Rapid and sensitive LC/MS/MS analysis of the novel tyrosine kinase inhibitor ZD6474 in mouse plasma and tissues

ZD6474 (N-(4-Bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy] quinazolin-4-amine) is a tyrosine kinase inhibitor with anti-angiogenic and anti-tumor activity that is currently undergoing human trials for cancer treatment. Pharmacokinetic studies in animal models are an important component in clinical development of this agent to relate pre-clinical models to patient treatment. A liquid chromatography tandem mass spectrometry method was developed for the determination of ZD6474 levels in mouse plasma and tissues. Plasma (0.05 mL) and tissue homogenates (0.1 mL of 10 mg/mL) were extracted under alkaline conditions with ethyl acetate:pentane (1:1, v/v) after addition of the internal standard (trazodone, 2-[3-[4-(3-chlorophenyl)-1-piperazinyl]propyl]-1,2,4-triazolo[4,3-a]pyridine-3(2H)-one). Separation was achieved on a C18, 50 mm × 2 mm column with quantitation by internal standard reference and multiple reaction monitoring of the ion transitions m/z 475 → 112 (ZD6474) and m/z 372 → 176 (trazodone). The calibration curve was linear from a range spanning 20-20,000 ng/mL in plasma and 10-320 ng/mg in tissue homogenates. Mean recoveries from plasma and tissue homogenates were 88 and 90%, respectively. The accuracy in plasma was 88% at the lower limit of quantitation (20 ng/mL with a 50 μL plasma sample) with high precision (R.S.D.% < 10%). Assay performance in liver and other tissue homogenates is also reported. The assay was applied to a pharmacokinetic study in mice to determine dosing schedules that would approximate therapeutic ZD6474 levels determined in humans.