## 3 Results and Discussion
### Method Validation and Application to Real Samples  
The method was submitted to a preliminary validation process and the analytical performances of linearity, selectivity, precision, and recovery have been determined by the analysis of blank and spiked calf urine samples. In order to verify the absence of matrix interfering peaks in the retention time-window of interest (\(\pm\)2.5% of the retention time of each \(\beta\)-agonist), 20 independent calf urine samples were analyzed, and an example of chromatogram of a blank urine sample is shown in Figure 4 and compared with that of a standard mix of clenbuterol, terbutaline, and fenoterol. The selectivity of the method toward endogenous compounds is demonstrated by the absence of interfering peaks at the retention times of the \(\beta\)-agonists. \begin{table}
\begin{tabular}{l c c c c c} \hline \multicolumn{5}{c}{**Potential (V vs. AgI AgCl)**} & \multicolumn{4}{c}{**Time (ms)**} \\ \hline
**Parameter** & **Explored Value** & **Optimized Value** & **Parameter** & **Explored Value** & **Optimized Value** \\ \hline E\({}_{\text{DET}}\) & +0.8 to +1.3 & +1.2 & t\({}_{\text{DET}}\) & 200 to 900 & 900 \\  & & & t\({}_{\text{DEL}}\) & 50 to 100 & 100 \\  & & & t\({}_{\text{INT}}\) & 150 to 800 & 800 \\ E\({}_{\text{OX}}\) & +2.0 & & t\({}_{\text{OX}}\) & 10 to 200 & 50 \\ E\({}_{\text{RED}}\) & \(-\)2.0 & & t\({}_{\text{RED}}\) & 10 to 200 & 50 \\ \hline \end{tabular}
\end{table} 
Table 1: Optimized potential-time parameters of the triple-step PAD waveform. Table 2 shows reported data relevant the performance parameters obtained for all the investigated analytes. A good linearity was found for the \(\beta\)-agonists determination in the range 0.5-100 \(\upmu\)g/L, with correlation coefficients higher than 0.9984. The goodness of fit of the data to the calibration curve is obtained in terms of response factor distribution (signal to concentration ratio, yi/xi) whose reference range is (y/x) mean \(\pm\)10%. Furthermore, any systematic instrumental bias can be ruled out since the intercept includes the zero value at a 95% confidence level. From the regression line parameters of the calibration curves obtained from standard solutions and spiked blank urine samples, instrumental and method LODs and LOQs have been calculated as 3.3 s\({}_{\mathrm{y/x}}\)/S and 10 s\({}_{\mathrm{y/x}}\)/S respectively, where S is the slope and s\({}_{\mathrm{y/x}}\) is the standard deviation of residuals. The values were in the range 0.1-0.2 \(\upmu\)g/L and 0.2-0.5 \(\upmu\)g/L, respectively, which allow a reliable quantification considering the MRL of 0.5 \(\upmu\)g/kg, established for clenbuterol. The method was tested for intraday precision by replicate injections of a spiked calf urine sample at 100 \(\upmu\)g/L, and the performance parameters in terms of signal-to-noise ratio (S/N) and relative standard deviations (RSD) of 10 replicate injections of the analytes are given in Table 3. The RSD down to 7% demonstrate that PAD assures good signal reproducibility. For comparison, Table 3 also shows data obtained under DC electrochemical detection at +1.2 V, pointing out the response decrease that occurs when repeated injections are performed at a constant potential (RSD higher than 13%). The lower response stability for constant potential detection is also confirmed by a higher variability of the noise levels and concomitant lower signal-to-noise ratios with respect to PAD. Recoveries of \(\beta\)-agonists were evaluated by comparing the concentration of spiked samples determined by interpolation on the calibration curve with the nominal concentration of the fortification level. As an example, Figure 5 reports the comparison of a spiked calf  
Figure 4: Comparison of chromatograms of a mixed standard solution of \(\beta\)-agonists, each at a concentration of 100 \(\upmu\)g.L\({}^{-1}\) (blue) and a calf urine sample purified by SPE (grey).