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Cypex 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,
l(ex) = 270 nm; l(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,
l(ex) = 252 nm; l(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.

 

Details of a plate reader assay for CYP2D6 activity using MAMC 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.

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