## 2 Results and Discussion
### Optimization of Extraction Conditions  
To achieve the highest sensitivity, some important factors affecting the extraction efficiency were studied, including the sampling volume, sampling rate, acetonitrile content in the sample, and desorption time [39]. When the peak area tends to be constant with the increase in the sample volume, the extraction equilibrium and the largest extraction efficiency will be obtained. However, an excessive sampling volume consumes more extraction and desorption time. As shown in Figure 7a, the peak areas of the analytes, especially for hekestrol and diethylslitbestrol, increased with the change of the sampling volume from 30 to 80 mL. The increase in the peak areas was unremarkable for other compounds when the sampling volume was more than 60 mL. Compromising the extraction efficiency and time, 60 mL was selected as the optimal sampling volume. After the same sampling volume of 60 mL was loaded, the sampling rate was investigated in the range of 0.75-2.50 mL/min. It can be seen in Figure 7b that the peak areas of the six analytes changed little when increasing the sampling rate from 0.75 to 2.50 mL/min. While the sampling rate increases beyond 1.00 mL/min, the downward trend occurs for hekestrol and diethylslitbestrol. In order to obtain better extraction efficiency and quick analysis for the six analytes, 2.00 mL/min was selected as the optimal sampling rate for overall consideration. Organic solvents can promote the solubility of hydrophobic analytes in an aqueous sample and improve analysis repeatability. Herein, acetonitrile was selected as the organic solvent to investigate the effect of organic compounds in the sample on the extraction process. The content of acetonitrile was varied from 0 to 4% (_v/v_), and the peak areas of the analytes were investigated. As shown in Figure 7c, except for hekestrol and diethylslitbestrol, the extraction efficiency of the other four analytes decreased with the addition of acetonitrile in the sample. The peak area of diethylslitbestrol has a little increasing trend  
Figure 6: Chromatograms of the target compounds using four kinds of fibers with different surface wettabilities (**Left**) and the comparative peak area for each target compound using 4 kinds of wires for in-tube SPME (**Right**). Symbol identification: (a) pristine wire, (b) superhydrophobic wire, (c) superhydrophobic wire, (d) superhydrophobic wire after UV irradiation. Conditions: sampling volume, 60 mL; sampling rate, 2.00 mL/min; desorption time, 2.0 min. Peak identification: (1) bisphenol A, (2) estradiol, (3) ethylnylestradiol, (4) estrone, (5) diethylslitbestrol, and (6) hekestrol. Mobile phase: Water/acetonitrile (53:47, _v/v_); Detection wavelength, 220 nm, room temperature. from 1 to 2% and then a decreasing trend from 2 to 4%. In order to obtain satisfactory extraction efficiency, no acetonitrile was added to the samples. A full desorption of all extracted analytes can be obtained after adequate desorption time, which thus reduces the impact of residual analytes on the next extraction. After the extraction was completed, acetonitrile-water (53:47) as the mobile phase flowed through the extraction tube at 1.00 mL/min. The analytes absorbed by the sorbent in the extraction tube were eluted and the desorption process was carried out. The effect of desorption time was varied in the range of 0.20-3.00 min. As shown in Figure 7d, the six estrogen peak areas exhibited an upward trend with the increase of the desorption time. Therefore, 2.00 min was selected as the optimal desorption time. Based on the above discussion, the optimal conditions of in tube SPME for estrogens are: 60 mL sampling volume, 2 mL sampling rate, 2 min desorption time at a flow rate of 1 mL/min. Under the optimized extraction conditions, the expected extraction performance of superhydrophilic wire (b) for the target analytes was achieved. As shown in Figure 8, the estrogen analytes were not detected for the direct injection of a sample spiked at 10 \(\upmu\)g/L.