LC–MS/MS assay for the simultaneous quantitation of the ATM inhibitor AZ31 and the ATR inhibitor AZD6738 in mouse plasma

Abstract

The ATM kinase inhibitor AZ31 and ATR kinase inhibitor AZD6738 are in various phases of preclinical and clinical evaluation for their ability to potentiate chemoradiation. To support the preclinical evaluation of their pharmacokinetics, we developed and validated an LC–MS/MS assay for the simultaneous quantification of AZ31 and AZD6738 in mouse plasma. A “dilute and shoot” method was used to precipitate proteins from a sample volume of 50μL. Chromatographic separation was achieved using a Phenomenex Polar-RP column and a gradient mobile phase consisting of methanol–water with 0.1% formic acid. Detection was accomplished using a Waters Quattro Micro mass spectrometer in positive ionization mode. The assay utilizing 50μL sample was linear from 10 to 5000ng/mL and determined to be both accurate (−8.2 to 8.6%) and precise (<5.4% CV) and achieved the criteria for U.S. FDA guidance for bioanalytical method validation. Quantification was achieved in mouse tissue homogenate using a separate 200μL sample preparation. This LC–MS/MS assay will be essential for determining the tissue distribution and pharmacokinetics in future mouse studies.