Background: For management and treatment of secondary hypertension, plasma renin activity (PRA) assay is considered an essential diagnostic tool. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to PRA offering improvements in laboratory workflow and throughput. During development, we observed a substantial number of clinical samples that have strong degradation activity toward angiotensin (Ang) I during generation. A preliminary characterization of this degradation activity was performed, and we provide here a method by which this degradation can be monitored via the addition of an isotope-labeled degradation standard.
Methods: Automated online sample extraction coupled with HPLC was used to isolate Ang I and internal standard from plasma. The effluent from the analytical column was directed to a triple quadrupole MS operated in selected reaction monitoring mode, monitoring the a(5) and b(5) product ions from the [M+3H](+3) precursors. Routine analysis could be achieved with as little as 150 μL plasma.
Results: We identified both C-terminal and N-terminal degradation products of Ang I using isotope-labeled peptides as controls and substrates. In 2%-5% of patient samples, the degradation essentially eliminated any Ang I produced during generation.
Conclusions: Our method requires reduced sample handling when compared with an RIA and eliminates the need for extended generation times for samples with low renin activity. Degradation of Ang I during generation appears to be a confounding variable in the interpretation of results from some clinical samples. Samples with profound degradation activity can be identified using a degradation standard that is added at the start of generation.