For each ADC, a series of experiments using the same parameters except for CE were performed. For ADC1 we observed the signature payload MS2 ion intensity reached plateau at around 100 eV and slightly decreased at 120 eV (Figure ), whereas for trastuzumab emtansine DM1-specific MS2 ion intensity reached its peak at 60 eV then significantly dropped as CE continued to increase (Figure ). The product ion monitored for trastuzumab emtansine is potentially further fragmented at higher CE. Hence, CE was selected at 60 eV. Precursor or product ions can also be summed to enhance sensitivity (Table ). We can establish calibration curve with r≥0.985 and QCs meeting acceptance criteria defined in ICH M10 by quantification with single payload signature product ion. For some DAR species, summing precursor or product ions can increase sensitivity by lowering the LLOQ by 1.5 to 12-fold, improving r value and QCs passing rate. Additionally, peak integration parameters, such as MS2 XIC width (Figure ), S/N integration threshold, Gaussian smooth width, and noise percentage, can be adjusted on a case-by-case basis for each ADC with different linker-payload and DAR distribution.