What is Hemoglobin A1c? An Analysis of Glycated Hemoglobins by Electrospray Ionization Mass Spectrometry

Hemoglobin A1c (HbA1c) is a stable minor Hb variant formed in vivo by posttranslational modification by glucose, originally identified by using cation exchange chromatography, and containing primarily glycated N-terminal beta-chains. However, the structure(s) of the quantified species has not been elucidated, and the available methods lack a reference standard. We used electrospray ionization mass spectrometry to determine the extent of glycation of samples separated by boronate affinity and/or cation exchange chromatography. Analyses of clinical samples were consistent with the curvilinear relationship of patient glucose and HbA1c. As glycation increased, the ratio of beta-chain to alpha-chain glycation increased, and the number of glycation sites on the beta-chain increased, although these were relatively minor components. We found several glycated species that cochromatographed with HbA1c on cation exchange, including species with both glycated alpha- and beta-chains, nonglycated alpha- and glycated beta-chains, and multiply glycated beta-chains. The combined use of affinity and cation exchange chromatography with structural confirmation by electrospray ionization mass spectrometry was found to be useful in producing samples of sufficient purity for the standardization of glycohemoglobin clinical assays.