An Improved SPE-LC-MS/MS Method for the Quantification of Bradykinin in Human Plasma Using the ionKey/MS System

Significance of the Topic

Bradykinin is a low-abundance, rapidly metabolized nonapeptide that plays a central role in inflammatory and cardiovascular processes. Accurate quantification in human plasma at picogram per milliliter levels is challenged by ex vivo generation, matrix interferences, and limited sample volume, making sensitive and selective bioanalytical methods indispensable for biomarker studies and drug development.

Study Objectives and Overview

This study aimed to develop and validate an improved solid-phase extraction (SPE) LC-MS/MS method for bradykinin in human plasma using the ionKey/MS microflow platform. Key goals included achieving a lower limit of quantitation (LLOQ) of 2.5 pg/mL with only 100 μL of plasma, optimizing sample handling to prevent artifactual bradykinin formation, and demonstrating high accuracy, precision, and throughput.

Methodology and Instrumentation

The workflow combined mixed-mode SPE with Oasis WCX 96-well μElution plates and microflow UPLC separations integrated into the ionKey/MS system:

• Sample preparation: 100 μL plasma spiked with internal standard ([Lys-des-Arg9]-bradykinin), alkaline dilution, and SPE cleanup (conditioning, loading, washing, μElution with 1% TFA in 75:25 ACN:H₂O).
• Chromatography: ACQUITY UPLC M-Class with 2D trap/back-flush, iKey Peptide BEH C18 300 Å, 1.7 μm, 150 μm × 50 mm, 2.5 μL/min at 75 °C, 10 μL injection, 12 min runtime.
• Mass spectrometry: Xevo TQ-S with ionKey source; ESI positive; MRM transitions m/z 354.18→419.18 and 354.18→408.18 for bradykinin, 344.94→386.03 for IS; optimized cone voltage and collision energy; data managed by MassLynx 4.1 and TargetLynx.

Results and Discussion

The ionKey/MS microflow platform delivered a 7–10× improvement in signal-to-noise versus 2.1 mm analytical flow, enabling:

• A 2× reduction in required plasma volume (100 μL) and 50× solvent savings.
• An LLOQ of 2.5 pg/mL with linearity from 2.5 to 8000 pg/mL (R²>0.99), accuracy 92.7–104.0%, and precision CV<5%.
• Fast SPE sample prep (<30 min) compatible with high-throughput 96-well automation.
• Pre-analytical evaluation showing BD P100 and P700 collection tubes preserved native bradykinin, while K₂EDTA alone led to artifactual increases upon freeze/thaw and storage.

Applications and Practical Benefits

This method offers:

• High sensitivity and selectivity for low-level peptide biomarkers in preclinical and clinical studies.
• Reduced sample and solvent consumption, lowering cost and environmental impact.
• Multiple injections for incurred sample reanalysis compliance.
• Robust automation potential for routine QA/QC and pharmacokinetic assays.

Future Trends and Potential Applications

Emerging opportunities include:

• Expansion of microflow LC-MS platforms to multiplexed peptide panels.
• Integration with high-resolution MS for deeper proteomic profiling.
• Point-of-care microfluidic systems for rapid clinical decision support.
• Broader adoption in biomarker discovery and large-cohort studies.

Conclusion

The optimized SPE-LC-MS/MS assay on the ionKey/MS system achieves unparalleled sensitivity (2.5 pg/mL), precision, and throughput for bradykinin quantification in human plasma, addressing critical challenges in peptide bioanalysis and ensuring reliable biomarker measurement.

Reference

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