High-Sensitivity Quantification of Angiotensin Oligopeptides Using Methoxyacetylation Coupled with LC-TIMS-qTOF/MS

Quantification of key effectors in the circulating renin-angiotensin system (RAS), including angiotensin (Ang) I, Ang (1-9), Ang II, Ang (1-7), and Ang A, is essential for understanding RAS-mediated blood pressure regulation. Herein, we present a highly sensitive and selective analytical method based on methoxyacetylation coupled with liquid chromatography-trapped ion mobility spectrometry-quadrupole time-of-flight mass spectrometry (LC-TIMS-qTOF/MS) operated in parallel reaction monitoring-parallel accumulation serial fragmentation (prm-PASEF) mode. Methoxyacetylation using an NHS ester reagent modulated protonation and fragmentation behavior, resulting in enhanced signal intensity and more concentrated product-ion formation. The TIMS parameters were optimized to achieve maximal sensitivity, with an inverse reduced mobility (1/ k 0 ) range of 0.7–1.1 cm 2 ·V -1 ·s -1 and accumulation and ramp times of 150 ms. Under these conditions, the method exhibited excellent analytical performance, with limits of detection ranging from 0.6 to 3.4 fmol/mL, precision below 9.3% (CV), and accuracy within 95.1–105.8%. Using [ 13 C 5 , 15 N]-labeled Ang II as an internal standard, all five target angiotensin peptides were successfully quantified from 100 μL of Sprague-Dawley rat plasma. The developed method provides a robust and sensitive platform for multiplexed quantification of angiotensin metabolites and enables detailed investigation of RAS dynamics in biological systems.