Cypex CYP2A13 QC Assays

Activity: Coumarin 7-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)
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 75/25 (v/v) (isocratic separation)
Flow rate: 1.0 ml/min
Run time: 12.5 min
Injection volume: 50 µl
Detection: fluorescence l(ex) = 324 nm, l(em) = 458 nm
Retention times:
       umbelliferone                5.2 min
     4-methylumbelliferone    7.2 min
     coumarin                      10.1 min

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.