The materials and methods were reported previously. Figure shows the workflow of experimental steps for sample preparation and LC-MS/MS data analysis. Briefly, 0.1 mg/mL AZD8205 (AstraZeneca, Gaithersburg, MD) was incubated for different lengths of time (0, 6, 24, 72 and 168 hr) in human or CD1 mouse plasma (BioIVT, Hicksville, NY) samples and then frozen at -80 °C until analysis. Biotinylated anti-idiotype (anti-ID) antibodies were immobilized via streptavidin on SMART IA beads (ThermoFisher Scientific, Waltham, MA). AZD8205 was enriched using anti-ID conjugated magnetic beads then digested at 70 °C after activation of the pre-immobilized trypsin on SMART IA beads. The solution containing tryptic digestion products was cleaned-up and concentrated using an Oasis HLB µElution plate (Waters Corporation, Milford, MA) following the manufacturer's protocol before being injected into Exion LC coupled with SCIEX 7600 ZenoTOF for LC-MS/MS analysis. Digested products were separated through a 74-minute LC gradient using a Waters Acquity BEH C18 column (Waters Corporation, Milford, MA). The settings of LC, MS and MS/MS were previously specified. To identify and analyze the AZD8205 biotransformation products, we employed the LC-MRM approach while applying two orthogonal fragmentation methods, CID and EAD. The successful confirmation of biotransformation uses a two-step mass fingerprinting approach. First, the monoisotopic m/z of the biotransformed species in MS spectrum was matched. Second, the fragment ions in MS/MS spectrum using biotransformed product as precursor ion were also matched.