Evaluation of Sample Preparation Options for the Simultaneous Extraction of Angiotensin and Aldosterone Prior to UHPLC-MS/MS Analysis

Importance of the Topic

Accurate measurement of aldosterone and angiotensin peptides in plasma is critical for diagnosing and managing secondary hypertension. The aldosterone‐to‐renin ratio guides clinical decisions, but traditional assays analyze these analytes separately, increasing sample requirements and processing time. A unified approach enhances throughput and delivers more comprehensive endocrine profiling.

Objectives and Study Overview

This study aimed to develop and compare sample preparation strategies for the simultaneous extraction of angiotensin I, angiotensin II and aldosterone from human plasma, followed by sensitive UHPLC‐MS/MS analysis. Key goals included:

• Minimizing matrix effects and non‐specific adsorption.
• Optimizing solid‐phase extraction (SPE) protocols.
• Validating method linearity, sensitivity and reproducibility.

Methodology and Instrumentation

Sample Preparation

• Plasma incubation: 250 µL plasma with 50 µL buffer at 37 °C for 1 hour; reaction quenched with 10 µL 10% formic acid.
• SPE sorbents: EVOLUTE® EXPRESS ABN, CX and AX (30 mg, 96‐well format).
• Key steps: conditioning, sample loading, pH‐dependent washing, sequential elution fractions for steroids and peptides.
• Evaporation: 40 °C under inert conditions to minimize plastic binding.
• Reconstitution: 200 µL 0.2% acetic acid in 50:50 water/methanol.

Instrumentation

• UHPLC: Shimadzu Nexera with ACE EXCEL C18, 1.7 µm, 50×2.1 mm column; mobile phase A: 0.2 mM NH4F; B: methanol; flow 0.3 mL/min; gradient 60:40 to 20:80.
• MS/MS: Shimadzu 8060 triple quadrupole; ESI positive/negative; MRM transitions optimized for each analyte; heat block 500 °C; interface 400 °C.

Main Results and Discussion

Evaporation Effects

• Significant peptide/steroid losses occurred due to adsorption during drying; optimized solvent ratios mitigated this.

SPE Optimization

• ABN sorbent delivered slightly higher recoveries for both aldosterone (>85%) and angiotensin peptides (>80%) compared to CX.
• pH and wash solvent composition enabled fractionation: steroids eluted with ethyl acetate mixtures; peptides with aqueous methanol.
• Phospholipid removal exceeded 90%, improving chromatographic cleanliness.

Anticoagulant Impact

• Performance was consistent across K2/K3-EDTA, citrate and heparin plasma, with minor signal variations.

Calibration and Sensitivity

• Calibration curves in plasma (10–10000 pg/mL) achieved r2 > 0.999 for all analytes.
• Limits of quantification were below clinical requirements.

Benefits and Practical Applications

This workflow reduces sample volume and processing steps by combining peptide and steroid extraction. High recoveries and low matrix interference support reliable measurement in clinical and research laboratories. The method is suitable for high‐throughput applications in endocrinology, pharmacokinetics and biomarker discovery.

Future Trends and Possibilities

Advancements may include:

• Automation of SPE steps to enhance throughput and reproducibility.
• Integration with multiplexed assays for broader hormone panels.
• Miniaturized extraction formats to reduce solvent consumption.
• AI‐driven data processing to streamline quantitative analysis.

Conclusion

A robust, simultaneous SPE‐UHPLC‐MS/MS method for angiotensin I, angiotensin II and aldosterone has been established. Optimized ABN protocols yield excellent recovery, sensitivity and linearity while minimizing matrix effects. This approach supports efficient clinical testing and research workflows.