Micromass quattro micro mass spectrometer

Urine was collected from restrained C57BL/6 mice 2 weeks after i.v. injection of B16F10 and supplemented with indomethacin (10 μg/ml in DMSO; Sigma-Aldrich). Urine was centrifuged at 10,000 g for 15 minutes at 4°C. Five microliters of urine was mixed with 50 μl 6-methoxysalicylate (internal standard, 10 μM) and 1 ml formic acid (10 mM). To measure salicyluric acid (SUA), a 5-μl sample was injected onto the HPLC (Waters 2695) equipped with a Micromass Quattro Micro Mass spectrometer (Waters). Separation was achieved using a Kinetex XB (2.6 μm, 2.1 × 50 mm) column maintained at 35°C with eluent A (10 mM formic acid) and eluent B (acetonitrile), using a flow rate of 0.25 ml/min and a gradient of 8%–50% B over 4 minutes. SUA was detected using electrospray in negative mode with tandem mass spectrometry with a capillary voltage of 1.2 V at 194–150 (cone voltage 20 V) and internal standard at 166.9–123 (cone voltage 20 V). Concentrations were calibrated against SUA [N-(2-hydroxybenzoyl)glycine, Apollo Scientific].

A mixture of acenocoumarol (1 μM) and liver microsomes (2 mg protein/ml) from wild-type (WT), hepatic P450 reductase–null (HRN), Cyp2c KO, Cyp2c/Cyp2d/Cyp3a KO, or CYP2C9 humanized mice in phosphate buffer (100 mM KH₂PO₄ pH7.4, 3.3 mM MgCl₂) was incubated in a water bath for 5 minutes at 37°C prior to the start of the reaction by addition of NADPH regenerating system (final concentrations: 1.3 mM NADPH, 4 mM glucose-6-phosphate, and 2 IU/ml glucose-6-phosphate dehydrogenase). The reaction mixture aliquots were taken at specified time points and mixed with an equal volume of acetonitrile containing warfarin (50 ng/ml) as an internal standard. The mixture was placed on ice for at least 20 minutes, centrifuged at 16.1 krcf for 15 minutes at +4°C, and 100 μl of the supernatant was transferred to a 96-well plate for analysis. The concentrations of S-acenocoumarol were measured by LC-MS/MS. Chromatographic separation was performed on a Kinetex C18 100 Å column (2.6 μm; 5.0 × 2.1 mm) (Phenomenex) using an injection volume of 10 μl and a run time of 7 minutes. The detector used was a Micromass Quattro Micro mass spectrometer (Waters Corporation) run in electrospray positive ion mode. The multiple reaction monitoring parameters for S-acenocoumarol and warfarin were 354.06 and 309.1 (parent ions) and 163.08 and 251.15 (collision ions), respectively.