Development and validation of a UPLC–MS/MS method for the novel folate-targeted small molecule drug conjugate EC1456 and its metabolites in tumor homogenates from mice

Product ion spectra of EC1456 and its metabolites tubulysin B hydrazide and tubulysin B. [Display omitted] •A validated LC–MS/MS method for measuring tumor levels of EC1456 and metabolites.•Precision, accuracy, stability, and selectivity determined based on FDA guidelines.•Concentrations of the analytes in KB tumor bearing mice dosed with EC1456 reported. EC1456 is a novel folate-targeted small molecule drug conjugate of tubulysin B hydrazide being developed as an anticancer agent for patients with advanced solid tumors expressing the folate receptor. To try and correlate circulating systemic levels of EC1456 and its metabolites to tumor concentrations and potentially develop a PK/PD model, a sensitive bioanalytical method was developed and validated for the quantitation of the analytes in KB tumor homogenates. The method involved homogenizing tumors with buffer containing N-maleoyl-β-alanine, mannitol and acetic acid, precipitation of the homogenate with acetone followed by heating at 55°C for 1h to convert tubulysin B hydrazide to its corresponding hydrazone. The extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC–MS/MS). The method demonstrated good inter-day (3 runs, n=18) accuracy (−2.3% to 7.3%) and precision (1.7% to 10.3%) for all three analytes. Stability was established for three freeze-thaw cycles, 4h on the bench-top on ice, 20h in the autosampler at 8°C and for at least 46days frozen at −70°C. This method was successfully used to determine concentration of EC1456 and its metabolites tubulysin B hydrazide and tubulysin B in tumor homogenates in preliminary experiments with KB tumor bearing mice dosed intravenously with EC1456.