The total haemoglobin (Hb) concentration in blood is one of the most frequently measured analytes in clinical medicine because of its significance for evaluating the health state of a human. The spectrophotometric cyanmethaemoglobin (HiCN) method is the internationally accepted conventional reference method to determine this biomarker. It is frequently used in clinical routine diagnostics but is not traceable to the International System of Units and thus does not meet highest metrological demands. A further critical issue is the toxicity of the necessary potassium cyanide. Different methods to solve these problems are reported here. They all were validated against the HiCN method in an interlaboratory comparison by measuring the total Hb concentration present in the certified reference material JCCRM 912-2M. Methods considered were the spectrophotometric alkaline haematin detergent (AHD) method as well as several isotope dilution (ID)-based approaches. The latter include inductively coupled plasma mass spectrometry (ICP-MS), species-specific (SS) ICP-MS, organic MS and Raman spectrometry. Graphical abstract ᅟ.

Abstract. The main objective of this study is the development of reference measurement procedures for the determination of total haemoglobin (Hb) in blood based by ID-ICPMS. First of all, a characterization of a Hb standard is carried out via total iron analysis by double ID-ICPMS. This standard will be used as a primary calibration solution in order to ensure the traceability of the measurement results. The validation of this method was conducted by the analysis of the CRM JCCRM 912-1 (ReCCS) certified in total Hb and taking into account the stoichiometry of the molecule (4Fe(Hb)). Furthermore, elemental speciation strategies are carried out through the use of hybrid techniques (e.g. HPLC-ICPMS). The separation of Hb is performed by liquid chromatography using cation or anion exchange columns. The detection is conducted by measuring the iron contained in the haeme-group of the protein, using a sector field ICPMS at medium resolution. In order to develop a primary reference method, isotope dilution analysis is carried out for the determination of total Hb by species-specific mode. For that purpose, the synthesis of an isotopically labelled standard Hb (Hb-57 Fe) is performed. This ID strategy is compared with the species-unspecific mode in terms of accuracy and precision.

The authors would like to call the reader's attention to the fact that unfortunately there was a mistake in the authorship of reference 27 in the original article. The correct reference is given below: 27. Sargent M., Harte R., Harrington C. Guidelines for achieving high accuracy in isotope dilution mass spectrometry (IDMS). Cambridge: Royal Society of Chemistry; 2002.