Cypex CYP2C18 QC Assays

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, l = 280 nm
Retention times:
      4'-hydroxydiclofenac          4.9 min
      diclofenac                       12.0 min

The metabolite, 4'-hydroxydiclofenac is available from Cypex Ltd

Effect of phosphate concentration on diclofenac turnover by CYP2C18LR Bactosomes
Diclofenac 4'-hydroxylase activity is inhibited at phosphate concentrations above 50 mM.

This assay was carried out at 200 µM diclofenac at a P450 concentration of 5 pmol per 200 µl.

Made under licence from BTG International Ltd (AU730155, EP0914446, US6566108 and other patents pending).

United States Patent Nos. 5,420,027 or 5,240,831, Canada Patent No. 2100245 and other patents pending.