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withdrawn and evaporated, and then redissolved with five times volume of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.