An intact LC-HRMS assay was developed to characterize ADC biotransformation species from in vitro and in vivo samples. This method allows a more specific identification of various biotransformation species as well as unbiased quantification. For each sample, the ADC concentration was first measured with the LBA-LC-MRM assay. Plasma concentrations were then adjusted to achieve 8.3 µg/mL ADC with a 120 µL aliquot enabling capture of 1 µg ADC. For certain samples with low concentrations where 1 µg of ADC was not achievable, the maximum volume of original plasma available was used (Table ) in capture step. The plasma sample and 75 µL SMART IA magnetic beads conjugated with 12.5 µg biotinylated anti-human Fc (a-HuFc) or anti-payload antibody were incubated for approximately 30 min at ambient temperature to capture the ADC and its biotransformed species. After the removal of the supernatant following the capture step, the beads were then washed twice with PBS and then twice with water (250 µL each wash step). Finally, the ADC and biotransformed species were eluted off the beads by incubating the beads for 5 min with 45 µL 1% FA in water with cytochrome C. The samples were not deglycosylated or reduced to preserve the maximal information for identification of biotransformation species. The eluted samples were injected onto Shimadzu Nexera LC. The separation was performed on a Waters BioResolve RP mAb polyphenyl column (PN186009017) with 1% FA, 0.01% TFA in water/ACN as mobile phases with flow rate of 0.5 mL/min at 80°C (example chromatogram in Figure ). Under the denaturing conditions of reversed phase liquid chromatography, DAR8 ADC light chain and heavy chain would separate due to the replacement of inter-chain disulfide bonds with linker-payloads. A shallow gradient was applied in the reversed phase separation to resolve the various species and the major parent molecule. After LC, the separated species were then ionized and acquired in full scan mode with either SCIEX 6600 Triple TOF or 7600 Zeno TOF system.