Either 5 or 10 μL of the supernatant was injected for tissue or plasma samples, respectively. Calibration curves and QC samples were prepared

in both brain and liver, for tissue see more sample analysis. The Selleck Belnacasan working ranges for liver and brain were 0.125–100 and 0.125–25 ng/mL, respectively. Equipment High performance liquid chromatography was carried out on an Agilent 1100 system (Agilent Technology, Palo Alto, CA), coupled with a single-quadrupole mass spectrometer, utilizing electrospray ionization in positive mode. Samples were cooled to 4°C in a thermostated autosampler and the column compartment, containing a Waters SymmetryShield RP8 column (2.1 × 50 mm, 3.5 μm), was maintained at 35°C. Samples were eluted using a gradient mobile phase, comprised of 10 mM ammonium acetate with 0.1% formic acid and methanol, running at a flow rate of 0.35 mL/min for 10 min, including re-equilibration. Mass spectrometric conditions were as follows: fragmentor, 150 V; gain, 2; drying gas flow, 10 L/min; drying gas temperature, 300°C; nebulizer pressure, 40 Ipatasertib in vitro psi; and capillary voltage, 1500 V. Selected-ion monitoring

was accomplished at m/z 494.2 for imatinib and m/z 213.1 for the internal standard. The chromatographic data were acquired and analyzed using the Chemstation software package (Agilent). Validation procedures Calculation of accuracy and precision was carried out according to procedures reported in detail previously [17]. Calibration samples were prepared fresh each

day in the relevant matrix and frozen QC samples were defrosted and analyzed. A 1/x2 weighting scheme was employed in the generation of standard curves to account for concentration dependent variance. Detector response for plasma was found to be linear in the imatinib concentration range of 10–1000 ng/mL. Plasma accuracy and precision were evaluated with QC samples. Overall, the assay was found to be accurate (deviation of less than 10% for QCs) and precise (within run precision <10%, between run precision <12.6%) for plasma, liver, and brain. Animals All experiments were performed on six-week old, male, SSR128129E Balb/C mice obtained from Charles River Laboratories (Wilmington, MA). The mice weighed approximately 15 to 20 g at the time of study. All mice were allowed unlimited access to water and rodent chow prior to, and during the experiment. Blank mouse liver and brain samples were harvested from surplus mice following euthanasia. NCI-Frederick is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the “”Guide for Care and Use of Laboratory Animals”" (National Research Council; 1996; National Academy Press; Washington, DC). The study design and protocol were approved by the NCI Animal Care and Use Committee (Bethesda, MD).