (Millipore). 2.2. Apparatus Electrochemical experiments were performed with an Autolab PGSTAT 128 N potentiostat, controlled by GPES 4.9 electrochemical software from Eco-Chemie (The Netherlands). The raw data were also treated in all SW voltammetric measurements with the aid of Savitzky and Golay filter (level 2) of the GPES software, followed by the moving average baseline correction with a peak width of 0.01 V. The three electrode systems used in this study contained BDD as a working electrode (Windsor Scientific Ltd.; Ø: 3 mm, diameter), Ag/AgCl (3.0 mol/L NaCl) as a reference electrode (Model RE-1, BAS, USA) and platinum wire as a counter electrode. The measurements were carried out in a standard 10-mL one-compartment voltammetric cell, at a laboratory temperature (20 ± 5 °C). The pH values of solutions were measured using a WTW inoLab pH 720 meter with a combined electrode (glass-reference electrodes). Firstly, before starting experiments, the BDD electrode was polished with 0.01 μm wet alumina powder and copiously rinsed with water. After this mechanical treatment, BDD electrode surface was pre-treated by applying a potential of +2.0 V for 180 s in the blank supporting electrolyte without stirring. Afterwards, the electrode was transferred to the measuring cell containing the supporting electrolyte and melatonin, and polarized under stirring at + 2.0 V for 60 s for each run. The convective transport was provided with a magnetic stirring of 900 rpm. Following this method, excellent electrode response and reproducibility were systematically observed for melatonin determination.

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sample. The sample tubes were vortexed for 1 min and then centrifuged for 10 min at 5000 rpm to remove the unknown endogenous chemicals (an unspiked sample of the same urine was kept as a blank). Appropriate volumes of the supernatant (20 μL) were mixed with the BR buffer (pH 3.0) in the voltammetric cell and analyzed like conditions described in Section 2.2.

3. Results and discussion 3.1. Cyclic voltammetric behavior of melatonin on BDD electrode The electrooxidation of melatonin was examined using the case of BDD electrode in potential range of + 0.0 to +1.5 V at scan rate of 100 mV/s. The repetitive CV of melatonin in BR buffer at pH 3.0 is presented in Fig. 1. During the scan, one well-defined oxidation peak was appeared at +0.85 V. According the literature data, melatonin presents two well-defined oxidation processes at ca. + 0.76 and +0.93 V and a reduction process at +0.38 V when the first CV scan is recorded in 0.1 M HClO4 at carbon paste electrode [8]. A reduction peak was obtained at a potential of −1.26 V that was not accompanied by an anodic peak indicating the irreversible nature of melatonin electrode reaction at a hanging mercury drop electrode in a acetate buffer (pH 5.0) [32]. The melatonin anodic peak was observed at about + 0.65 V in BR buffer solution (pH 6.6) at activated glassy carbon electrode which indicated a totally irreversible electrochemical oxidation with adsorption characteristics [30]. In another study, the melatonin oxidation peak was observed at +0.61 V in phosphate buffer (pH 7.5) at multi-wall carbon nanotube film coated glassy carbon electrode [33]. As seen in the literatures, using the modified electrode, the voltammograms of melatonin were almost similar to our results obtained on BDD electrode under different conditions and cases requiring modification. The effect of the scan rate at BDD electrode was investigated under the conditions above. From the CVs(Fig. 2.), it was found that the initial oxidation peak current of melatonin gradually increased and a positive shift in the peak potential existed with increasing scan rate. The current response (log ip) was linearly proportional to the scan rate (log v) within the range 10–250 mV/s according to the relationship and the Eq. (1); suggesting a typical adsorption-controlled process [43]. log ipðμAÞ ¼ 0:72 log νðmV=sÞ 0:4929 ðcorrelation coefficient; r ¼ 0:997n ¼ 5Þ

ð1Þ

2.3. Preparation of tablet and urine samples Melatonin tablets were bought from Oxy Life Nutritional Supplements, Turkey. Each tablet contains a dose of 3.00 mg melatonin. Ten tablets were weighted, powdered and mixed well. A portion equivalent to 23.23 mg of melatonin was transferred into 100 mLvolumetric flask, and dissolved in water. The contents of the flask were sonicated for 30 min, and then filtered through a filter paper (Whatman No. 1) for obtaining clear filtrate. Appropriate volume (20 μL) was mixed with the BR buffer (pH 3.0) in the voltammetric cell and analyzed using the conditions described in Section 2.2. to use in calibration graph. Blank urine samples were obtained from a healthy and nonsmoking donor (male, age 30 years). An aliquot volume of sample was fortified with melatonin dissolved in methanol to achieve final concentration of 4.0 × 10 4 M, and treated with 4.00 mL acetonitrile then the volume was completed to 10.00 mL with the same urine

Fig. 1. The repetitive CVs of 5.0 × 104 M melatonin solutions in 0.1 M BR buffer, pH 3.0 for BDD electrode. Scan rate, 100 mV/s. The black line represents the first scan, and the blue line the second one, while dashed line represents background current.

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Fig. 2. CVs of 5.0 × 104 M melatonin in 0.1 M BR buffer, pH 3.0 at different scan rates.

For an irreversible electrode process, the peak potential (Ep) and scan rate (v) were defined by the following (2) equation [44];   0 0 Ep ¼ E þ ð2:303RT=αnFÞ log RTk =αnF þ ð2:303RT=αnFÞ logv

ð2Þ

peaks with nice shape and high currents were recorded under acidic conditions (Figs. 3A–4A) in comparison with the peaks in the other supporting electrolytes (Figs. 3B–4B). In addition, BR buffer was employed over a broad pH range (2.0–11.0). As can be seen in Figs. 3A–4A, current strengths increased with acidity as oxidation potentials shifted slightly to less positive values. These results were in accordance with those observed in Figs. 3B–4B. The most welldefined oxidation peak and the highest anodic current density was obtained in BR buffer at pH 3.0, which was chosen for further experiments and development of the methodology. Pulse voltammetric technique such as SWV is effective and rapid electroanalytical technique with well-established advantages, including good discrimination against background currents and low detection limits. Due to the poorly resolved signal obtained by CV with a decrease in pH above 6.0, the effect of solution basicity on oxidation process was studied using SWV. When the pH was more than 6.0, the oxidation peak of current melatonin was low, and voltammogram shape was not well-defined. This result is entirely different from the previous studies related to melatonin [30,33]. In this study, when the pH was increased, the peak potentials shifted towards less positive values, indicating proton participation in the oxidation process (Fig. 4): Ep ðVÞ ¼ −0:0369 pH þ 0:9719 ðr ¼ 0:9929Þ

where α is charge transfer coefficient, n is electron transfer number, and R, T, and F have their usual meanings. Thus the value of αn can be easily calculated from the slope of Ep versus log v (Ep = 0.0523 log v + 0.7512, n = 5, r = 0.998). Herein, the slope is 0.0523 and the αn was calculated to be 1.13. Generally, α is assumed to be 0.5 in totally irreversible electrode process. So, n value was found to be 1.87 (≈2), which is in agreement with the results explained for the oxidation process of melatonin in other electrodes [27,30,33]. Though the elucidating the mechanism of melatonin electrochemical oxidation is beyond the aim of this study, a short comment can be made. From the CVs at BDD electrode and considering the voltammetric behavior of melatonin at carbon paste electrode in acidic aqueous solution [27,8], at multi-wall carbon nanotube film coated glassy carbon electrode in phosphate buffer solution (pH 7.5) [33], at activated glassy carbon electrode [8] besides Radi and Bekheit studied electrochemical and oxidation properties of melatonin in detail [27], we may assume that the oxidation peak could be due to the two-electron oneproton transfer which corresponds to the formation of cation at position 5 of indole ring. The reaction pathway for this step can be written as in Scheme 1: 3.2. Effect of pH The choice of the supporting electrolyte is an important stage in electroanalytical studies because its composition and pH affect the properties of the solution as well as the electrode–solution interface, modifying the thermodynamics and kinetics of the charge transfer process, and the adsorption at the electrode surface [45]. Effects of various supporting electrolytes and pH were studied on the oxidation of melatonin by SWV mode at BDD electrode. Fig. 3 depicts the SW voltammograms of 5.0 × 10 6 M melatonin in different medias. The voltammograms recorded in all solutions within the potential range +0.4 V to +1.3 V exhibited a single oxidation peak. Oxidation H N

H3CO

CH 3

O N H

ð3Þ

The evolution of peak potential with pH shows one linear and two almost constant segments Fig. 4A. The linear one is between (~pH 4.0–9.0) and the other two segments are constant at (~ pH 2.0–4.0) and (~pH 9.0–12.0). In Eq. (3), the slope of 0.0369 mV pH 1 obtained on BDD electrode was almost similar to that shown in previous electrochemical investigations [30,33] and it is suggested that the number of electrons transferred in the oxidation of melatonin is double that of protons [27,30,33]. 3.3. Effect of square-wave voltammetric parameters Square-wave voltammetry is a pulse technique that offers the advantages of fast speed and good sensitivity for reversible as well as irreversible reactions. The excitation signal is obtained applying a series of forward and reverse pulses superimposed onto a constant height potential staircase to the working electrode. The current is sampled twice in each wave cycle: once at the end of the forward pulse and once at the end of the reverse pulse. The signal is given by the difference between these two currents. As a consequence, an important step in the development of the electroanalytical methodology is the optimization of the parameters (analysis with n = 3) that can influence the voltammetric response. Hence, the dependence of the SWV responses on parameters such as frequency, step potential and pulse amplitude was analyzed in order to optimize the experimental set-up for melatonin determination [24]. Further optimization of the analytical signal focused on studying the effect of preconcentration/stripping conditions, such as accumulation potential (Eacc) and accumulation time(tacc). The influence of the Eacc either at open-circuit potential or at a potential range from +0.1 V to +0.8 V was studied with tacc of 120 s in stirred 2.0 × 10 6 M melatonin solution. The maximum peak current at an Eacc = + 0.5 ± 0.04 V is because of an increased accumulation rate, due to more favorable

H 3CO

4 5

3

H +, 2e -

6 7

Scheme 1. The proposed mechanism for oxidation of melatonin.

N

1

2

H N

CH 3

O

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Fig. 3. SW voltammograms of 5.0 × 106 M melatonin solutions in different supporting electrolytes and at different pHs. A: 0.1 M Britton-Robinson buffer pH 2–11; B: pHs of different supporting electrolytes. Electrode, BDD; 120 s at open circuit condition. SWV parameters: frequency, 50 Hz; scan increment, 12 mV; pulse amplitude, 40 mV.

alignment of the molecule by the electric field at the electrodesolution interface. After fixing the Eacc at this value, the deposition time was varied between 15 and 360 s. The increasing of peak currents was obtained at a fast rate up to about 60 s and at tacc = 60 ± 0.05 s a plateau was attained. For further analytical studies, the deposition stage was carried out at + 0.5 V for a pre-concentration time of 60 s. Final optimization of the analytical signal centered upon varying the SW parameters such as the frequency, step potential and pulse amplitude (data not shown). The effect of frequency was studied in the range 25 to 200 Hz. The peak currents increased with the frequency due to the increase in the effective scan rate but the peak shape and baseline were distorted at frequencies higher than 150 Hz. This was attributed to the greater contribution of the capacitive current at higher frequencies. The influence of step potential was investigated between 4 and 20 mV. The peak height increased up to 12 mV because the effective scan rate was increased, but the peak heights decreased at higher values of step potential. The analytical signal was dependent on the pulse amplitude even if this parameter seems to be less important than the frequency. Pulse amplitude was examined in the range from 10 to 60 mV. Peak heights increased upon increase of the pulse amplitude. However, the peak shape became wider at higher pulse amplitudes than 40 mV. Thus, the frequency of 150 ± 0.03 Hz, the step potential of 12 ± 0.05 mV and the pulse amplitude of 40 ± 0.03 mV were selected for all subsequent work. 3.4. Effect of surfactants It has been reported that surfactant media induce two important effects on the electrochemical reactions. In the first place, the surfactant can stabilize radical ions and other reaction intermediates, having this way an effect on the mechanism of the electrode reaction.

Secondly, the presence of surfactant molecules modifies the double layer structure [25,46,47]. The effect of three typical surfactants, cetyltrimethylammonium bromide (CTAB; cationic type), Tween-20 (non-ionic type) and sodium dodecylbenzene sulfonate (SDS; anionic type), over the adsorption of melatonin 2.0 × 10 6 M under the optimum conditions for SWV was investigated in detail. The results showed that the addition of SDS 2.0 × 10 4 M would cause about 15% loss of the melatonin peak current, while CTAB 2.0 × 10 4 M and Tween-20 2.0 × 10 4 M significantly reduced the magnitude of current to 43.5 and 33.7%, respectively. In addition, the peak potential shifted to positive values by about 30, 40 and 60 mV in the presence of SDS, Tween-20 and CTAB, respectively. This indicates that the surfactants interact with melatonin adsorbed on the surface of BDD electrode, so that competitive adsorption would reduce the incorporation of melatonin [30]. 3.5. Effect of interferences In order to evaluate the selectivity of the proposed method, increasing concentrations of the possible interfering agents such as some metal ions and small biomolecules which are usually present in the biological fluids and pharmaceuticals were added to a solution with a fixed amount of melatonin 2.0 × 10 6 M, and the corresponding voltammograms were recorded. The tolerance limit was defined as the maximum concentration of potential interfering substance that causes a relative error less than ±5% for determination of 2.0 × 106 M melatonin. At about 100-fold excess, Ca 2+, K +, Na +, Fe 3+, Cu2+, Pb 2+, Mg2+, Zn2+, α-tocopherol, folic acid, L-histidine and nicotinic amid did not significantly influence the height of the peak currents at BDD electrode. In the presence of compounds that presented oxidation signals, namely L-tryptophan (+0.98 V), ascorbic acid (+0.91 V), uric acid (+0.95 V), pyridoxamin (+1.08 V), estradiol (+1.08 V) and

Fig. 4. Effect of the pH on the melatonin peak potential and peak current in different supporting electrolytes using SWV. A: BR buffer pH 2–11; B: pHs of different supporting electrolytes. Melatonin concentration, 5.0 × 106 M. Other operating conditions as indicated in Fig. 3.

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Fig. 5. (A): SW voltammograms at BDD electrode in 0.1 mol/L BR buffer (pH 2.0) containing different concentrations (0.5, 1.0, 1.5, 2, 2.5, 3.0, 3.5, 4.0, 4.5 μM) of melatonin. (B): Calibration curve for the quantitation of melatonin. SWV parameters: tacc, 60 s; Eacc, 0.5 V; frequency, 150 Hz; scan increment, 12 mV; pulse amplitude, 40 mV.

indole-3-acetic acid (+0.92 V), severe interferences on the voltammetric peak of melatonin (0.88 V) were shown. However, the oxidation peaks of estradiol (+1.08 V) and pyridoxamin (+1.10 V) were well distinguished from that of melatonin (+0.88 V). Furthermore, high concentration (more than 1.5 × 105 M) of L-tryptophan, ascorbic acid, uric acid, and indole-3-acetic acid did not interfere with determination of melatonin. The proposed method was therefore found to be quite satisfactory for the selective determination of melatonin. 3.6. Analytical application The described method was validated according to the standard procedures [48], in terms of linearity, limits of detection (LOD) and quantification (LOQ), precision and accuracy. Calibration was performed on BDD electrode for the determination of melatonin using the following SWV conditions–frequency: 150 Hz; step potential: 12 mV; pulse amplitude: 40 mV in 0.1 M BR (pH 3.0). The proposed method on BDD electrode offered well-defined concentration dependence. Fig. 5A presents SW voltammograms obtained by successive additions of melatonin over the 5.0 × 10 7 M–4.0 × 10 6 M (0.12– 0.93 μg/mL) concentration range. The peak current at a potential of +0.88 V increased proportionally with the melatonin concentration (Fig. 5B) to yield a highly plot according to linear calibration (4). Relative standard deviation (RSD, n = 3) value of the slope at the linearity range was found to be 2.14%, indicating the repeatability of the calibration curve of proposed method. ip ðμAÞ ¼ 0:4367 CðμMÞ–0:0815 ðr ¼ 0:998; n ¼ 8Þ

ð4Þ

The precision of the method was evaluated by replicated determination of inter-day and intra-day reproducibility. Intra-day repeatability was determined by replicated measurement of standard solutions of melatonin 2.0 × 10 6 M 10 times on the same day. Interday reproducibility was determined by analysis of standard solutions on three different days. RSD% of intra- and inter-day for standard solutions of melatonin 2.0 × 10 6 M were calculated 2.06 and 2.75, respectively. These results indicate that the BDD electrode achieves a high degree of precision, and reproducibility. The practical application of the present system was tested by measuring the concentration of melatonin in tablet formulation. After the extraction process, which was explained in Section 2.3; appropriate volume (20 μL) of the final extract was transferred to a voltammetric cell containing 10 mL of 0.1 M BR buffer (pH 3.0) and the voltammetric procedure was followed. Quantifications were performed by means of calibration curve method. Five replicate determinations were made; satisfactory results were obtained for the drug and were in good agreement with the label claims (Table 1). It was found that melatonin amount can be quantitatively recovered by the proposed method, being thus a guarantee of the accuracy of the voltammetric determination of melatonin in tablet formulation. The results were reproducible with less RSD values than 3%. Besides, developed method at BDD electrode was successfully applied to human urine samples. It is well known that urine is a complex matrix and the determination of it is not an easy task. Urine is composed essentially of water, salts, proteins, uric acid and etc.. Under the optimum experimental conditions, the representative voltammograms for the urine samples are illustrated in Fig. 6. Dashed line shows the

The sensitivity of the proposed method was checked in terms of the LOD and LOQ values. LOD and LOQ were calculated according to the formulas 3 s/m and 10s/m, respectively, where s is the standard deviation of the response (blank) (nine runs), and m the slope of the calibration curve. LOD and LOQ were found to be 1.1 × 10 7 M (0.025 μg/mL) and 3.7 × 10 7 M (0.086 μg/mL), respectively. Table 1 Melatonin content in the melatonin tablet obtained by proposed voltammetric method. Sample label claim (mg/tablet) Melatonin determineda Recovery (%) ± RSDb (%) 1 2 3 4 5 a b

3.00 3.00 3.00 3.00 3.00

2.95 2.91 3.02 3.06 2.97

98.33 ± 2.45 97 ± 2.63 100.67 ± 2.27 102 ± 2. 57 99 ± 2. 77

Values reported are the average of three independent analysis of each sample. RSD: Relative standard deviation.

Fig. 6. SW voltammograms obtained for the recovery analysis of melatonin in spiked human urine sample. The sample spiked at a melatonin levels of (a) 1.0 × 106 M, (b) 1.5 × 106 M, (c) 2.0 × 106 M (d) 2.5 × 106 M. Dashed lines represent blank human urine. Other operating conditions as indicated in Fig. 5.

A. Levent / Diamond & Related Materials 21 (2012) 114–119 Table 2 Results of the recovery analysis of melatonin in human urine samples. Melatonin added (mol/L)

Melatonin determineda (mol/L)

Recovery (%) ± RSDb (%)

1.0 × 106 1.5 × 106 2.0 × 106 2.5 × 106

1.05 × 106 1.45 × 106 1.97 × 106 2.53 × 106

105 ± 2.72 96.67 ± 3.14 98.5 ± 2.24 101.2 ± 2.37

a b

Values reported are the average of three independent analysis of each spiked sample. RSD: Relative standard deviation.

SW voltammogram of blank urine sample, and solid lines depicts the SW voltammograms of melatonin in sample after addition of a certain concentrations of melatonin standard solution. It is clearly shown that the observed peak at about 0.88 V is assigned to the oxidation of melatonin since this peak height increases while adding melatonin standard solution. To check the accuracy of the proposed method, the spike/recovery experiments were also performed. Recovery of melatonin was calculated by comparing the concentration obtained from the spiked mixtures with those of the pure melatonin. Table 2 shows the added quantities, as well as the values found with their RSD values and the obtained recovery percentages. A good agreement between the spiked and found quantities was observed, with an average recovery of 100.3% 4. Conclusions BDD electrode was successfully used in combination with the SWV technique to develop a novel and alternative electroanalytical method for melatonin determination in the tablet formulation and human urine. When compared with the other electrochemical methods in the literature, the proposed approach has similar sensitivity or more than the previous studies, but the most prominent advantages are simplicity (not electrode modification), and excellent precision. Moreover, the determination of melatonin in tablet formulation and human urine was successfully achieved without sample pretreatment. Therefore, this confers credibility and feasibility to the methodology proposed for the determination of melatonin in tablet and urine samples. In addition, due to the simplicity of the preparation procedures, fast routine determinations can also be achieved in the direct electrochemical analysis of melatonin and other hormones in pharmaceutical preparations. Acknowledgements The author would like to thank to English lecturer, İhsan Pilatin, for his valuable contributions to corrections of this manuscript. References [1] R.J. Reiter, News Physiol. Sci. 1 (1986) 202–205. [2] R.J. Reiter, Endocrinol. Rev. 12 (1991) 151–180.

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