Abstract
We describe a selective and highly sensitive high-performance liquid chromatography-electrospray ionization-collision induced dissociation-tandem mass spectrometry (HPLC-ESI-CID-MS/MS) assay for the pan-antiapoptotic Bcl-2 family inhibitor, obatoclax, in human plasma. Fifty μL plasma specimens were prepared by addition of extraction solution consisting of Mobile Phase A (10mM ammonium formate (aq) titrated to pH of 3.0 with formic acid): Mobile Phase B (100% methanol) (20:80, v,v) and internal standard followed by centrifugation. HPLC separations were performed on a Waters, YMC-Pack™, ODS-AQ™ S-3 analytical column with LC mobile phase A and B. Linearity and sensitivity was assessed over a linear range of 0.04-25ng/ml at eleven concentrations. The lower limit of quantification for obatoclax was 0.04ng/mL. The intra-day precision based on the standard deviation of replicates of quality control (QC) samples ranged from 0.9 to 5.1% and the accuracy ranged from 98.9 to 106.8%. Stability studies performed replicate sets of QC samples (0.1ng/mL, 2.5ng/mL, and 15ng/mL) showed that obatoclax in human plasma was stable at room temperature for 24h as well as at -80°C for 1m and 2y. Stability was also demonstrated after 3 freeze/thaw cycles (RT to -80°C). The analytical method showed excellent sensitivity, precision, and accuracy. This method is robust and has been successfully employed in a Children's Oncology Group Phase 1 Consortium study of obatoclax in children with cancer.