Absolute Quantification of Total Hemoglobin in Whole Blood by High-Performance Liquid Chromatography Isotope Dilution Inductively Coupled Plasma-Mass Spectrometry

Accurate and traceable measurement of hemoglobin (HGB) is of great importance in clinical testing. Although the HiCN method is the internationally accepted conventional reference method for this biomarker and frequently used in clinical routine diagnostics, the HiCN method cannot be traceable to the International System of Units (SI) and thus does not meet highest metrological demands. In this study, an absolute quantitative approach for total HGB in a whole blood sample is proposed based on the determination of natural Fe and S present in the heme-group of HGB by HPLC isotope dilution ICP-MS. IRMM/IFCC-467 is used for method validation, and then clinical blood samples are measured by the established strategy and HiCN method. The measurable ranges of total HGB were 10.0-240.0 g L-1. Limits of detection via Fe and S were 0.01 and 0.07 g L-1, respectively. The intra-assay imprecision CVs via Fe and S were 0.89-1.35 and 0.99-1.56%, and the interassay CVs were 1.19-2.15 and 1.55-2.55%, respectively. Good agreement was achieved in the method validation. In the comparison with HiCN experiments, the ID-ICP-MS assays via Fe and S showed correlations of r2= 0.991 and 0.970 against HiCN methods. Moreover, the concentration of transferrin (Tf) was also simultaneously measured. This strategy has potential to serve as a reference measurement procedure for total HGB in whole blood, which could be traceable to SI and does not require toxic derivation reagent.