Improving sensitivity and selectivity of human ACTH[1-39] quantitation using online size exclusion chromatography and
B R Jones1, L Shan2, A Spytko3
1Bioanalysis and Biomarkers, Crinetics Pharmaceuticals Inc., San Diego, CA, USA.
Background:
Quantitation of adrenocorticotropic hormone (ACTH) in human plasma by clinical immunoassays is prone to selectivity challenges, whereas quantitation by mass spectrometry assays (MSA) is limited by sensitivity. The use of nano-liquid chromatography (nano-LC) addresses the MSA sensitivity gap but commonly suffers from poor robustness due to LC system backpressure issues, which is mitigated by incorporation of online size exclusion chromatography (SEC).
Research Design And Methods:
An antibody-free, multidimensional nano-LC, high-resolution mass spectrometry method was designed and implemented to measure intact ACTH[1-39] in human plasma. Online SEC separation was performed at relatively high flow rates prior to trap and elute reversed phase separations at sub-µL/min flow rates. ACTH[1-39] was detected using a high-resolution orbitrap mass analyzer.
Results And Conclusions:
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.
