title: Modified HPLC-electrospray ionization/mass spectrometry method for HbA1c based on IFCC reference measurement procedure.
abstract: 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.
authors.affiliation: Reference Measurement Laboratory, Instand e.V., Düsseldorf, Germany. kaiser@instand-ev.de
authors.forename: Patricia
authors.initials: P
authors.lastname: Kaiser
authors.forename: Theodorus
authors.initials: T
authors.lastname: Akerboom
authors.forename: Petra
authors.initials: P
authors.lastname: Molnar
authors.forename: Hans
authors.initials: H
authors.lastname: Reinauer
mesh_terms: 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
chemical_list: D002021:Buffers; D005561:Formates; D006442:Glycated Hemoglobin; D014269:Trifluoroacetic Acid; C517652:hemoglobin A1c protein, human; C030544:formic acid