## 3 Results  
### Characterization of DCF Electrochemical Behavior  
Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV) were used to study the electrochemical reactivity of DCF on the surfaces of the activated electrodes and to evaluate the best pH and medium conditions for its determination. The electrochemical activation, previously developed with GCE in order to enhance the acetaminophen (APAP) signals [20], was optimized for SPCEs. The best procedure was described in the experimental section. By this electrochemical activation, it is possible to obtain a systematic increase in the DCF peak currents, but the effect is quite moderate compared to that achievable for APAP. Moreover, the shift towards lower potential values of the APAP oxidation peak was not observed for DCF. An example of the voltammograms obtained from the DCF and acetaminophen solutions with SPCE and aSPCE is reported in Figure 1.  
In order to study the pH effect on the DCF electrochemical signals, CV scans were carried out with aGCE on solutions of 20 \(\upmu\) L\({}^{-1}\) DCF at different pH and medium: pH 2.0, with 1 \(\times\) 10\({}^{-2}\) mol L\({}^{-1}\) hydrochloric acid; pH 2.0, with 2.5 \(\times\) 10\({}^{-2}\) mol L\({}^{-1}\) phosphoric acid; pH 4.7, with 2.5 \(\times\) 10\({}^{-2}\) mol L\({}^{-1}\) acetic buffer; pH 7.0, with 2.5 \(\times\) 10\({}^{-2}\) mol L\({}^{-1}\) phosphate buffer. The signals obtained are reported in the Supporting Material File, Figure S1. The maximum values of the peak current were obtained with hydrochloric acid and phosphoric acid at pH 2.0. At this pH, the voltammograms show three well-defined peaks, the first one, corresponding to a non-reversible process, at about 0.85 V, and the other two, corresponding to a quite reversible process, in the potential range 0.3-0.4 V. Two very weak further peaks can be seen at about 0.62 V. Figure 2a shows the result of the first and the second CV scans on the same solution of 20 \(\upmu\)g L\({}^{-1}\) DCF, in 1 \(\times\) 10\({}^{-2}\) mol L\({}^{-1}\) phosphoric buffer, pH = 2.0. In the first oxidative scan, only the peak at 0.85 V is visible; therefore, the other peaks are \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline \multirow{2}{*}{**Parameter a**} & \multicolumn{3}{c}{**aGCE**} & \multicolumn{3}{c}{**aSPCE1**} \\ \cline{2-9}  & \multicolumn{3}{c}{**Level**} & \multicolumn{3}{c}{**Level**} & \multirow{2}{*}{**Optimized**} & \multicolumn{3}{c}{**Level**} & \multirow{2}{*}{**Optimized**} \\ \cline{2-9}  & \(-\mathbf{1}\) & \(\mathbf{0}\) & \(\mathbf{1}\) & & \(-\mathbf{1}\) & \(\mathbf{0}\) & \(\mathbf{1}\) & \\ \hline \(E_{\text{pulse}}\) (mV) & 30 & 90 & 140 & 115 & 50 & 100 & 150 & 100 \\ \(I_{\text{pulse}}\) (ms) & 20 & 40 & 60 & 50 & 10 & 30 & 50 & 10 \\ SC (mV/s) & 10 & 25 & 40 & 25 & 10 & 25 & 40 & 25 \\ \hline \hline \end{tabular}  
* \(E_{\text{pulse}}\): pulse amplitude; \(I_{\text{pulse}}\): pulse length; SC: scan rate.  
\end{table} 
Table 2: Values of the applied measurement parameters the Design of Experiments and optimized parameters. probably due to DCF oxidation by-products. The reaction steps involved were named E and C to distinguish, respectively, electrochemical and chemical reactions and were depicted in Figure 2b for the main peaks. A chemical reaction occurs after the oxidation of DCF. This chemical reaction generates an electroactive species T and two distinct redox reactions therefore occur. The product of the chemical reaction T generates the electrochemical signals observable on the second scan at potentials between 0.3 and 0.4 V. Scheme 1 shortly describes the whole process:  
Figure 1: DPV signals of acetaminophen (APAP) and DCF obtained by non-activated and activated SPCE1. DPV signals of 11.5 mg L\({}^{-1}\) acetaminophen in 4.5\(\cdot\)10\({}^{-2}\) mol L\({}^{-1}\) PB, pH 5.0, \(E\) vs. Ag/AgCl, _E_pulse 100 mV, _t_pulse 20 ms, _E_step 4 mV, scan rate 5 mV/s.