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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, λ = 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), λ = 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.

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