Development of an isotope dilution mass spectrometry assay for HbA1c based on enzyme-cleaved peptide analysis

HbA1c is an index of control in diabetes patients. We report a highly reproducible measurement method for HbA1c based on analysis of the enzyme-cleaved peptide by electrospray ionization mass spectrometry using deuterium-labeled synthetic peptides as internal standards. Intra- and inter-assay coefficients of variation for the novel method ranged from 1.23 to 1.99% for samples with high and low HbA1c. Using this method, we clarified the extent of discrepancies among the indices of diabetes measured by conventional methods and the ESI method for clinical samples including those from patients with Hb variants. High-performance liquid chromatography (HPLC) methods for most samples with variants underestimate the true HbA1c value, although a few variants give a positive error for HbA1c. Immunoassays may also underestimate the values, if the reactivity of the antibody is low against the glycated N-terminal of the variant beta-chains by conformational change. The method proposed here is an important step to establish a candidate definitive method, and is also useful in assessing specific HbA1c test systems using samples containing Hb variants.