Modified HPLC-Electrospray Ionization/Mass Spectrometry Method for HbA1c Based on IFCC Reference Measurement Procedure

Abstract

Abstract Background: Monitoring of hemoglobin A1c (HbA1c) is important in the management of diabetes. The IFCC reference measurement procedure for HbA1c is based on the ratio of glycated to nonglycated N-terminal hexapeptides of the β-chains of hemoglobin after digestion with Glu-C endoproteinase. We developed a modification of the original reference measurement procedure with HPLC-electrospray ionization/mass spectrometry (ESI/MS). Method: We performed chromatographic separation of the hexapeptides using a C12 reversed-phase column and a binary gradient system consisting of a mixture of H2O/acetonitrile/formic acid. Results: Using this method, we obtained higher signal intensities and improved system stability compared with the reference measurement procedure. In the range of 3% to 14% HbA1c, intralaboratory CVs were 0.71% to 1.86%. Deviations from IFCC target values were −0.87 to 1.00 relative %. These values fulfill acceptability criteria for HbA1c determination set by the IFCC Working Group on HbA1c Standardization. Conclusions: This procedure for the determination of HbA1c improves the existing reference measurement procedure.