Substrate: Testosterone
Incubation conditions
Assay buffer: 0.1 M Tris-HCl pH 7.5 (at 37°C), 5 mM MgCl2, 10 µg/ml alamethicin (for kinetics only)
Incubation volume: 200 µl (time/protein linearity) or 1000 µl (kinetics)
Stop reagent: 1 M hydrochloric acid (20 µl for time/protein linearity; 100 µl for kinetics)
HPLC conditions
Column: HyperClone BDS-C18 (3 µm) 100 x 4.6 mm
Temperature: 40°C
Mobile phase: Water/Acetonitrile/Methanol/0.05% orthophosphoric acid, 35/15/30/20 (v/v/v/v) (initial)
Separation type: Gradient (acetonitrile) [0.5 min 15%; 3.5 min 45%; hold at 45% to 5.4 min; 5.5 min 15%]
Flow rate: 1.0 ml/min
Run time: 10 min
Injection volume: 80 µl
Detection: UV, λ = 245 nm
Retention times:
Testosterone glucuronide 4.0 min
Testosterone 5.0 min