Similarly, we also observed the formation of THT GSH adduct. XICs across different timepoints and mass spectrum at 168 hr are shown in Figures and, respectively. Interestingly, we observed four peaks with different RT for THT GSH adduct in XIC. The CID MS/MS spectrum in Figure confirms the gluthathionylation of this biotransformed product, where a peak represents THTC-GSH is found in MS/MS spectrum using CID fragmentation. Similarly, we discovered a unique peak representing THT + 1 Hydrolyzed Linker-PL in EAD MS/MS spectrum, as shown in Figure . The maleimide exchange reaction that forms thiol adducts may be one plausible metabolism pathway for cysteine-conjugated ADCs. Within the family of reactive circulating thiol-containing molecules, we also observed homocysteine and Cys-Gly adduct formation on THT peptide backbone from Hc at very low concentration (not shown). Hence, it is possible that other than confirmed adduct species (Cys, GSH, homoCys and Cys-Gly), additional molecules bearing free thiol groups may react with liberated cysteines from linker-payload deconjugation. To our surprise, we did not observe Cys or GSH adduct formation on GEC and SCDK peptides, possibly due to low abundance, small size and/or hydrophilic nature of these adducts, preventing their effective separation using current approach.