SIMULTANEOUS MEASUREMENT OF ALDOSTERONE AND PLASMA RENIN ACTIVITY IN HUMAN SERUM USING LIQUID CHROMATOGRAPHY COUPLED TO TANDEM MASS SPECTROMETRY (LC-MS/MS) FOR CLINICAL RESEARCH

Importance of Topic

The renin–angiotensin–aldosterone system (RAAS) plays a vital role in blood pressure regulation by controlling fluid balance and vascular resistance. Quantitative measurement of aldosterone and plasma renin activity (PRA) is critical for assessing RAAS status in clinical research, guiding the development of novel antihypertensive therapies and deepening our understanding of cardiovascular physiology.

Objectives and Study Overview

This application note describes the development and validation of a single LC-MS/MS method for concurrent measurement of aldosterone and PRA (through angiotensin I quantification) in human serum. The primary goals were to:

• Establish a robust assay with high precision, accuracy, and sensitivity.
• Assess matrix effects and stability under typical sample handling conditions.
• Compare automated versus manual sample preparation workflows.

Methods and Instruments Used

The analytical workflow comprised:

• Sample Preparation: Solid-phase extraction of human serum followed by derivatization for angiotensin I measurement.
• Chromatography: Ultra-performance liquid chromatography (UPLC) separation employing a Waters XBridge column.
• Detection: Tandem mass spectrometry (Xevo TQ-S) with multiple reaction monitoring (MRM) transitions optimized for aldosterone and angiotensin I.
• Matrix Evaluation: Raincloud plot analysis to quantify signal enhancement in surrogate versus human matrices.
• Stability Testing: Freeze/thaw cycles and incubation at controlled temperatures over 20 days.
• Automation Comparison: Sample prep on Hamilton MicroLab Star versus manual extraction.

Main Results and Discussion

Key findings include:

• Linearity and Sensitivity: Limits of quantification met the requirements for clinical research, with clear, well-resolved peaks and retention time shifts ≤0.20 s for angiotensin I.
• Precision and Accuracy: Across 20 replicates per time point over five days, both analytes showed total CV ≤15% and accuracy within acceptable recovery ranges.
• Matrix Effects: Minimal signal suppression/enhancement when transitioning from surrogate to human serum, confirming assay robustness.
• Method Comparison: Passing–Bablok regression for 58 aldosterone samples yielded y = 0.96 x – 2.49 (mean bias –6.0%); for 61 PRA samples, y = 1.26 x – 0.02 (mean bias 21.4%).
• Stability: Analyte concentrations remained stable over multiple freeze/thaw cycles and 20 days of storage, with reproducible results across biological replicates.
• Automation Benefits: Automated extraction reduced %CV and improved throughput compared to manual preparation.

Benefits and Practical Applications

This single-run LC-MS/MS assay offers:

• Improved Laboratory Efficiency: Simultaneous analysis of two RAAS biomarkers reduces instrument time and consumable usage.
• Enhanced Data Quality: High reproducibility and minimal matrix effects support reliable longitudinal studies.
• Cost Savings: Consolidation of assays on a single platform lowers operational expenses and simplifies method maintenance.

Future Trends and Applications

Potential developments include:

• Expansion to Additional RAAS Components: Integration of other peptides (e.g., angiotensin II) for comprehensive pathway profiling.
• Clinical Diagnostic Adoption: Transitioning the validated method into regulated diagnostic workflows pending further clinical validation.
• Advanced Automation: Full integration with laboratory information management systems (LIMS) to streamline sample tracking and reporting.
• High-throughput Screening: Application in large-scale epidemiological studies and drug discovery programs targeting cardiovascular disorders.

Conclusion

The presented LC-MS/MS method enables reliable, simultaneous measurement of aldosterone and plasma renin activity in human serum. It demonstrates excellent analytical performance, stability under routine handling, and workflow efficiencies through automation. This approach supports high-quality RAAS research and offers a pathway toward broader clinical application.

Reference

Oliveiro G, Burgos R, Foley D, Bosh G, Davey L. Simultaneous measurement of aldosterone and plasma renin activity in human serum using LC-MS/MS for clinical research. Waters Technology Application Note. 2024.