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).


METHODS
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.
Kaiser
Patricia
P
Reference Measurement Laboratory, Instand e.V., Düsseldorf, Germany. kaiser@instand-ev.de
Akerboom
Theodorus
T
Molnar
Petra
P
Reinauer
Hans
H
D002021:Buffers; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods / Q000592:standards; D005561:Formates; D006442:Glycated Hemoglobin / Q000032:analysis*; D006801:Humans; D012015:Reference Standards; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000592:standards; D014269:Trifluoroacetic Acid
D002021:Buffers; D005561:Formates; D006442:Glycated Hemoglobin; D014269:Trifluoroacetic Acid; C517652:hemoglobin A1c protein, human; C030544:formic acid