CANDIDATE REFERENCE METHODS FOR HEMOGLOBIN A(1C) BASED ON PEPTIDE-MAPPING

A reference method that specifically measures hemoglobin (Nb) A(1c) is an essential part of the reference system for the international stand ardization of Hb A(1c)/glycohemoglobin. We have developed a new method far quantification, based on the specific N-terminal residue of the h emoglobin beta-chains. Enzymatic cleavage of the intact hemoglobin mol ecule with endoproteinase Glu-C has been optimized to obtain the beta- N-terminal hexapeptides of Hb A(1c) and Hb A(0). These peptides have b een separated by reversed-phase HPLC and quantitated by electrospray i onization-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 curre ntly used protein separation systems for Wb A(1c).