CYP2D6LR Bactosomes (low reductase)

CYP2D6 QC Assays

Activity: Dextromethorphan O-demethylase

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: Hypersil BDS-C18 (3 µm) 100 x 4 mm
Temperature: 30°C
Mobile phase: 0.05% orthophosphoric acid/acetonitrile 78/22 (v/v) (isocratic)
Flow rate: 1.0 ml/min
Run time: 13.5 min
Injection volume: 50 µl
Detection: fluorescence, λ(ex) = 270 nm; λ(em) = 312 nm

Retention times:
dextrorphan 2.4 min
dextromethorphan 10.9 min

Activity: Bufuralol 1′-hydroxylase

Incubation conditions
Assay buffer: 0.05 M potassium phosphate pH 7.4, 5 mM MgCl₂
Incubation volume: 200 µl
Stop reagent: 70% perchloric acid (10 µl)
Internal standard: none

HPLC conditions
Column: Hypersil BDS-C18 (3 µm) 100 x 4 mm
Temperature: 30°C
Mobile phase: Water/10 mM perchloric acid/acetonitrile, 56/20/24 (v/v/v) (initial)
Run type: gradient (24 – 80% acetonitrile from 2.5 to 3.5 min)
Flow rate: 1.0 ml/min
Run time: 12.5 min
Injection volume: 10 µl
Detection: fluorescence, λ(ex) = 252 nm; λ(em) = 302 nm

Retention times:
1′-hydroxybufuralol 2.8 min
bufuralol 5.4 min

Effect of phosphate concentration on dextromethorphan turnover by CYP2D6R Bactosomes:

Dextromethorphan turnover by CYP2D6 is sensitive to the concentration of phosphate in the incubation.

This assay was carried out at 10 µM dextromethorphan at a P450 concentration of 0.2 pmol per 200 µl.