The electrochemical measurements were performed with an Autolab potentiostat/galvanostat (PGSTAT 302N, Eco Chemie, the Netherlands). The experimental conditions were controlled with General Purpose Electrochemical System (GPES) software. A conventional three electrode cell was used at 25 ± 1°C. An Ag/AgCl/KCl (3.0 M) electrode, a platinum wire, and 2FF/Cu-TiO₂/IL/CPE were used as the reference, auxiliary and working electrodes, respectively. A Metrohm 710 pH meter was used for pH measurements.

All solutions were freshly prepared with double distilled water. Droxidopa, tryptophan and all of the other reagents were of analytical grade and were obtained from Merck (Darmstadt, Germany). The buffer solutions were prepared from orthophosphoric acid and its salts in the pH range of 2.0-9.0.

TiO₂ and Cu doped TiO₂ nano powder was prepared by the controlled sol-gel method with titanium(IV) n-butoxide Ti(OCH₂CH₂CH₂CH₃)₄ as raw materials. All the chemicals were purchaced from Merck (Darmstadt, Germany) and were used without any further purification. Deionized water obtained with a Milli-Q purification system (Millipore, Bedford, MA, USA), and filtered through 0.45 μm Millipore solvent filter, was used throughout. The dopant starting material was metallic copper sulfate. In a typical process, 5 mL of Ti(OBu)₄ was dissolved in 15 mL of absolute ethanol in a dry atmosphere and ultrasonically dispersed to produce a mixture (solution A). Meanwhile, 5 mL of water and 1 mL of HNO₃ (65%) were added to another 20 mL of absolute ethanol in turn to form an alchol/acid/water solution (solution B). After the two resulting solutions were stirred, respectively, the solution A was slowly added drop wise to the solution B under vigorously stirring to carry out a hydrolysis. Subsequently, the roughly stirring was conducted so that the temperature was raised from room temperature to 80°C at the end of the reaction. The gel was dried in the air for about 24 h at 85°C and subsequently the resulting material was powdered and then calcined in an electric muffle furnace at 450°C for 2 h to obtain crystalline powders of TiO₂. Cu doped TiO₂ nanoparticles were synthesized using almost the same method. The molar amount of transition metal ion dopant (Cu²⁺) was calculated in order to substitute 1% of titanium ions in TiO₂ and was solubilized in an appropriate amount of ethanol/nitric acid/water solution prior to the hydrolysis. The remaining procedures were the same as described above. After hydrolysis, the greenish transparent sol was obtained. A typical SEM image of the synthesized Cu-TiO₂ nanocomposite is shown in Fig. 1.

PdCl₂(PPh₃)₂ (29 mg) and CuI (7.6 mg) were added to a stirred solution of aryl halides (1.0 mmol, 0.26 g) and ethynylferrocene (1.2 mmol, 0.25 g) in Et₃N (5 mL) and DMF (5 mL) and the resulting mixture was stirred at reflux temperature for 3 h. The progress of the reaction was monitored by TLC. After addition of 200 mL of H₂O and stirring for 30 min, the resulting crude product was filtered. The resulting raw material was dissolved in CH₂Cl₂, then washed with diluted HCl and saturated NaCl solution. After drying with MgSO₄ and solvents removal, the crude products was purified by column chromatography on silica gel using hexane-CH₂Cl₂ as eluent to afford the pure product in 87% yield.

Red solid; mp: 147-148°C; IR: ν (cm^-1)= 750, 1602, 1712 (C=O), 2201 (C=C); ¹H NMR (500 MHz, CDCl 3): d(ppm)= 4.20-4.30 (pseudo s, 7H), 4.54 (s, 2H), 7.30-7.76 (m, 7H); ¹³C NMR (125 MHz, CDCl 3 ): d(ppm)= 64.62, 69.04, 70.00, 71.48, 85.03, 90.51, 120.20, 120.45, 124.36,124.88, 127.02, 129.12, 134.16, 134.27, 134.81, 137.19, 142.94, 144.07, 193.11; Anal. Found: C, 76.98; H, 3.96. Calc. for C₂₅H₁₆FeO: C, 77.34; H, 4.15%.

The Cu/TiO₂-IL-2FF/CPEs were prepared by hand mixing 0.04 g of Cu/TiO₂ nanocomposite, 0.01 g of 2FF with 0.95 g graphite powder and 0.2 mL ionic liquid with a mortar and pestle. Then, ~ 0.7 mL of paraffin oil was added to the above mixture and mixed for 20 min until a uniformly-wetted paste was obtained. The paste was then packed into the end of a glass tube (ca. 3.4 mm i.d. and 15 cm long). A copper wire inserted into the carbon paste provided the electrical contact. When necessary, a new surface was obtained by pushing an excess of the paste out of the tube and polishing with a weighing paper.

For comparison, unmodified CPE in the absence of Cu/TiO₂ nanocomposite, 2FF and ionic liquid were also prepared in the same way.

Urine samples were stored in a refrigerator immediately after collection. Ten millilitres of the samples were centrifuged for 15 min at 2,000 rpm. The supernatant was filtered out by using a 0.45 μm filter. Next, different volumes of the solution was transferred into a 25 mL volumetric flask and diluted to the mark with PBS (pH 7.0). The diluted urine samples were spiked with different amounts of droxidopa and tyrosine. The droxidopa and Tryptophan contents were analysed by the proposed method by using the standard addition method.

The serum sample was centrifuged, and after filtering, diluted with PBS (pH 7.0) without any further treatment. The diluted serum sample was spiked with different amounts of droxidopa and Tryptophan. The droxidopa and tyrosine contents were analysed by the proposed method by using the standard addition method.

2FF/Cu-TiO₂/IL/CPE was prepared and its electrochemical properties were studied in a PBS (pH 7.0) using CV. It should be noted that one of the advantages of 2FF as an electrode modifier is its insolubility in aqueous media. Experimental results showed reproducible and well-defined CVS. Anodic and cathodic peak potentials were 0.35 and 0.25 V vs. Ag/AgCl/KCl (3.0 M) respectively. The observed peak separation potential, ΔE_p = (E_pa − E_pc) of 100 mV, was greater than the value of 59/n mV which is expected for a reversible system [34], suggesting that the redox couple of 2FF in 2FF/Cu-TiO₂/IL/CPE has a quasi-reversible behavior in aqueous medium.

In addition, the long-term stability of the 2FF/Cu-TiO₂/IL/CPE was tested over a three-week period. When CVs were recorded after the modified electrode was stored in atmosphere at an ambient temperature, the peak potential for droxidopa oxidation was unchanged and the current signals showed less than 2.6% decrease relative to the initial response. The antifouling properties of the modified electrode toward droxidopa and its oxidation products were investigated by recording the CVs of the modified electrode before and after use in the presence of droxidopa. CVs were recorded in the presence of droxidopa after having cycled the potential 15 times at a scan rate of 10 mV s^-1. The peak potentials were unchanged and the currents decreased by less than 2.3%. Therefore, at the surface of 2FF/Cu-TiO₂/IL/CPE, not only the sensitivity increases, but the fouling effect of the analyte and its oxidation product also decreases.

The electrochemical behavior of droxidopa is dependent on the pH value of the aqueous solution, whereas the electrochemical properties of Fc/Fc⁺ redox couple are independent on pH. Therefore, pH optimization of the solution seems to be necessary in order to obtain the electrocatalytic oxidation of droxidopa. Thus the electrochemical behavior of droxidopa was studied in 0.1 M PBS in different pH values (2.0-9.0) at the surface of 2FF/Cu-TiO₂/IL/CPE by voltammetry. It was found that the electrocatalytic oxidation of droxidopa at the surface of 2FF/Cu-TiO₂/IL/CPE was more favored under neutral conditions than in acidic or basic medium. This appears as a gradual growth in the anodic peak current and a simultaneous decrease in the cathodic peak current in the cyclic voltammograms of 2FF/Cu-TiO₂/IL/CPE. Thus, the pH 7.0 was chosen as the optimum pH for electrocatalysis of droxidopa oxidation at the surface of 2FF/Cu-TiO₂/IL/CPE.

Fig. 2 depicts the CV responses for the electrochemical oxidation of 300.0 μM droxidopa at an unmodified CPE (curve b), Cu-TiO₂/CPE (curve d), 2FF/IL/CPE (curve e) and 2FF/Cu-TiO₂/IL/CPE (curve f). As it is seen, while the anodic peak potential for droxidopa oxidation at the Cu-TiO₂/CPE, and unmodified CPE are 600 and 645 mV, respectively, the corresponding potential at 2FF/Cu-TiO₂/IL/CPE and 2FF/IL/CPE is about 350 mV. These results indicate that the peak potential for droxidopa oxidation at the 2FF/Cu-TiO₂/IL/CPE and 2FF/IL/CPE electrodes shift by ~ 250 and 295 mV toward negative values compared to Cu-TiO₂/CPE and unmodified CPE, respectively. However, 2FF/Cu-TiO₂/IL/CPE shows much higher anodic peak current for the oxidation of droxidopa compared to 2FF/IL/CPE, indicating that the combination of Cu-TiO₂ nanocomposite and the mediator (2FF) has significantly improved the performance of the electrode toward droxidopa oxidation. In fact, in the absence of droxidopa 2FF/Cu-TiO₂/IL/CPE exhibited a well-behaved redox reaction (Fig. 2, curve c) in 0.1 M PBS (pH 7.0). However, there was a drastic increase in the anodic peak current in the presence of 300.0 μM droxidopa (curve f), which can be related to the strong electrocatalytic effect of the 2FF/Cu-TiO₂/IL/CPE towards this compound [34]. According to these results, we suggest an EC′ catalytic mechanism [34] for the electrochemical oxidation of droxidopa at 2FF/Cu-TiO₂/IL/CPE, as shown in in Scheme 3. It has been proposed that in a catalytic reaction (C), droxidopa is oxidized by the oxidized form of 2FF which is produced during an electrochemical reaction (E) at the electrode surface.

The effect of potential scan rate on the electrocatalytic oxidation of droxidopa at the 2FF/Cu-TiO₂/IL/CPE was investigated by linear sweep voltammetry (LSV) (Fig. 3). As can be observed in Fig. 3, the oxidation peak potential shifted to more positive potentials with increasing scan rate, confirming the kinetic limitation in the electrochemical reaction. Also, a plot of peak height (I_p) vs. the square root of scan rate (ν^1/2) was found to be linear in the range of 5-500 mV s^-1, suggesting that, at sufficient over potential, the process is diffusion rather than surface controlled (Fig. 3A). A plot of the scan rate-normalized current (I_p/v^1/2) vs. scan rate (Fig. 3B) exhibits the characteristic shape typical of an EC' process [34].

Fig. 4 shows the Tafel plot for the sharp rising part of the voltammogram at the scan rate of 5 mV s^-1. If deprotonation of droxidopa is a sufficiently fast step, the Tafel plot can be used to estimate the number of electrons involved in the rate determining step. A Tafel slope of 0.096 V was obtained which agrees well with the involvement of one electron in the rate determining step of the electrode process [34], assuming a charge transfer coefficient, α of 0.38.

Chronoamperometric measurements of droxidopa at 2FF/Cu-TiO₂/IL/CPE were carried out by setting the working electrode potential at 0.4 V for the various concentrations of droxidopa in PBS (pH 7.0) (Fig. 5). For an electroactive material (droxidopa in this case) with a diffusion coefficient of D, the current observed for the electrochemical reaction at the mass transport limited condition is described by the Cottrell equation [34]. Experimental plots of I vs. t^-1/2 were employed, with the best fits for different concentrations of droxidopa (Fig. 5A). The slopes of the resulting straight lines were then plotted vs. droxidopa concentration (Fig. 5B). From the resulting slope and Cottrell equation the mean value of the D was found to be 1.5 × 10^-6 cm²/s.

The electrocatalytic peak current of droxidopa oxidation at the surface of the modified electrode can be used for determination of droxidopa in solution. Therefore, DPV experiments were performed using modified electrode in PBS (pH 7.0) containing various concentration of droxidopa (Fig. 6). The mediated oxidation peak currents of droxidopa at the surface of a modified electrode were proportional to the concentration of the droxidopa within the ranges 5.0 × 10^-8 to 4.0 × 10^-4 M with slope of 0.0548 μA μM^-1 in the DPV. The detection limit (3σ) was 30.0nM.

To our knowledge, no paper has used the modified Cu-TiO₂ nanocomposite electrode and specially modified Cu-TiO₂ nanocomposite carbon paste electrode for simultaneous determination of droxidopa and tryptophan and this is the first report for simultaneous determination of droxidopa and tryptophan using modified Cu-TiO₂ nanocomposite carbon paste electrode. The electrochemical determination of droxidopa using bare electrodes suffers from interference by tryptophan, because the oxidation potential for tryptophan is fairly close to that of droxidopa.

Determination of two compounds was performed by simultaneously changing the concentrations of droxidopa and tryptophan, and recording the DPVs (Fig. 7). The voltammetric results showed well-defined anodic peaks at potentials of 315 and 715 mV, corresponding to the oxidation of droxidopa and tryptophan, respectively, indicating that simultaneous determination of these compounds is feasible at the 2FF/Cu-TiO₂/IL/CPE as shown in Fig. 7.

The sensitivity of the modified electrode towards the oxidation of droxidopa was found to be 0.055 μA μM^-1. This is very close to the value obtained in the absence of tryptophan (0.0548 μA μM^-1, see Section 3.4), indicating that the oxidation processes of these compounds at the 2FF/Cu-TiO₂/IL/CPE are independent and therefore, simultaneous determination of their mixtures is possible without significant interferences.

The influence of various substances as compounds potentially interfering with the determination of droxidopa was studied under optimum conditions. The potentially interfering substances were chosen from the group of substances commonly found with droxidopa in pharmaceuticals and/or in biological fluids. The tolerance limit was defined as the maximum concentration of the interfering substance that caused an error of less than ±5% in the determination of droxidopa. According to the results, L-lysine, glucose, NADH, acetaminophen, uric acid, L-asparagine, L-serine, L-threonine, L-proline, histidine, glycine, tryptophan, phenylalanine, lactose, saccarose, fructose, benzoic acid, methanol, ethanol, urea, Mg²⁺, Al³⁺, NH₄⁺, F^-, SO₄^2- and S^2- did not show interference in the determination of droxidopa.

In order to evaluate the analytical applicability of the proposed method, also this method was applied to the determination of droxidopa and tryptophan in human blood serum and urine samples. The samples were found to be free from droxidopa and tryptophan. Therefore different amounts of droxidopa and tryptophan were spiked to the sample and analyzed by the proposed method. The results for determination of the two species in real samples are given in Table 1. Satisfactory recovery of the experimental results was found for droxidopa and tryptophan. The reproducibility of the method was demonstrated by the mean relative standard deviation (R.S.D.).