Quantitation of hepcidin in dried blood spots by microfluidic LC-chip-MS/MS

Microfluidic LC on a chip coupled to tandem mass spectrometry has been shown to be a sensitive tool for the quantitative analysis of small molecules and peptides. A major advantage of microfluidic separation techniques is the small requirements in terms of sample volume. This feature is particularly valuable for applications in the context of studies on small laboratory animals like rodents, or less-invasive clinical routine tests in patients, especially for paediatric applications.
We previously developed a quantitation method for hepcidin, a peptide biomarker, in human plasma using µ-SPE (miniaturised solid-phase extraction) as sample preparation technique. The developed method allowed the quantitation of hepcidin using only 50 µL plasma with an excellent sensitivity. However, this efficient technique is quite expensive and time-consuming. To further reduce sample volume needs and simplify sample preparation, we developed a preparation method based on dried blood spot (DBS) analysis. In this case, a precise volume of blood (< 20 µL) is collected with a micro-capillary, dispensed on a paper card and dried. The obtained blood spot is then punched from the card, extracted in an appropriate solvent, and analysed by microfluidic LC-chip coupled to mass spectrometry.
Method development was assisted by experimental design to optimise blood deposition and extraction conditions to ensure maximal analyte recovery while reducing interference co-extraction. Different ways of internal standard addition were also evaluated.
In this study, the results obtained using µ-SPE and DBS are compared in terms of ease-of-use, throughput, sample requirements, cost, sensitivity and robustness.