3.3. Solid-Phase Extraction (SPE) Procedure

Overall solid-phase extraction procedures were performed according to manufacturer’s instruction with slight modification. Two hundred microliter of collected plasma were transferred into 2 mL centrifuge tube, followed by addition of 5 µL of internal standard (IS) solutions (1 µg/mL). Then, plasma samples were diluted with 800 µL of 0.2 M acetate buffer at pH 5.2. SPE cartridges filled with C18 sorbent (Agilent, Santa Clara, CA, USA), which were sequentially pre-conditioned with 1 mL of MeOH and 1 mL of DW. After sample loading, the SPE cartridges were washed with 1 mL of DW, followed by dryness under vacuum (–30 kPa). Finally, all target steroids were eluted from dried SPE sorbents using 1 mL of EA.