Modified HPLC-electrospray ionization/mass spectrometry method for HbA1c based on IFCC reference measurement procedure.

BACKGROUND Monitoring of hemoglobin A(1c) (HbA(1c)) is important in the management of diabetes. The IFCC reference measurement procedure for HbA(1c) is based on the ratio of glycated to nonglycated N-terminal hexapeptides of the beta-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 H(2)O/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% HbA(1c), 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 HbA(1c) determination set by the IFCC Working Group on HbA(1c) Standardization. CONCLUSIONS This procedure for the determination of HbA(1c) improves the existing reference measurement procedure.