UPLC-MS/MS Analysis of Aldosterone in Plasma for Clinical Research

Importance of the topic

The regulation of blood pressure by the mineralocorticoid hormone aldosterone is critical in clinical research and diagnostics. Traditional immunoassays for aldosterone involve hazardous radioisotopes and suffer from cross-reactivity, manual extraction steps, and limited specificity. The development of an automated UPLC-MS/MS workflow addresses these challenges by offering enhanced analytical sensitivity, selectivity, and throughput, supporting accurate measurement of low physiological concentrations of aldosterone in plasma.

Objectives and Study Overview

This study aims to establish and validate an automated sample preparation and UPLC-MS/MS method for quantifying aldosterone in human plasma. Key goals include minimizing sample handling, achieving baseline separation from structurally related steroids, and demonstrating robust method performance across a broad concentration range for clinical research applications.

Methodology and Used Instrumentation

Sample preparation involved spiking 200 µL plasma with a deuterated internal standard, zinc sulfate, and phosphoric acid. Automated solid-phase extraction was performed on a Tecan Freedom Evo 100/4 liquid handler using an Oasis MAX µElution plate. Chromatographic separation was achieved on an ACQUITY UPLC I-Class system equipped with a CORTECS UPLC C18 column at 45 °C and a 0.40 mL/min gradient over 4 minutes. Detection was carried out on a Xevo TQ-S tandem quadrupole mass spectrometer operating in negative ESI mode with MRM transitions for aldosterone (359.2>189.2 quantifier, 359.2>297.2 qualifier) and the 4H2 internal standard (363.2>190.2). Data acquisition and processing used MassLynx with TargetLynx.

Used Instrumentation

• ACQUITY UPLC I-Class System
• Oasis MAX µElution Plate
• CORTECS UPLC C18 Column
• Xevo TQ-S Tandem Quadrupole Mass Spectrometer
• MassLynx Software with TargetLynx
• Tecan Freedom Evo 100/4 Liquid Handler

Main Results and Discussion

No interferences were observed from nine related steroids, demonstrating clean chromatograms and baseline resolution from 18-hydroxycorticosterone and prednisone. The lower limit of quantification was 42 pmol/L with signal-to-noise ratios above 10:1. Total precision across low, mid, and high QC levels showed RSD ≤9.8%, and repeatability was ≤8.2%. Linearity was confirmed from 42 to 4161 pmol/L with r2 >0.996. No carryover was detected, and dilution integrity at 1:2 over-range samples yielded 99% accuracy. Matrix effects were minimal (mean raw effect 1.10, net 1.00), indicating reliable quantification. Method comparison with an independent LC-MS/MS assay produced a Deming regression of y = 1.07x – 22.94 and a Bland-Altman mean bias of –4.9%, confirming agreement and lack of systematic bias.

Benefits and Practical Applications

• High sensitivity enables accurate measurement at low physiological aldosterone levels.
• Automated SPE reduces hands-on time, improves throughput, and minimizes operator error.
• Selectivity via UPLC and SPE removes interferences, enhancing method robustness.
• Minimal sample volume requirement (200 µL) suits clinical research settings.

Future Trends and Potential Applications

Advances in extraction chemistries and microfluidic automation may further decrease sample volume and processing time. High-resolution mass spectrometry could enable multi-steroid profiling in a single run. Integration with laboratory information systems and artificial intelligence–driven data analysis will enhance workflow efficiency. The methodology may extend to point-of-care platforms and expand to larger hormone panels for comprehensive endocrinology research.

Conclusion

An automated UPLC-MS/MS method for plasma aldosterone was developed and validated, offering high sensitivity, selectivity, and throughput with minimal sample volume. The workflow demonstrates robust precision, linearity, and agreement with existing methods, supporting its application in clinical research laboratories.

Reference

• Foley D, Calton L. UPLC-MS/MS Analysis of Aldosterone in Plasma for Clinical Research. Waters Corporation; 2015.