Optimization and clinical validation of a simplified workflow for renin-angiotensin-aldosterone system hormones and...

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is widely used for quantifying renin-angiotensin-aldosterone system (RAAS) hormones and adrenal steroids. However, pre-analytical factors frequently compromise analyte stability and measurement accuracy. This study evaluated the stability of key RAAS-related analytes under routine pre-analytical conditions, optimized a fixed-baseline calculation for plasma renin activity (PRA), and validated a simplified protocol for clinical adoption. This three-phase study comprised: a discovery cohort (n = 2661) to optimize the fixed pre-incubation angiotensin I (Ang I) baseline value; a stability study using blood samples from 40 healthy volunteers, in which analyte stability was evaluated in whole blood before centrifugation (4 ℃ or 25 ℃, 0-8 h) and in plasma after centrifugation (4 ℃, 24-72 h; -20 ℃, 15 days; or three freeze-thaw cycles at -80 ℃); and a validation cohort (n = 1230) to assess the clinical reclassification rate of aldosterone-to-renin ratio (ARR). For PRA, the pre-incubation Ang I baseline was fixed at a value determined through sensitivity optimization in the discovery cohort. Storage conditions markedly affected Ang I stability, while steroid analytes remained unchanged. Whole blood samples met acceptance criteria for up to 4 h at 25 ℃ or 8 h at 4 ℃ prior to centrifugation. After centrifugation, plasma samples remained stable for up to 24 h at 4 ℃, 15 days at -20 ℃, or after three freeze-thaw cycles at -80 ℃. The simplified PRA model showed excellent concordance with the measured values (R² = 0.999) and negligible bias in Bland-Altman analysis. This approach resulted in minimal reclassification of ARR results (0.16%, 2/1230), with all discordant cases located near the screening cut-off. This study establishes practical stability limits for RAAS-related analytes and supports a simplified PRA calculation method. This standardized approach minimizes pre-analytical variability and enhances laboratory efficiency without compromising clinical diagnostic integrity.