CYP1A2 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 λ(ex) = 324 nm, λ(em) = 458 nm

Retention times:
umbelliferone 5.2 min
4-methylumbelliferone 7.0 min
7-ethoxycoumarin 10.3 min

Effect of phosphate concentration on 7-ethoxycoumarin turnover by CYP1A2R Bactosomes:
ECOD activity is relatively insensitive to the concentration of phosphate in the incubation.

This assay was carried out at 20 µM 7-ethoxycoumarin at a P450 concentration of 0.5 pmol per 200 µl.

Activity: Phenacetin 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: 3-acetamidophenol (10 µl of 10 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: 18.5 min
Injection volume: 50 µl
Detection: UV, λ = 250 nm

Retention times:
4-acetamidophenol 4.2 min
3-acetamidophenol 6.1 min
phenacetin 12.5 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 λ(ex) = 530 nm, λ(em) = 580 nm

Retention times:
resorufin 4.0 min

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