Activity: Diclofenac 4′-hydroxylase
Incubation conditions
Assay buffer: 0.05 M potassium phosphate pH 7.4, 5 mM MgCl2
Incubation volume: 200 µl
Stop reagent: methanol (200 µl)
HPLC conditions
Column: Hypersil ODS (5 µm) 250 x 4 mm
Temperature: 30°C
Mobile phase: 20 mM potassium phosphate pH 7/methanol/acetonitrile 60/22.5/17.5 (v/v/v) (isocratic)
Flow rate: 1.0 ml/min
Run time: 13.5 min
Injection volume: 100 µl
Detection: UV, λ = 280 nm
Retention times:
4′-hydroxydiclofenac 4.9 min
diclofenac 12.0 min
The metabolite, 4′-hydroxydiclofenac is available from Cypex Ltd