Improving sensitivity and selectivity of human ACTH[1–39] quantitation using online size exclusion chromatography and antibody-free LC-HRMS

Nanoelectrospray allows measurement of ACTH[1-39] to a clinically relevant concentration of 3-5 pg/mL, with low bias and high precision across the concentration range interrogated. SEC reduces microfluidic backpressure effects commonly observed with the use of nano-LC for bioanalytical applications. Eliminating antibody capture reduces susceptibility to endogenous antibody interferences. This format can be readily adapted for multiplexed measurement of other low abundance peptide hormones in biological samples.