## 3 Results and Discussion
### Method Performance and Application  
In order to assess the analytical efficacy of the proposed methodology, several significant parameters were tested, namely the linear dynamic range, correlation coefficient, limit of detection (LOD), limit of quantification (LOQ), reproducibility, recovery, and robustness. The insignificance of the matrix effects of groundwater and tap water is evident from the findings presented in Table S5. Consequently, the examination of selected steroids spiked in ultrapure water and steroids-free urine samples was carried out to generate calibration curves and analyze the validation parameters of the developed method. As summarized in Table 4, the linear dynamic ranges of BES, TES, and PRO in water/steroid-free urine are 0.899-10\({}^{4}\)/1.357-10\({}^{4}\), 1.124-10\({}^{4}\)/1.337-10\({}^{4}\), and 0.573-10\({}^{4}\)/0.929-10\({}^{4}\) ug L\({}^{-1}\), respectively. The correlation coefficients of determination (R2) exceeded 0.994 for all steroids in both tested matrixes, indicating a robust linear relationship between concentration and response. The LOD/LOQ values were calculated to be 0.269/0.899 \(\upmu\)L\({}^{-1}\) for BES, 0.337/1.124 \(\upmu\)g L\({}^{-1}\) for TES, and 0.171/0.573 \(\upmu\)g L\({}^{-1}\) for PRO in spiked water samples. In spiked steroid-free urine samples, the LOD/LOQ were 0.407/1.357 \(\upmu\)g L\({}^{-1}\), 0.401/1.337 \(\upmu\)g L\({}^{-1}\), and 0.278/0.929 \(\upmu\)g L\({}^{-1}\) for BES, TES, and PRO, respectively. Table 4 shows good repeatability and reproducibility with RSDs ranging from 1.43% to 5.09% and 1.98% to 7.11%, respectively. The robustness results presented in Table 5 revealed slight discrepancies in the collected data. They confirmed that the test conditions did not significantly affect the results of the HDE-SBME-HPLC-DAD analysis. The recovery yields of the selected steroids exceeded 91.7% in water and steroid-free urine matrixes. Figure 6: RSM plots showing the effect of selected steroid hormones EE% in process variables on (**A**) the number of HDE-SBME devices and time of extraction, (**B**) the number of HDE-SBME devices and volume of elution, and (**C**) the number of HDE-SBME devices and elution time. After the optimization and validation conditions were established, tests were performed on actual samples to demonstrate the practical application of the current methodology. For this purpose, the developed HDE-SBME-HPLC-DAD method was applied to measure the selected steroids in urine samples and real water samples such as groundwater and tap water. The lack of significant interference in the retention times of selected steroids in both the real water and urine matrices, as shown in Figure 7, indicates that the developed method does not interfere with other pre-existing components and can be effectively identified and measured from the selected analytes independently. The presence of steroid hormones was not discerned in any water samples. Furthermore, the method's efficacy was assessed on biological samples, specifically urine samples. Two urine samples were procured in the morning from a healthy young boy and a woman in the fifth month