Method for Hemoglobin A1c Measurement Based on Peptide Analysis by Electrospray Ionization Mass Spectrometry with Deuterium-labeled Synthetic Peptides as Internal Standards

[摘要] Hemoglobin A1c (HbA1c), which is defined as Hb that is irreversibly glycated at the N-terminal valine of the β-chain, is an important index in the monitoring of glucose control in patients with diabetes (1). Considerable discrepancies in the measured values of HbA1c have been observed among methods and among laboratories (2)(3). To standardize HbA1c methods, Kobold et al. (4) proposed a high-level reference method based on liquid chromatography combined with electrospray ionization mass spectrometry (ESI-MS) analysis of the glycated and nonglycated N-terminal hexapeptides of the Hb β-chains, which are released by enzymatic cleavage of the intact Hb molecule by endoproteinase Glu-C. Their method is now the officially approved IFCC reference method (5).In our laboratory, Hb variants have been measured by ESI-MS (6)(7). Specimens containing Hb variants showed unexpected HbA1c values, especially when analyzed by HPLC. To correctly estimate the degree of Hb glycation in such specimens, we have applied the peptide method using ESI-MS as proposed by Kobold et al. (4), but the ion signals of peptides occasionally fluctuate, depending on the MS conditions in our laboratory.We devised a method with a stable-isotope-labeled internal standard to obtain more reproducible values in laboratories where mass spectrometers are used for multiple purposes. We also used synthetic nonlabeled peptides to calibrate the measurement, as reported previously (6)(7). For HbA1c standardization, we used the calibrators provided by the IFCC working group. The calibrators were prepared by mixing isolated HbA and HbA1c (8).Four hexapeptides containing the six N-terminal amino acids of the Hb β-chain were chemically synthesized by Peptide Institute Inc. (Osaka, Japan): a nonglycated, unlabeled hexapeptide (Val-His-Leu-Thr-Pro-Glu; HD0, lot no. 749-901201), 1-deoxyfructosyl-hexapeptide …

[学科分类] 过敏症与临床免疫学