Simultaneous Extraction of Aldosterone and Angiotensin I and II from Human Plasma Using EVOLUTE® EXPRESS ABN Prior to LC/MS Analysis

Significance of the Topic

Accurate quantification of aldosterone and angiotensin peptides in human plasma is critical for diagnosing and managing disorders of the renin–angiotensin–aldosterone system, including primary aldosteronism and hypertension. Traditional workflows often analyze these analytes separately, adding complexity, time, and cost. A streamlined, simultaneous extraction method enhances laboratory throughput, reduces sample volume requirements, and improves consistency for clinical and research applications.

Study Objectives and Overview

This application note presents a method for concurrent extraction of aldosterone, angiotensin I, and angiotensin II from human plasma using EVOLUTE EXPRESS ABN solid phase extraction (SPE) plates prior to LC-MS/MS analysis. The goals are to demonstrate robust analyte recovery (> 80%), low variability (RSD < 5%), minimal phospholipid interference, and reliable quantitation over a broad concentration range.

Sample Preparation Methodology

Pre-treatment steps:

• Spike 250 µL plasma with aldosterone-D4 internal standard (10 µL at 500 pg/mL).
• Add incubation buffer (50 µL) containing Tris, EDTA, PMSF, and SBTI to inhibit angiotensin conversion; incubate at 37 °C for 1 h.
• Quench with 10 µL 10% formic acid.

EVOLUTE EXPRESS ABN SPE:

• Condition with methanol (1 mL) and equilibrate with 0.1% NH₄OH (aq) (1 mL).
• Load sample (300 µL) under positive pressure (~3 mL/min).
• Wash with 0.1% NH₄OH (aq) (1 mL) to remove weak interferences.
• Elute angiotensins with 400 µL 2% NH₄OH in 50/50 MeOH/H₂O; dry.
• Elute aldosterone with 500 µL ethyl acetate; dry.
• Reconstitute in 250 µL 30% MeOH with 0.4% formic acid for analysis.

Used Instrumentation

• UPLC: Shimadzu Nexera UHPLC, Restek Biphenyl column (50×2.1 mm, 1.7 µm).
• MS: Shimadzu 8060 triple quadrupole with ESI interface.
• Mobile phases: A = 0.2 mM ammonium fluoride, B = methanol; gradient 60/40 to 20/80 over 4 min, 0.3 mL/min, column at 35 °C.
• MS settings: positive and negative ion modes, MRM transitions for each analyte; typical interface 400 °C, DL 150 °C, heat block 500 °C.

Main Results and Discussion

• Analyte recoveries exceeded 80% with RSD < 5% across replicates.
• Phospholipid removal was confirmed by monitoring the m/z 184 product ion. Ethyl acetate elution for aldosterone effectively eliminated phospholipid signals compared to protein precipitation.
• Calibration curves were linear from 10 to 10 000 pg/mL for angiotensin I and II, and < 25 pg/mL LOQ for aldosterone, with r² > 0.999 for all analytes.
• Representative chromatograms showed clear separation and sensitivity at 1 ng/mL spiking levels.

Benefits and Practical Applications

• Simultaneous extraction reduces assay time and sample consumption compared to separate workflows.
• The method delivers high-throughput compatibility for clinical and pharmacodynamic studies.
• Enhanced clean-up minimizes matrix effects, improving reproducibility and instrument uptime.

Future Trends and Potential Applications

• Extension to additional renin–angiotensin–aldosterone axis biomarkers or other small peptides in plasma.
• Automation of SPE steps and integration into robotic platforms for large-scale clinical trials.
• Adapting the protocol to dried blood spots or other low-volume matrices for point-of-care testing.

Conclusion

The described SPE-LC-MS/MS workflow enables reliable, simultaneous extraction and quantitation of aldosterone and angiotensin I/II from human plasma. High recoveries, low variability, and effective phospholipid removal demonstrate its suitability for clinical research and diagnostic laboratories seeking to streamline analysis of renin–angiotensin–aldosterone biomarkers.

Reference

AN894 Application Note, Biotage, 2018