Approved IFCC Reference Method for the measurement of HbA1c in human blood

A reference method that specifically measures hemoglo-bin (Hb) A1c is an essential part of the reference system for the international standardization of Hb A1c /glycohe-moglobin. We have developed a new method for quan-tification, based on the specific N-terminal residue of the hemoglobin b-chains. Enzymatic cleavage of the intact hemoglobin molecule with endoproteinase Glu-C has been optimized to obtain the b-N-terminal hexapep-tides of Hb A1c and Hb A0 . These peptides have been separated by reversed-phase HPLC and quantitated by electrospray ionization-mass spectrometry (method A) or by capillary electrophoresis (method B). With these peptides and hyphenated separation techniques, it has been possible to overcome the insufficient resolution of currently used protein separation systems for Hb A1c .