Optima grade water

Optima grade water + 0.1% formic acid, Optima grade acetonitrile +0.1% formic acid, L-ascorbic acid, hydroquinone, and acetone were purchased from Fisher Scientific (Fair Lawn, NJ). All chemicals were ACS reagent grade or higher. MassPREP™ Digestion Standard Protein Expression Mixture 2 and enolase digestion standard were obtained from Waters Corporation (Milford, MA).

R-warfarin, S-warfarin, racemic warfarin, warfarin metabolites (4’-, 6-, 7-, 8- and 10-OH-warfarin), and warfarin-d₅ (deuterated phenyl ring; used as internal standard [IS]) were purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). Ammonium acetate and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Optima-grade water, acetonitrile, methanol, and acetic acid were obtained from Fisher Scientific (Pittsburgh, PA, USA). Blank human plasma (collected in K₂-EDTA tubes) was purchased from Innovative Research (Novi, MI, USA). Recombinant human CYP3A4 and CYP2C9 Supersomes™, microsomes prepared from baculovirus-infected insect cells expressing human CYP enzymes and NADPH-cytochrome P450 reductase, were purchased from BD Gentest (Woburn, MA, USA).

Sequencing grade Asp-N was purchased from Promega (Madison, WI). Triethylammonium bicarbonate, and iodoacetamide were purchased from Supelco. DTT was from Sigma. Formic acid, optima grade water and acetonitrile are from Fisher Scientific. In-Gel digestion was carried out following manufacturer’s protocol. Briefly, gel was cut into pieces <1 mm³, destained with 50% acetonitrile (ACN) in water, dehydrated with ACN, then rehydrated with 25 mM DTT, incubated at 65 °C for 20 min, washed, dehydrated with ACN. Samples were rehydrated with 50 mM iodoacetamide, incubated at room temperature in the dark for 25 minutes, washed with water, and dehydrated with ACN. Gel was then rehydrated with 10 ng μl⁻¹ Asp-N solution, and incubated overnight at 37 °C. Peptides were extracted twice by sequential addition of 100 μl 50% ACN, 100 μl 20% Formic acid, and 100 μl. Extracts were combined and concentrated with speedvac to dryness.