Calibration curve standards and quality control samples were prepared in blank pooled CD-1 mouse plasma using reference standard AZD8205. Calibration range was 0.100-15.0 µg/mL. 50 µL of AZD8205 sample was then enriched by immunoaffinity capture using 30 µL SMART IA streptavidin beads conjugated to 3 µg biotinylated anti human-Fc with approximately 2 h incubation at ambient temperature. After separating the beads from supernatant and extensive washing of the beads, SMART IA digestion buffer with stable isotope-labeled internal standard was added to beads for tryptic digestion at 70 °C for 2 h. After trypsin digestion, one fraction of the supernatant was used for total antibody and intact antibody assay. The other fraction was further digested using 0.5 mg/mL papain (overnight at 37 °C) to release AZ14170132 for ADC assay. The characteristic peptides were quantified as surrogate analytes for the total antibody (heavy chain peptide -GLEWIGEINHSGSTSYNPSLK) and intact antibody (light chain peptide -NDVGWYQQKPGK) concentrations and the released AZ14170132 served as the surrogate analyte for the ADC concentration. Internal standards used were stable isotope labeled peptides (terminal lysine 13 C6, 15 N2) and payload ( 2 H5). The ADCs were immunocaptured using the heavy chain. Total antibody assay monitors heavy chain peptide whereas intact antibody monitors light chain peptide. The presence of light chain confirms that the antibody is intact since there is no interchain disulfide bond in these DAR8 ADCs. The ADC concentration included all species of biotransformed molecules with payload in a DAR-sensitive manner, regardless of linker biotransformations. All three assays were analyzed on the SCIEX Triple Quadrupole 6500+ mass spectrometer coupled with a Shimadzu liquid chromatography system. Chromatographic separation was performed using Waters ACQUITY UPLC BEH C18 Column (PN186002350). Mobile phases were A: 0.1 % formic acid in water and B: 0.1 % formic acid in acetonitrile with flow rate of 0.5 mL/min at 60°C. Data were acquired and analyzed with Analyst (v1.7) and MultiQuant (v3.0.3863) software, respectively.