Characterization of the Impact of Drug Metabolism on the Gas-Phase Structures of Drugs Using Ion Mobility-Mass Spectrometry

Conventional strategies for drug metabolite identification employ a combination of liquid chromatography-mass spectrometry (LC-MS), which offers higher throughput but provides limited structural information, and nuclear magnetic resonance spectroscopy, which can achieves the most definitive identification but lacks throughput. Ion mobility-mass spectrometry (IM-MS) is a rapid, two-dimensional analysis that separates ions on the basis of their gas-phase size and shape (reflected by collision cross section, CCS) and their mass-to-charge (m/z) ratios. The rapid nature of IM separation combined with the structural information provided by CCS make IM-MS a promising technique for obtaining more structural information on drug metabolites without sacrificing analytical throughput. Here, we present an in vitro biosynthesis coupled with IM-MS strategy for rapid generation and analysis of drug metabolites. Drug metabolites were generated in vitro using pooled subcellular fractions derived from human liver and analyzed using a rapid flow injection–IM-MS method. We measured CCS values for 19 parent drugs and their 37 metabolites generated in vitro (78 values in total), representing a wide variety of metabolic modifications. Post-IM fragmentation and computational modeling were used to support metabolite identifications and explore the structural characteristics driving behaviors observed in IM separation. Overall, we found the effects of metabolic modifications on the gas-phase structures of the metabolites to be highly dependent upon the structural characteristics of the parent compounds and the specific position of the modification. This in vitro biosynthesis coupled with rapid IM-MS analysis workflow represents a promising platform for rapid and high-confidence identification of drug metabolites, applicable at a large scale.