Cypex CYP2B6 QC Assays

Activity: Diazepam N-demethylase

Incubation conditions
Assay buffer: 0.05 M potassium phosphate pH 7.4, 5 mM MgCl₂
Incubation volume: 200 µl
Stop reagent: acetonitrile (100 µl)
Internal standard: none

HPLC conditions
Column: Hypersil BDS-C18 (3 µm) 100 x 4 mm
Temperature: 30°C
Mobile phase: 100 mM potassium phosphate pH 2/acetonitrile 74/26 (v/v) (isocratic)
Flow rate: 1.0 ml/min
Run time: 11.5 min
Injection volume: 80 µl
Detection: UV, l = 236 nm
Retention times:
      N-desmethyldiazepam       4.3 min
      diazepam                         9.0 min

Activity: Bupropion 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: none

HPLC conditions
Column: HyperClone BDS-C18 (3 µm) 125 x 4 mm
Temperature: 30°C
Mobile phase: 100 mM potassium phosphate pH 2/acetonitrile/water 20/16/64 (v/v/v) (isocratic)
Flow rate: 1.0 ml/min
Run time: 17.5 min
Injection volume: 100 µl
Detection: UV, l = 196 nm
Retention times:
      hydroxybupropion         6.3 min
      bupropion                   12.4 min

Activity: 7-ethoxy-4-trifluoromethylcoumarin (7EFC) O-deethylase

Incubation conditions
Assay buffer: 0.1 M potassium phosphate pH 7.4, 5 mM MgCl₂
Incubation volume: 200 µl
Stop reagent: 1 M hydrochloric acid (25 µl)
Internal standard: none

HPLC conditions
Column: Hypersil ODS (5 µm) 250 x 4 mm
Temperature: 35°C
Mobile phase: 0.05% orthophosphoric acid/methanol 35/65 v/v (isocratic)
Flow rate: 1.0 ml/min
Run time: 12.8 min
Injection volume: 50 µl
Detection: fluorescence l(ex) = 324 nm, l(em) = 458 nm
Retention times:
       7-hydroxy-4-trifluoromethylcoumarin        4.8 min
     7-ethoxy-4-trifluoromethylcoumarin         10.3 min

Effect of phosphate concentration on 7EFC turnover by CYP2B6LR Bactosomes
7EFC O-deethylation by CYP2B6 is relatively insensitive to the concentration of phosphate in the incubation.

This assay was carried out at 20 µM 7EFC at a P450 concentration of 2 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.