Activity: Progesterone 17α-hydroxylase
Incubation conditions
Assay buffer: 0.05 M potassium phosphate pH 7.4, 5 mM MgCl2
Incubation volume: 200 µl
Stop reagent: 1 M hydrochloric acid (20 µl)
HPLC conditions
Column: Hypersil ODS (5 µm) 250 x 4 mm
Temperature: 40°C
Mobile phase: 0.05% orthophosphoric acid/methanol/acetonitrile; 25/70/5 (v/v/v) (isocratic separation)
Flow rate: 1.0 ml/min
Run time: 9.5 min
Injection volume: 100 µl
Detection: UV, λ = 240 nm
Retention times:
17α-hydroxyprogesterone 4.1 min
Progesterone 6.1 min