The drug concentration was quantified by rat plasma using high-performance liquid chromatography–tandem mass spectroscopy (LC/MS/MS). Standard stock solutions of PHA666859 were prepared in acetonitrile and diluted to the working standard solutions (in acetonitrile). The internal standard used was buspirone, which was prepared in methanol (50 ng/mL). All standard and QC stocks were stored at −20°C.
Proteins were precipitated with acetonitrile/methanol (4:1, vol:vol) containing 0.1% formic acid. The LC-MS/MS system consisted of pumps (model LC10AD; Shimadzu, Columbia, MD), with an autosampler (CTC-PAL; LEAP Technologies, Carrboro, NC) and a cool stack and triple quadrupole mass spectrometer (Sciex API 4000; Applied Biosystems, Inc. [ABI], Foster City, CA). A gradient elution method was used to perform chromatography on a reversed-phase column (XBridge C18, 3.5 μm, 50 × 2.1 mm; Waters, Waltham, MA) at a flow rate of 500 μL/min (mobile phase A consisted of 100% HPLC grade water with 0.1% formic acid, and mobile phase B contained 100% acetonitrile with 0.1% formic acid). The mass spectrometer was operated under the following conditions: positive ion turbo-ionspray mode, ionspray potential, 5.0 kV; interface temperature, 400°C; curtain gas, 20; CAD gas, 6; GS1, 70; and GS2, 30. The conditions for PHA666859 were as follows: MS/MS transition, m/z 416.16→340.94; declustering potential, 91; and collision energy, 41. The conditions for buspirone (internal standard) were as follows: MS/MS transition, m/z 386.3→122.1; declustering potential, 80; and collision energy, 40. Peak areas were then determined (Analyst software, ver. 1.4.1; ABI). Quantitation was performed by linear regression with a 1/x 2 weighting. The lower limit of quantitation for the assay was 1 ng/mL. The upper limit of quantitation for the assay was 5000 ng/mL.