This technical note demonstrates the comprehensive characterization and confident identification of glucuronide metabolites from hepatocyte incubations of midazolam. An orthogonal fragmentation mechanism was applied to generate diagnostic fragment ions for confident identification of glucuronide metabolites using electron activated dissociation (EAD). Several key glucuronide conjugations were identified, including aromatic/aliphatic hydroxylation, o-glucuronide conjugation and N-dealkylated midazolam N-glucuronide. A streamlined workflow was developed to efficiently characterize and identify conjugated structures during drug metabolism studies.
Glucuronide conjugation can be challenging to characterize thoroughly by MS/MS alone, as the glucuronic acid bond is often labile, both in the ionization source and the collision cell of mass spectrometers.3 One of the significant challenges when implementing a soft-spot analysis approach is the ability to produce data promptly in alignment with the pace of the drug discovery process. This technical note demonstrates a quick and robust soft-spot identification procedure using a novel orthogonal fragmentation mechanism, EAD, on the ZenoTOF 7600 system (Figure 1). Sites of midazolam glucuronide conjugation were predicted using Mass-MetaSite software. Structural determination was performed using unique EAD fragments.
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