Substrate: Trifluoperazine (N-glucuronide formation)
Incubation conditions
Assay buffer: 0.1 M Tris-HCl pH 7.5 (at 37°C), 5 mM MgCl2
Incubation volume: 200 µl (time/protein linearity) or 1000 µl (kinetics)
Stop reagent: 1 M hydrochloric acid (20 µl for time/protein linearity; 100 µl for kinetics)
HPLC conditions
Column: HyperClone BDS-C18 (3 µm) 100 x 4.6 mm
Temperature: 30°C
Mobile phase: Water/acetonitrile/0.5% trifluoroacetic acid, 22/38/40 (v/v/v)
Separation type: Isocratic
Flow rate: 1.0 ml/min
Run time: 7.0 min
Injection volume: 100 µl
Detection: fluorescence λ(ex) = 264 nm, λ(em) = 476 nm
Retention times:
Trifluoperazine N-glucuronide 3.6 min
Trifluoperazine 4.5 min