Figure shows the mass spectra of [GEC+succinimide+R2+3H] 3+ of ADC1 with 0 h (black line) and 168 h (blue line) incubation. After 168 h incubation, the signal intensity of [GEC+succinimide+R2+3H] 3+ (observed monoisotopic m/z 485.8744, -11 ppm difference) dropped significantly compared to the sample with 0 h incubation. To be specific, the absolute intensity of the monoisotopic peak of [GEC+succinimide+R2+3H] 3+ dropped from 2.9×10 4 to 7.2×10 2 cps after 168 h of incubation. Instead, we noticed a notable increase in peaks representing GEC+succinimide+R2 with +18 Da shift (observed monoisotopic m/z 491.8779, z=3, -11 ppm difference). Thus, the hydrolysis of succinimide over 168 h was confirmed. Figure is the extracted ion chromatogram (XIC) of [GEC+succinimide+R2+3H] 3+ (m/z 485.8799) in 0 h and 168 h incubated sample. Figure shows the XIC of [GEC+succinimide+H2O+R2+3H] 3+ (m/z 491.8834) at different incubation times. Similarly, the XICs support the conclusion that most of the succinimide conjugated to GEC is hydrolyzed over 168 hours of incubation. Table in the supporting information gives theoretical and observed monoisotopic m/z values of discussed ions. The hydrolysis of thio-succinimide creates a pair of constitutional isomeric products, as shown in Figure and reported previously. In Figure , two distinctive peaks with different retention times observed in XIC confirm the presence of GEC+succinimide+H2O+R2 isomers. The peak with RT = 45.9 minute is denoted as peak 1, whereas the peak with RT = 46.4 minute is denoted as peak 2. In order to elucidate the chemical structure corresponding to each peak, we employed two orthogonal dissociation methods, CID and EAD, on [GEC+succinimide+H2O+R2+3H] 3+ . Figures show the CID MS/MS spectra of precursor ion from peak 1 and 2, respectively. However, we did not find any distinctive peaks by comparing these two spectra. Hence, CID does not provide useful information in distinguishing thio-succinimide hydrolysis isomers.