UGT1A9 QC Assays
Substrate: 4-methylumbelliferone (4-MU)
Incubation conditions
Assay buffer: 0.1 M Tris-HCl pH 7.5 (at 37°C), 5 mM MgCl2, 10 µg/ml alamethicin (for kinetics only)
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 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: 15.0 min
Injection volume: 10 µl
Detection: fluorescence λ(ex) = 315 nm, λ(em) = 375 nm
Retention times:
4-MU glucuronide 3.8 min
4-methylumbelliferone 8.9 min
Substrate: Propofol
Incubation conditions
Assay buffer: 0.1 M Tris-HCl pH 7.5 (at 37°C), 5 mM MgCl2, 10 µg/ml alamethicin (for kinetics only)
Incubation volume: 200 µl (time/protein linearity) or 1000 µl (kinetics)
Stop reagent: 70% perchloric acid (10 µl for time/protein linearity; 50 µl for kinetics)
HPLC conditions
Column: HyperClone ODS (5 µm) 250 x 4 mm
Temperature: 30°C
Mobile phase: Water/Acetonitrile/50 mM perchloric acid, 50/30/20 (v/v/v) (initial)
Separation type: Gradient (acetonitrile) [0 min 30%; 5.5 min 57.5%; 6 min 80%; hold at 80% to 8 min]
Flow rate: 1.0 ml/min
Run time: 20 min
Injection volume: 100 µl
Detection: UV, λ = 200 nm
Retention times:
Propofol glucuronide 5.8 min
Propofol 9.9 min
Note: Residual UDP-glucuronic acid in the assay samples can interfere with HPLC quantitation of the propofol glucuronide. Samples are therefore left in the HPLC vials overnight at room temperature prior to injection to allow the UDP-glucuronic acid to degrade.
