A modified 174 polarographic analyzer (EG & G, USA) was used for application of the cyclic voltammetric and differential pulse voltammetric excitation potential regimes and measurement the resulting current. Data acquisition was performed through a GPIB (IEEE, USA) interface interfaced to a computer and controlled by an in-house modified LabVIEW (IEEE, USA) software. All the experiments were performed using a home-made 3-electrode H-shape electrochemical cell where the working electrode compartment was separated from the reference and auxiliary electrode compartment by a fine glass frit. The two compartments of the cell were equipped with inlets and outlets for purging and blanketing with oxygen-free nitrogen gas, rinsing, furnishing the cell with pure supporting electrolyte solution and introduction of the standards and dopamine samples. A 0.5 mm polycrystalline platinum wire was used as a working electrode (Aldrich 99.99% minimum purity certified reagent, USA). The electrode surface area was controlled by rendering the immersed part of the electrode had a U-shape and the shorter part of the electrode was touching the surface of the solution from underneath the level of the solution. A silver/silver chloride was used as a quasi-reference electrode (QRE) and all the reported potentials in this work are referenced to this electrode. The auxiliary electrode was a 0.5 mm polycrystalline platinum wire (Aldrich, certified 99.99% minimum purity, USA). The wire was spiral-shaped to provide a large surface area. All chemicals used were of analytical grade (AR) and used as received without further purification. Sulfuric acid (95–97%) was supplied from Merck (USA), Dopamine (99% pure) was purchased from Across Organics (USA.USA), potassium iodide was purchased from Sigma-Aldrich (USA). Milli-Q water (Millipore, Aldrich, USA) was used for preparation of all solutions. Nitrogen gas (5G grade, 99.999% minimum purity) was supplied by The International Jordanian Gas Company (Jordan).

Phosphate buffer solutions (PBS) with different pH values were prepared by mixing a 0.1M of Na₂HPO₄ with 0.1M of NaH₂PO_4. The pH was measured using (Jenway 3030 pH meter, USA) and the pH was adjusted to the desired value using 0.100 M HCl solution. A 0.100 mM dopamine stock solution was prepared from dopamine hydrochloride (Thermo Fisher Scientific, USA, 99% pure) and the above-mentioned PBS solution. Standard solutions of lower concentrations of dopamine were prepared by successive dilution from the stock solution.

Preparation of iodine-coated platinum electrode was described elsewhere (Hourani and Amayreh 2018). Briefly, the platinum working electrode was cleaned by immersion in a hot freshly prepared chromic acid solution followed by extensive rinsing with Milli-Q water. The electrode was transferred to an electrochemical cell containing 1.0 M H₂SO₄ solution where the electrode potential was scanned between the cathodic and anodic potential limits as an “electrochemical cleaning”. The potential cyclization was continued until the well-known cyclic voltammogram of polycrystalline platinum was reproduced (Bard and Faulkner 2001).

The potential scan was stopped in the double layer region on the positive-going scan where we believe that the electrode is not loaded with hydrogen or oxygen in this region. The electrode was exposed to 1.0x10^-2 M potassium iodide solution for 5 minutes. The electrode potential was cycled in a 0.5M H₂SO₄ solution between -0.2V and +0.85V at 50 mv/s to verify the completeness of the coating process. The cyclic voltammogram of the iodine-coated platinum electrode between the cathodic potential limit and about 0.85 V shows complete absence of all the characteristic voltammetric features of platinum electrode. The absence of oxygen and hydrogen adsorption-desorption peaks from the recorded cyclic voltammograms of an iodine-coated platinum electrode is an indication of complete coverage of platinum electrode surface with a monolayer of iodine(Hourani and Amayreh 2018). The electrode potential was maintained between the cathodic potential limit at -0.2 V and the onset of iodine desorption from the platinum surface at ca. 0.85 V. The iodine-coated platinum electrode acts as a real polarizable electrode in the potential window between ~-0.2 and ~ +0.85 V. Validation of the electrochemical system cleanliness and coating of the platinum electrode with iodine was ensured before any experiment. It is also worth mentioning that scanning of the electrode potential was limited to the potential range from -0.2 V to 0.85 V at most to avoid desorption of iodine from the platinum surface.

Blood serum and urine samples from various healthy volunteers were collected by The University of Jordan Hospital personnel. The samples were stored in a freezer at -20 °C until the time of analysis which didn’t exceed one week. A volume of 50 µL of human blood serum was diluted to the mark in a 20.00 ml volumetric flask with pH 7 PBS solution. The solution was introduced to the working electrode compartment in the electrochemical cell. The differential pulse voltammograms were recorded by application of a potential scan between -0.2 and 0.80 V vs. QRE.

Fig. 1 shows a representative cyclic voltammogram for the iodine-coated platinum electrode in 0.10 M PBS containing 1.00 mM dopamine. The recorded cyclic voltammogram shows a prominent anodic peak for dopamine oxidation at a potential of~ 0.4 V with a counter peak centered at 0.2 V.

Figure 1. 

Cyclic voltammogram of an iodine-coated platinum electrode in 0.1 M PBS solution containing 1.0 mM dopamine. Experimental conditions: dE/dt = 100 mV/s, pH = 7. This anodic peak was considered for quantification of dopamine in urine and serum samples.

The effect of pH on the oxidation peak current and peak potential of dopamine at iodine-coated platinum electrode was investigated within the pH range 3.0–7.0. Fig. 2A, B show the effect of pH on the peak potential and peak current respectively. Fig. 2A shows a sharp change in peak potential upon changing the pH from 4 to 7.

Figure 2. 

The effect of pH on (A) anodic peak potential and (B) anodic peak current. Anodic peak potentials and currents were extracted from cyclic voltammograms of iodine-coated platinum electrode in 0.1 M PBS solution containing 1.0 mM dopamine at different pH values. dE/dt = 100 mV/s.

The balanced equation for the oxidation is

C₈H₁₁NO₂ → C₈H₉NO₂ + 2H⁺ + 2e-

Which dictates a shift towards less positive potential with increasing pH as manifested in Fig. 2A. The explanation relies on the fact that increasing the pH of the solution decreases the concentration of hydrogen ions which shifts the equilibrium in the forward direction and stabilizes the products. Thus the increase in the pH makes The results here coincide with others’ work on electrooxidation of dopamine (Barham et al. 2009). Peak current (Fig. 2B) shows a more complicated trend between the pH and the anodic peak current. Seemingly, there is more than one factor affecting the rate of the oxidation of dopamine. At low pH values, the dopamine exists in the protonated form which is more stable and less reactive towards oxidation. With increasing pH, the easily oxidizable deprotonated dopamine form prevails over the protonated form. This is reflected in observation of higher peak current at higher pH because the current is an index of the rate of the electrochemical reaction. The highest peak current was recorded at pH 7 and higher pH values were avoided because of the instability of iodine coating in basic solutions (Hourani and Hijaz 2019). Thus, the optimal pH value is 7 and all of our experiments were performed at this pH for this reason.

Fig. 3 shows the effect of scan rate on the anodic peak current in the linear sweep voltammograms. . An excellent linearity between anodic peak current and the square root of scan rate ((scan rate)^1/2) has been indicated (R² = 0.9977). This indicates that the oxidation of dopamine at iodine-coated platinum electrode over the investigated range (10–100 mV s^-1) is controlled by diffusion. In absence of surface processes and adsorption and at one hand and the diffusion-controlled oxidation of dopamine at the iodine-coated platinum electrode on the other hand makes this electrode nearly ideal for voltammetric analysis of dopamine.

Figure 3. 

The effect of scan rate on the anodic peak current for oxidation of dopamine at iodine-coated platinum electrode. A. Cyclic voltammograms of dopamine electrooxidation at iodine-coated electrode in 0.1 M PBS (pH 7) solution containing 1.00 mM dopamine. Scan rate = 100 mV/s B. A plot of anodic peak current vs. (scan Rate)^1/2, where n is the scan rate of the recorded voltammograms in (A).

Fig. 4A shows the differential pulse voltammograms of the iodine-coated platinum electrode at different concentrations ranging from 1.00 µM to 100 µM dopamine solutions. The calibration curve for the analysis (Fig. 4B) shows excellent linearity with a coefficient of determination, R² = 0.9977. The calibration equation is

Figure 4. 

A. Differential pulse voltammograms of an iodine-coated platinum electrode in 0.1 M PBS (pH 7) containing different concentrations of dopamine. scan rate: 5 mV s^-1, modulation amplitude = 50 mV. pulse repeat time: 1 s. B. A plot of the concentration of dopamine vs. the peak currents extracted from the corresponding voltammograms (in Fig. 4A).

i_p(µA) = 0.0064C_dopamine + 0.2064

where C_dopamine is in micromolar units.

The limit of detection (LOD) and the limit of quantitation (LOQ) for determination of the dopamine by differential pulse voltammetry at iodine coated platinum electrode were estimated based on the equations LOD=3s_b/m, and LOQ = 10s_b/m respectively where s_b is the standard deviation for 20 measurements of the anodic peak current for a blank solution and m is the slope of the calibration curve (calibration sensitivity). The estimated LOD and LOQ are 0.29 µM and 0.96 µM respectively. The values of LOD and LOQ attest to the sensitivity of the developed method.

The interday and intraday precision were estimated by extracting the peak currents from the differential pulse voltammograms for electrooxidation of dopamine at the iodine-coated electrodes for the 25.0 µM dopamine standard solution. Ten differential pulse voltammograms were reproduced for the above-mentioned solution within one day for the intra-day and within ten days for the interday precision evaluation. The measurements were equally spaced by one hour for the intraday precision evaluation and 24-hour for the interday precision evaluation. The coefficient of variation was 2.66% and 4.34% for the intra-day and the inter-day respectively. The measured values of standard deviation confirm the high precision of the developed method.

Method selectivity is of prime importance in chemical analysis. The selectivity of the developed method was tested by application of the method to analysis of dopamine in presence of two potential interferences ascorbic acid and uric acid spiked in analyzed samples.

Fig. 5A shows the effect of presence of ascorbic acid on the voltammetric analysis of dopamine. The results indicate that there is no effect of presence of ascorbic acid even at concentrations of tenfold or higher of ascorbic acid. A little shift in the anodic peak potential is observed but it does not affect the accuracy of analysis.

Figure 5. 

A. Differential pulse voltammograms for the iodine coated electrode in PBS containing (A) (blue) 0.05 mM dopamine, (red) 0.05 mM dopamine + 0.5 mM ascorbic acid, and (green) 0.05 mM dopamine + 2.5 mM ascorbic acid. B. (blue) 0.05 mM dopamine, (green) 0.05 mM dopamine + 0.1 mM uric acid and (orange) 0.05 mM dopamine + 1.75 mM uric acid.

Experimental conditions for all voltammograms: scan rate: 5 mV s-1, pulse repeat time: 1 s, modulation amplitude: 50 mV.

Similarly, presence of uric acid at large concentrations of uric acid has no effect on the peak current of dopamine. On the other hand, presence of relatively very large concentrations of uric acid leads to a shift in the anodic peak of dopamine electrooxidation towards higher positive potential. The peak current, however, is not affected by the presence of uric acid as shown in Fig. 5B. This indicates that quantification of dopamine in presence of a large concentrations of uric acid is still possible.

The recovery results can be taken as evidence for the absence of interference and absence of determinate errors upon analysis of the spiked samples.

Calculated volumes of 1.0 mM dopamine standard solution were spiked separately into the serum and urine samples (three of each) to produce solutions with nominal concentrations of 20 and 40µM dopamine in serum and urine matrices. The spiked samples were analyzed for their dopamine concentration and the recoveries were calculated. Fig. 6 shows representative differential pulse voltammograms for human urine samples before and after spiking the samples such that their nominal concentrations were 20.0 and 40.0 µM. The voltammograms unequivocally indicate an increase in the peak current with increasing dopamine spiked volume.

Figure 6. 

Differential pulse voltammograms for an iodine coated electrode in 0.1 M PBS (pH 7) of spiked with (A) spiked with urine sample only (B) spiked with 20 µl of dopamine (C) spike with 40 µl of dopamine. Scan rate = 5 mV s^-1, modulation amplitude = 50 mV, pulse repeat time = 1 s.

Table 1 shows the recoveries for 20 and 40µM dopamine spiked samples in urine and serum matrixes. The recoveries of the analyzed spiked samples ranged from 94.4 to 104.5 104.5%. Thus the recoveries lie within 5% of the nominal values which eliminates the possibility of determinate errors regardless of the complexity of urine and serum samples.