Characterization of metabolites in rat and liver microsomes after intravenous injection of MMAF by liquid chromatography/tandem mass spectrometry

A rapid, sensitive and reliable method using pMRM-IDA-EPI scans with QTRAP 5500 UPLC-MS/MS and Lightsight software was established for the identification of metabolites in rat and liver microsomes from different species after intravenous injection of MMAF (monomethyl auristatin F). MMAF is a new auristatin derivative, which has been employed as the payloads in a number of antibodydrug conjugates (ADCs). Separation of the metabolites of MMAF was achieved by column chromatography in this study. Using our approach, a total of five metabolites were identified from both in vivo and in vitro models. The possible metabolic pathway was proposed, including O-demethylation, amide hydrolysis, N-demethylation, oxidation and demethylation + oxidation. In summary, we developed a simple, sensitive and reliable approach for metabolite identification, which is expected to contribute to our better understanding of MMAF metabolism behavior in vivo and in vitro. Furthermore, it provides support for the process optimization, mechanism-based research, new antibody conjugate technology and preclinical study of ADCs with conjugated MMAF.