Preparation and characterisation of an 57Fe enriched haemoglobin spike material for species-specific isotope dilution mass spectrometry

Abstract

Haemoglobin (HGB) and its various isoforms are powerful markers for diagnostic and treatment control. For the first time a species-specific isotope dilution mass spectrometry (IDMS) method for the quantification of the whole protein without peptide digestion was developed in this study. The required species-specific HGB spike material was prepared by substituting the entire protohaem containing ^natFe for the same group enriched with ⁵⁷Fe. The spike material was characterised regarding isotopic enrichment and structural modification compared to natural HGB. Two different separation methods using a MonoQ® ion exchange chromatography column and a size exclusion column (SEC), respectively, have been developed for the separation of HGB from the matrix and their results for the quantification using IDMS were compared. For this, a high-performance liquid chromatography (HPLC) system was linked to an inductively coupled plasma mass spectrometer (ICP-MS) and HGB was quantified via its iron content. The IRMM/IFCC-467 and JCCRM 912-2 reference materials were used as control samples and for comparison of both separation techniques (MonoQ® and SEC). The HGB values obtained for both methods were found to be identical within the expanded uncertainty.