Simultaneous UPLC-MS/MS quantification of angiotensin (I and II) and eight adrenal steroids for the assessment of plasma renin activity and secondary hypertension subtyping

Early screening and precise subtyping are crucial for the clinical management of secondary hypertension. However, comprehensive analytical methods for profiling multiple related biomarkers remain limited. This study aimed to develop and validate a multiplex ultra-performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of angiotensin (I and II) and eight adrenal steroids (aldosterone, 18-oxocortisol, 18-hydroxycortisol,cortisol,cortisone,corticosterone,11-deoxycortisol, and 21-deoxycortisol) in human plasma. Plasma samples were prepared utilizing solid-phase extraction and chromatographic separation was achieved on a BEH C8 column (2.1 × 50 mm, 1.7 μm). Quantitative analysis was performed using a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source. The plasma renin activity was indirectly determined by quantifying the generation of angiotensin I following a 3-hours sample incubation. The method demonstrated satisfactory linearity for all analytes (r > 0.99). The within-run and total coefficients of variation were 1.4%-8.4% and 1.6%-10.5%, respectively. Extraction recoveries ranged from 80.0% to 108.3%, and matrix effects were evaluated between 80% and 120% with negligible carryover. All validation parameters met the acceptance criteria. Ultimately, this method was successfully applied to clinical plasma samples, providing a promising analytical tool for the early screening, diagnosis, and subtyping of secondary hypertension.