5α-reductase inhibition assay

DPC were seeded at a cell density of 1 × 10⁵ cells/ml onto 96-well plates (100 μl of 10,000 cells/well). After 24 h, the cells were separately treated (in a total volume of 200 μl) with the final concentrations of testosterone (18 μg/ml), test compounds (18 μM) and <1% Methanol or 1% DMSO as the negative control. After 48 h, the culture medium of each treatment was collected in Eppendorf tubes, and the attached cells were lysed using 200 μL of 0.1 N sodium hydroxide (NaOH) in each well. The cell lysates were transferred into the respective Eppendorf tubes (containing the culture medium) and extracted with the 1 mL of ethyl acetate twice with a brief vortex to ensure homogeneity between each extraction for 30 s. The ethyl acetate layer was then evaporated to dryness and reconstituted using 100 μL of methanol. The internal standard (IS) chosen for this assay was mesterolone. Samples were then injected into LC-MS/MS for subsequent analysis.

Liquid chromatography was performed on a Agilent 1200 series liquid chromatography (Germany). The separation was carried on a Agilent ZORBAX Eclipse Plus C18 column (3.0 × 150 mm, 5 μm) protected with a Zorbax-SB C18 guard column maintained at room temperature. The mobile phase composition was 75% of methanol (with 0.1% formic acid) and 25% of water (with 0.1% formic acid) in isocratic mode at a flow rate of 0.5 ml/min. The injection volume was 10 μl. An Agilent 6400 series triple quadrupole mass spectrometer equipped with Turbo Ion Spray interface operating in the positive electrospray ionization (ESI) mode was used for the detection. Operating conditions optimized by Mass Hunter optimizer for DHT and IS were: dry gas temperature 350°C; nebulizer pressure 35 psi; nitrogen gas flow rate 10 ml/min; capillary voltage 4,000 V and fragmentor voltage 130 V for DHT and 140 V for IS. Product ions (of DHT) resulting from transition of 291.2 → 255.2 (collision energy 12 eV) and product ions (of IS) 305.2 → 93.1 (collision energy 36 eV) were monitored at retention time of 5.5 min (for DHT) and 6.3 min (for IS).