Introduction:
The combination of primary aldosteronism (PA) and alterations in plasma renin activity (PRA) is a leading cause of secondary hypertension. An accurate measurement of Renin Angiotensin-Aldosterone System (RAAS) pathway improves the recognition of symptoms related to stroke, coronary disease, heart failure and metabolic syndrome.

Clinical Research Method
Previously, surrogate matrices have been used when demonstrating the analytical performance of LC-MS/MS clinical research methods for the measurement of aldosterone and angiotensin I. Here, we have developed an analytically sensitive and reliable analytical strategy for simultaneous measurement of Aldosterone and Angiotensin I in human matrices. In-house calibrators and quality control samples were prepared, containing varying ranges of both aldosterone (20-2000pg/mL; 55-5548pmol/L) and angiotensin 1 (0.6-240ng/mL; 0.46-185nmol/L). The samples were extracted using the Oasis™ MAX 96-well μElution plate and then separated through the ACQUITY™ UPLC I-Class FL using a XBridge™ Premier Peptide BEH C18 Column, over a run time of 3 minutes. Samples were then injected to Xevo™ TQ Absolute Mass Spectrometer, using both positive and negative acquisition modes. The downstream LC-MS/MS data analysis was performed using MassLynx™ MS Software. The analytical performance was demonstrated by extracting and quantifying two replicates of samples on two occasions per day over five separate days. Additionally, the stability of aldosterone and angiotensin I over the period of incubation (3-5hr) was evaluated, which is necessary when investigating the plasma renin activity in human Serum/Plasma samples.

Results
High reproducibility and repeatability were both determined, indicating total precision and repeatability (<15%CV) for both analytes and no significant changes were found in Aldosterone and Angiotensin I concentrations over 3-5hr of incubation. No significant system carryover (<10% of the lowest calibrator) from high concentration samples into subsequent blank injections was observed. In addition, calibration lines in human serum/plasma were linear, with coefficient of determinations (r2) >0.992 for all analyses at different incubation times. The analytical sensitivity of the clinical researchmethod allows for precise quantification at 55 pmol/L for aldosterone and 0.46 nmol/L/hr forangiotensin I, with both analytes providing S/N (PtP) ≥10:1 and %RSD of <15% at these concentrations.

Conclusions
An analytically sensitive and selective clinical research method has been developed for the analysis of aldosterone and angiotensin I, using the Xevo TQ Absolute Mass Spectrometer. The Xevo TQ Absolute Mass Spectrometer enables the simultaneous analysis of physiologically low levels of aldosterone and plasma renin activity in human samples, (20pg/mL; 55pmol/L), (0.6ng/mL; 0.46nmol/h/L). A single test that improves instrument utilization, reduces cost, and allows the user to expand their existing test menu on the same platform has been established.