## 3 Results and Discussion
### Triple Potential Step PAD Waveform Optimization  
Within a pulsed waveform, duration, and potential values of each step may significantly affect the sensitivity and reproducibility of response. Thus, to further increase the detection capability of the triple step PAD waveform, a systematic investigation was carried out to optimize the potential-time settings by varying each value, while all the other parameters were held constant. Waveform optimization was carried out by performing multiple injections of the same mixed standard solution of \(\beta\)-agonists in a C8 column,  
Figure 1: Chemical structures of clenbuterol, fenoterol, and terbutaline.  
Figure 3: Current-potential curves of 5 mM of terbutaline (blue line 1 st cycle, red line 10 th cycle) at a glassy carbon electrode in 20 mM of acetic acid/acetonitrile (55:54 \(v/v\)), pH 7.0 supporting electrolyte (black line). Scan rate: 100 mV\(s^{-1}\). Potential range: (**A**) \(-\)1.5/+1.5 V; (**B**) 0/+1.5 V.  
Figure 2: Current-potential curves of 5 mM of clenbuterol (blue line 1 st cycle, red line 10 th cycle) at a glassy carbon electrode in 20 mM of acetic acid/acetonitrile (55:45 \(v/v\)) at pH 7.0 supporting electrolyte (black line). Scan rate: 100 mV\(s^{-1}\). Potential range: (**A**) \(-\)1.5/+1.5 V; (**B**) 0/+1.5 V.  
under isocratic elution conditions at 1.0 mL/min. The potential waveform was applied to the glassy carbon working electrode, and the choice of the optimal potential-time values providing the maximum sensitivity was performed by the evaluation of the signal-to-noise ratio (average of three replicates) as a function of the parameter to be optimized. For each set of applied potential, the background noise was evaluated as the peak-to-peak value. The detection potential was increased from +0.8 to +1.3 V in 0.05 V intervals and the peak current increased as the potential increased, reaching a maximum at 1.2 V. Subsequent increments produced a higher noise, so +1.2 V was selected as the optimum detection potential. The effect of integration times was studied in the range 150-800 ms. Higher values are to be avoided to keep the overall cycle time short, enough to maintain peak integrity. It was observed that integration times lower than 800 ms determined a sensitivity decrease. Potential values of +2.0 V and \(-\)2.0 V, respectively, for the steps of oxidative cleaning and reductive electrode restoration, were essential for good response stability, ensuring the oxidation of the surface (to avoid the electrode fouling) and the reactivation of working electrode. The corresponding time parameters were varied from 10 to 200 ms. A value of 50 ms both for t\({}_{\text{OX}}\) and t\({}_{\text{RED}}\) gave the best results, since higher times produced a substantial decrease in the response. The potential-time parameters selected as the optimum are reported in Table 1. In order to demonstrate the efficiency of PAD to preserve the signal magnitude, repetitive injections of spiked calf urine sample were performed. No significant difference was found between the initial and final readings (_t_-test, 95% confidence, \(n\) = 12). The optimized waveform was also evaluated in terms of detection limit, using clenbuterol as the model compound. A LOD of 0.1 ug/kg was obtained, with respect to 0.4 ug/kg resulted by DC analysis. As far as we know, the LOD of the present method is approximately 40-100 fold lower than the values obtained by HPLC-UV [6; 35] and comparable (even slightly lower) with the detection capability of 0.15 ppb determined for clenbuterol in bovine muscle by the LC-MS screening method [36].