Clinical Chemistry
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Clinical Chemistry 54: 1018-1022, 2008. First published April 10, 2008; 10.1373/clinchem.2007.100875
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Right arrow Endocrinology and Metabolism
(Clinical Chemistry. 2008;54:1018-1022.)
© 2008 American Association for Clinical Chemistry, Inc.

Endocrinology and Metabolism

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

Patricia Kaiser1,a, Theodorus Akerboom2, Petra Molnar1 and Hans Reinauer1

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

aAddress correspondence to this author at: Instand e.V., Ubierstrasse 20, 40223 Duesseldorf, Germany. Fax +49 211 15921356; e-mail kaiser{at}instand-ev.de.

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.






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Copyright © 2008 by the American Association for Clinical Chemistry.