Received on November 27, 2007
Accepted on March 18, 2008

Endocrinology and Metabolism

Modified HPLC-Electrospray Ionization/Mass Spectrometry Method for HbA_1c Based on IFCC Reference Measurement Procedure

¹ Reference Measurement Laboratory, Instand e.V., Düsseldorf, Germany
² Institute of Clinical Chemistry and Laboratory Diagnostics, Heinrich-Heine University, Düsseldorf, Germany

^* To whom correspondence should be addressed. E-mail: kaiser{at}instand-ev.de.

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 -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 method. 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.