## 3 Results and Discussion
### Method Performance and Application
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline \multirow{2}{*}{**Parameters**} & \multicolumn{3}{c}{**Water**} & \multicolumn{3}{c}{**Urine**} \\ \cline{2-7}  & **BES** & **TES** & **PRO** & **BES** & **TES** & **PRO** \\ \hline Calibration curve & PA = 0.5917x \(-\) & PA = 1.2458x \(+\) & PA = 1.7164x \(-\) & PA = 0.5779x \(-\) & PA = 1.2149x \(+\) & PA = 1.6629x \(-\) \\ Correlation & 0.1226 & 0.0538 & 0.3684 & 0.1481 & 0.0359 & 0.3086 \\ coefficient (R\({}^{2}\)) & 0.999 & 0.997 & 0.998 & 0.995 & 0.996 & 0.994 \\ Standard & 1.07–3.44 & 2.13–3.42 & 1.54–4.08 & 1.52–3.81 & 2.76–3.90 & 1.82–5.13 \\ deviation, n = 3 & 1.07–3.04 & 1.90–3.67 & 1.43–4.21 & 3.51–4.32 & 2.22–4.46 & 2.13–5.09 \\ Inter precision (RSD\%, n = 5) & 2.71–3.04 & 2.71–5.83 & 1.98–5.11 & 2.71–7.11 & 3.06–6.21 & 2.78–6.53 \\ LOD (ug L\({}^{-1}\)) & 0.269 & 0.337 & 0.171 & 0.407 & 0.401 & 0.278 \\ LOQ (ug L\({}^{-1}\)) & 0.899 & 1.124 & 0.573 & 1.357 & 1.337 & 0.929 \\ Linear range (ug L\({}^{-1}\)) & 0.899–10\({}^{4}\) & 1.124–10\({}^{4}\) & 0.573–10\({}^{4}\) & 1.357–10\({}^{4}\) & 1.337–10\({}^{4}\) & 0.929–10\({}^{4}\) \\ \hline \hline \end{tabular}  
* A 5-point calibration line was estimated by regressing the peak area (PA) against drug concentration for each solute.  
\end{table} 
Table 4: Validation parameters * of HDE-SBME-HPLC-DAD method for selected steroids spiked in water and steroid-free urine samples. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Matrix**} & \multirow{2}{*}{**Level**} & **Spiked** & \multicolumn{2}{c}{**Mean Extracted**} & \multirow{2}{*}{**Accuracy**} & \multirow{2}{*}{**Recovery**} \\  & **Concentration** & & & **Concentration** \\  & **(ug L\({}^{-1}\), n = 3)** & & & **(ug L\({}^{-1}\), n = 3)** & **RE \%** \\ \hline \multirow{8}{*}{Water} & \multirow{8}{*}{2} & BES & 698.2 & \(-\)4.68 & 95.32 \\  & & 10 & TES & 880.3 & \(-\)2.58 & 97.41 \\  & & & PRO & 852.9 & \(-\)2.98 & 97.01 \\ \cline{2-6}  & & & BES & 704.3 & \(-\)3.84 & 96.16 \\  & & & TES & 890.9 & \(-\)1.43 & 98.58 \\  & & & PRO & 862.2 & \(-\)1.93 & 98.06 \\ \cline{2-6}  & & & BES & 687.8 & \(-\)6.09 & 93.91 \\  & & & TES & 876.6 & \(-\)2.99 & 97.00 \\  & & & PRO & 839.0 & \(-\)4.57 & 95.43 \\ \hline \multirow{8}{*}{Steroids-free urine} & \multirow{8}{*}{2} & BES & 675.6 & \(-\)7.75 & 92.24 \\  & & & TES & 852.3 & \(-\)5.68 & 94.31 \\ \cline{1-1}  & & & PRO & 814.2 & \(-\)7.38 & 92.61 \\ \cline{1-1} \cline{2-6}  & & & BES & 688.8 & \(-\)5.95 & 94.04 \\ \cline{1-1}  & & & TES & 862.4 & \(-\)4.57 & 95.42 \\ \cline{1-1}  & & & PRO & 828.7 & \(-\)5.73 & 94.26 \\ \cline{1-1} \cline{2-6}  & & & BES & 673.1 & \(-\)8.09 & 91.90 \\ \cline{1-1} \cline{2-6}  & & & TES & 845.1 & \(-\)6.48 & 93.51 \\ \cline{1-1}  & & & PRO & 806.2 & \(-\)8.29 & 91.70 \\ \hline \hline \end{tabular}  
* The pH = 7.20, extraction time 32 min, volume of methanol for elution 210 \(\mu\)L and ultrasonicated for 6 min. 2: The pH = 7.00, extraction time 30 min, volume of methanol for elution 200 \(\mu\)L and ultrasonicated for 5 min. 3: The pH = 6.80, extraction time 28 min, volume of methanol for elution 190 \(\mu\)L and ultrasonicated for 4 min. RE: Relative error.  
\end{table} 
Table 5: Robustness of the HDE-SBME-HPLC-DAD method in spiked water and steroids-free urine matrices with three operation levels. of gestation. The BES and PRO were detected in the pregnant woman's urine sample at 11.2 \(\upmu\)g L\({}^{-1}\) (RSD 4.31%) and 5.31 (RSD 5.22%) \(\upmu\)g L\({}^{-1}\), respectively. Only TES 1.79 \(\upmu\)g L\({}^{-1}\) (RSD 3.81%) was detected in a urine sample from a young healthy man. Figure 7: _Cont._