Cypex CYP1A1 QC Assays

Activity: 7-ethoxycoumarin O-deethylase

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)
Internal standard: 4-methylumbelliferone (10 µl of 0.1 µg/ml)

HPLC conditions
Column: Hypersil ODS (5 µm) 250 x 4 mm
Temperature: 30°C
Mobile phase: 0.05% orthophosphoric acid/acetonitrile (gradient separation)
Flow rate: 1.0 ml/min
Run time: 14.5 min
Injection volume: 50 µl
Detection: fluorescence l(ex) = 324 nm, l(em) = 458 nm
Retention times:
       umbelliferone                5.2 min
     4-methylumbelliferone    7.0 min
     7-ethoxycoumarin         10.3 min

Activity: 7-ethoxyresorufin O-deethylase

Incubation conditions
Assay buffer: 0.1 M potassium phosphate pH 7.4, 5 mM MgCl₂
Incubation volume: 200 µl
Stop reagent: methanol (100 µl)
Internal standard: none

HPLC conditions
Column: Hypersil ODS (5 µm) 250 x 4 mm
Temperature: 35°C
Mobile phase: 20 mM potassium phosphate pH 7/methanol 58/42 (v/v) (isocratic)
Flow rate: 1.0 ml/min
Run time: 5.5 min
Injection volume: 75 µl
Detection: fluorescence l(ex) = 530 nm, l(em) = 580 nm
Retention times:
     resorufin                         4.0 min

The substrate 7-ethoxyresorufin and the metabolite resorufin are both available from Cypex Ltd.

Important: By purchasing this product from Cypex you accept the standard terms and conditions of supply, details of which may be provided upon request.

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.