Cypex CYP2C19 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 76/24 (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

Effect of phosphate concentration on diazepam turnover by CYP2C19R Bactosomes
Diazepam N-demethylase activity is inhibited at phosphate concentrations above 100 mM.

This assay was carried out at 12.5 µM diazepam at a P450 concentration of 1.25 pmol per 200 µl.

Activity: S-mephenytoin 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)
Internal standard: none

HPLC conditions
Column: HyperClone BDS-C18 (3 µm) 125 x 4 mm
Temperature: 40°C
Mobile phase: 100 mM potassium phosphate pH 2/acetonitrile/water 70/7/23 (v/v/v)
Step to 100 mM potassium phosphate pH 2/acetonitrile/water 70/29/1 (v/v/v)
Flow rate: 1.2 ml/min
Run time: 10.5 min
Injection volume: 50 µl
Detection: UV (DAD), l = 197 nm
Retention times:
      4'-hydroxymephenytoin      3.0 min
      S-mephenytoin                  4.7 min

Effect of phosphate concentration on S-mephenytoin turnover by CYP2C19R Bactosomes
S-mephenytoin 4'-hydroxylase activity is inhibited at phosphate concentrations above 100 mM.

This assay was carried out at 30 µM S-mephenytoin at a P450 concentration of 10 pmol per 200 µl.

Effect of phosphate concentration on S-mephenytoin turnover by CYP2C19LR Bactosomes
S-mephenytoin 4'-hydroxylase activity is inhibited at phosphate concentrations above 100 mM.

This assay was carried out at 30 µM S-mephenytoin at a P450 concentration of 25 pmol per 200 µl.

Details of a plate reader assay for CYP2C19 activity using OMF 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.