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Pig CYP2C49 QC Assays

Activity: Tolbutamide 4-hydroxylase

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)

HPLC conditions
Column: HyperClone ODS (5 µm) 250 x 4 mm
Temperature: 30°C
Mobile phase: Water/100 mM potassium phosphate pH 2/methanol/acetonitrile, 50/10/35/5 (v/v/v/v) (initial conditions)
Run type: gradient (5 – 25% acetonitrile over 4 min)
Flow rate: 1.0 ml/min
Run time: 16 min
Injection volume: 100 µl
Detection: UV, λ = 230 nm

Retention times:
4-hydroxytolbutamide 5.9 min
(carboxytolbutamide 6.8 min)
tolbutamide 9.1 min

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