## 3 Results and Discussion
### Method Validation and Application to Real Samples
Column LiChroCART C8 LiChrospher 60 RP-select B column (25 cm \(\times\) 4 mm, 5 \(\upmu\)m LD., Merck) coupled with a guard column LiChroCART LiChrospher 60 RP-select B (4 mm \(\times\) 4 mm, 5 \(\upmu\)m LD., Merck). Mobile phase: 20 mM of acetic acid/acetonitrile (55:45), pH 7.0. Flow rate 1.0 mL_min\({}^{-1}\). Injection volume: 25 \(\upmu\)L. Detection: PAD at a glassy carbon electrode. urine sample at 10 \(\upmu\)g/L of each analyte and a standard solution at 100 \(\upmu\)g/L. Considering the 10-fold concentration factor introduced by the SPE purification step, a very good recovery is displayed, as well as a satisfactory separation of ferbutaline and fenoterol from an interference peak, which was present in all blank urine samples. Table 4 displays the recovery values obtained for all the analytes investigated at three spiking levels in the range 0.5-20 \(\upmu\)g/L. The results in the range 78.6-97.2% are in agreement with the reference values indicated by the European Commission for the validation of analytical methods [37]. \begin{table}
\begin{tabular}{c c c} \hline \hline \(\upmu\)**S-Agnist** & **Spiked Level** & **Mean Recovery \(\pm\) SD 1 ** \\ \cline{2-3}  & \(\upmu\)g.L\({}^{-1}\) & \% \\ \hline  & 0.5 & 79.1 \(\pm\) 9.5 \\ TERBUTALINE & 5.0 & 81.5 \(\pm\) 8.6 \\  & 20.0 & 95.3 \(\pm\) 9.5 \\ \hline  & 0.5 & 83.5 \(\pm\) 10.0 \\ FENOTEROL & 5.0 & 90.5 \(\pm\) 9.1 \\  & 20.0 & 97.2 \(\pm\) 8.9 \\ \hline  & 0.5 & 78.6 \(\pm\) 8.6 \\ CLENBUTEROL & 5.0 & 92.1 \(\pm\) 9.2 \\  & 20.0 & 93.4 \(\pm\) 8.4 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Recoveries from urine calf samples spiked with \(\upmu\)-Agonists at three different levels. \begin{table}
\begin{tabular}{l c c c c c c} \hline \hline \multirow{2}{*}{\(\upbeta\)-Agnist} & **Sensitivity** & **iLOD 1 ** & **iLQ 1 ** & **Linear Range** & **R** & **mLOD 2 ** & **mLOQ 2 ** \\ \cline{2-3}  & \(\upmu\)C/ppb** & & \(\upmu\)g/L & & & \(\upmu\)g/L \\ \hline TERBUTALINE & 3.2 \(\times\) 10\({}^{-4}\) & 0.23 & 0.50 & 0.5–100 & 0.9984 & 0.31 & 0.64 \\ FENOTEROL & 1.7 \(\times\) 10\({}^{-4}\) & 0.14 & 0.22 & 0.5–100 & 0.9984 & 0.19 & 0.38 \\ CLENBUTEROL & 4.9 \(\times\) 10\({}^{-5}\) & 0.10 & 0.20 & 0.5–100 & 0.9993 & 0.12 & 0.36 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Performance parameters of \(\upbeta\)-Agonists analyzed by LC-PAD at GCE. \begin{table}
\begin{tabular}{c c c} \hline \hline \multirow{2}{*}{**Parameter**} & \multirow{2}{*}{\(\upbeta\)**-Agnist**} & \multicolumn{2}{c}{**Detection Method**} \\ \cline{3-4}  & & **PAD** & **DC** \\ \hline  & TERBUTALINE & 340 \(\pm\) 200 & 340 \(\pm\) 250 \\ S/N 1  & FENOTEROL & 310 \(\pm\) 200 & 280 \(\pm\) 200 \\  & CLENBUTEROL & 62 \(\pm\) 37 & 95 \(\pm\) 63 \\ \hline  & TERBUTALINE & 7.0\% & 13.0\% \\ RSD 2  & FENOTEROL & 3.6\% & 14.0\% \\  & CLENBUTEROL & 4.0\% & 17.9\% \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Signal to noise ratio (S/N) and relative standard deviation of \(\upbeta\)-Agonists evaluated for PAD and DC detections in LC at GCE. The applicability and usefulness of the proposed method has been demonstrated by the analysis of blank and contaminated calf urine samples. Quantification was performed with the standard addition method. The quantitative determinations have been compared with the results obtained by the ELISA method (see Table 5), confirming the feasibility of the RP-LC/PAD method for quantitative screening purposes.