There are multiple factors that may contribute to DF-MDMS assay sensitivity. While both DAR8 ADCs (ADC1 and ADC2) with homogeneous conjugation showed excellent sensitivity that can reach as low as 50 ng/mL, ADC2 DAR4 and trastuzumab emtansine had much higher LLOQ, even though ADC2 has the same payload as ADC1. ADCs with stochastic heterogeneous conjugation inherently has distribution of DAR species on both light and heavy chain, and thus affecting the LLOQ of each species. DAR also has impact on LLOQ as moles of conjugated payload would impact signal intensity. Linker-payload intrinsic ionization and dissociation properties are also factors that can significantly affect assay sensitivity. For TOP1i payload (AZ14170132), there are multiple signature product ions that can be used, whereas for DM1 only has one unique MS2 product ion with low signal observed and used for quantification (Figure ). Therefore, suitability of ADC quantification using the DF-MDMS CID approach should be considered in the context of linker-payload structure and conjugation approach.