2.4 Analytical methods
The sample from the PS bioconversion (100 μL) was extracted with ten times volume of ethyl acetate by shaking for 3 min. The mixture was centrifuged at 13,000 rpm for 3 min, and 100 μL of supernatant was withdrawn and evaporated, and then redissolved with five times volume of ethanol. Steroid metabolites were quantitatively analyzed by high performance liquid chromatography (Agilent Tech, USA) equipped with the Elit Hypersil ODS2-C18 (4.6 mm×250 mm, 5 μm) column using the mobile phase (methanol/water, 80:20, v/v) at a flow rate of 1 mL/min and the detection wavelength was 254 nm.