Quantification of Hepcidin in Plasma Using the Immundiagnostik Hepcidin-25 LC- MS/MS Kit (Ruo) For Clinical Research

Importance of the Topic

Hepcidin is a key regulator of iron homeostasis and understanding its plasma concentration is critical for clinical research into disorders of iron metabolism. Quantitative analysis of this cysteine-rich peptide poses challenges due to its disulfide bonds, multiple charge states, and tendency to adsorb onto surfaces at low concentrations. Developing a robust LC-MS/MS method addresses these obstacles and enables reliable measurement of hepcidin in research settings.

Objectives and Study Overview

The primary aim of this work was to establish and validate a streamlined LC-MS/MS workflow for sensitive and accurate quantification of hepcidin-25 in citrate plasma, using the Immundiagnostik Hepcidin-25 LC-MS/MS Kit (RUO) in combination with Waters instrumentation. Key goals included optimization of sample preparation, selection of ideal precursor charge states, and demonstration of method performance across calibration, precision, accuracy, and comparability to a reference protocol.

Methodology and Instrumentation Used

The sample preparation employed solid-phase extraction on a Waters OASIS HLB µElution 96-well plate, low-bind consumables, and reagents from the Immundiagnostik kit. Citrate plasma samples, calibrators, and quality controls underwent protein binding reduction and two-step washing before elution and dilution. Chromatographic separation was carried out on an ACQUITY UPLC I-Class System with flow-through needle, using an XSelect CSH C18 column and proprietary mobile phases. Detection was performed on a Xevo TQ-S micro tandem quadrupole MS in positive ESI MRM mode. IntelliStart optimization guided the choice of 4+ and 5+ precursor charge states and specific transitions for quantification.

Key Results and Discussion

• Linearity: Excellent calibration response over 3.5–132.7 ng/mL with R² > 0.99 for both 4+ and 5+ precursors.
• Precision: Intra-assay CVs below 10% across low, medium, and high QC levels.
• Accuracy: Recoveries within 95–98% of target values for both charge states.
• Sensitivity: Limits of quantification extrapolated to 1.4 ng/mL (4+) and 1.8 ng/mL (5+) based on signal-to-noise criteria.
• Comparability: Bland-Altman analysis showed negligible bias against the reference Immundiagnostik method, confirming method equivalence.

Benefits and Practical Applications

• Simplified sample handling using low-binding tips and plates minimizes analyte loss at trace levels.
• High-throughput 96-well SPE format accelerates preparation for large clinical studies.
• Dual-charge-state monitoring provides flexibility and confirmation of results.
• Robust performance supports biomarker studies in iron-related disorders and therapeutic monitoring.

Future Trends and Opportunities

• Extension to other biofluids and additional hepcidin isoforms or post-translational variants.
• Integration with automation platforms for fully hands-off sample processing.
• Adoption of lower-volume calibrators and enhanced sensitivity reagents to push detection limits further.
• Combination with complementary proteomic workflows to explore peptide networks in iron metabolism.

Conclusion

This validated LC-MS/MS workflow combines the Immundiagnostik Hepcidin-25 kit with Waters UPLC and Xevo TQ-S micro instrumentation to deliver reproducible, accurate, and sensitive quantification of hepcidin in citrate plasma. The method meets rigorous clinical research requirements and provides a versatile platform for advancing studies of iron regulation.

References

1. Nemeth E, et al. Identification of Hepcidin, a Major Regulator of Iron Metabolism. J Clin Invest. 2004.
2. Ganz T. Hepcidin and Iron Regulation. J Clin Invest. 2012.
3. Rishi G, et al. Advances in Hepcidin Assay Methodologies. Front Physiol. 2019.
4. Smith JR, et al. Clinical Implications of Hepcidin Measurement. Haematologica. 2019.