Cypex CYP2C9 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: HyperClone 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 CYP2C9 substrate diclofenac and metabolite, 4'-hydroxydiclofenac are available from Cypex.

Activity: Tolbutamide 4-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: HyperClone ODS (5 µm) 250 x 4 mm
Temperature: 30°C
Mobile phase: Water/100 mM potassium phosphate pH 2/methanol/acetonitrile, 50/10/35/5 (v/v/v/v) (initial conditions)
Run type: gradient (5 - 25% acetonitrile over 4 min)
Flow rate: 1.0 ml/min
Run time: 16 min
Injection volume: 100 µl
Detection: UV, l = 230 nm
Retention times:
      4-hydroxytolbutamide        5.9 min
     (carboxytolbutamide          6.8 min)
      tolbutamide                     9.1 min

Effect of phosphate concentration on diclofenac turnover by CYP2C9HR Bactosomes
Diclofenac 4'-hydroxylase activity is inhibited at phosphate concentrations above 100 mM.

This assay was carried out at 50 µM diclofenac at a P450 concentration of 0.4 pmol per 200 µl.

Details of a plate reader assay for CYP2C9 activity using MFC can be found here.

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.