In Vitro Cytochrome P450 Assay

Potassium phosphate buffer (200 µl, 0.1 M, pH 7.4) containing 1 mM NADPH, 0.5 mg/mL human liver microsomes, and a cocktail assay of seven probe substrates (Phenacetin for CYP1A2, Paclitaxel for CYP2C8, Tolbutamide for CYP2C9, Omeprazole for CYP2C19, Dextromethorphan for CYP2D6, Chlorzoxazone for CYP2E1, Dextromethorphan and Testosterone for CYP3A) or a single substrate (≤Km) were incubated at 37°C for 15 min (Supplementary Table S1). The contents of organic solvent and DMSO in incubation mixture was under 1% (v/v) and 0.1% (v/v) respectively. Reactions were terminated by adding 200 µL of an ice-cold stop solution consisting of methanol containing Rutin (2 µg/ml) as internal standard. Samples were subsequently cooled in ice bath to precipitate proteins. Supernatants were collected into clean tubes after centrifugation at 12000 rpm at 4°C for 10 min prior to inject into LC-MS/MS.