title: Achieving comparability with IFCC reference method for the measurement of hemoglobin A1c by use of an improved isotope-dilution mass spectrometry method.
abstract: The development of reference measurement methods for hemoglobin A1c (HbA1c) is important for quality assurance in diabetes management. The IFCC reference method using purified proteins as calibration standards is the recommended accuracy-based reference method for the standardization of HbA1c measurement. We developed a highly precise and accurate liquid chromatography-isotope-dilution tandem mass spectrometry (LC-IDMS/MS) procedure, which can serve as an alternative accuracy-based method for HbA1c measurement. In this method, enzymatic proteolysis was applied to sample preparation, followed by LC-IDMS/MS measurement of hemoglobin A0 (HbA0) and HbA1c, using two "signature" hexapeptides for calibration. The concentrations of the signature hexapeptide calibration solutions were, in turn, determined using a hydrolysis method with HCl, followed by LC-IDMS/MS measurement using amino acid solutions as calibration standards. These solutions were gravimetrically prepared from pure amino acid certified reference materials (CRMs). The developed LC-IDMS/MS method was used in participation in an IFCC ring trial for reference laboratories (RELA 2013 and 2014) for HbA1c, where our results were compared with those using the IFCC reference method. The deviations were found to be 0.4-1.7 mmol mol(-1) [or 0.04-0.16% in National Glygohemoglobin Standardization Program (NGSP) units], revealing good comparability with the IFCC reference method. The relative expanded uncertainty of the LC-IDMS/MS was in the range of 2.6% to 2.8% (1.6% to 2.2% after converting to NGSP units). With excellent method precision, good comparability with the IFCC reference method, and a small measurement uncertainty, the developed LC-IDMS/MS method may be used as an alternative accuracy-based reference method for HbA1c measurement.
authors: Chemical Metrology Division, Applied Sciences Group, Health Science Authority, 1 Science Park Road, #01-05/06, The Capricorn, Singapore Science Park II, Singapore, 117528.
authors: Hong
authors: H
authors: Liu
authors: Chemical Metrology Division, Applied Sciences Group, Health Science Authority, 1 Science Park Road, #01-05/06, The Capricorn, Singapore Science Park II, Singapore, 117528.
authors: Lingkai
authors: L
authors: Wong
authors: Chemical Metrology Division, Applied Sciences Group, Health Science Authority, 1 Science Park Road, #01-05/06, The Capricorn, Singapore Science Park II, Singapore, 117528.
authors: Sharon
authors: S
authors: Yong
authors: Chemical Metrology Division, Applied Sciences Group, Health Science Authority, 1 Science Park Road, #01-05/06, The Capricorn, Singapore Science Park II, Singapore, 117528. LIU_Qinde@hsa.gov.sg.
authors: Qinde
authors: http://orcid.org/0000-0003-0769-4642
authors: Q
authors: Liu
authors: Chemical Metrology Division, Applied Sciences Group, Health Science Authority, 1 Science Park Road, #01-05/06, The Capricorn, Singapore Science Park II, Singapore, 117528.
authors: Tong Kooi
authors: TK
authors: Lee
mesh_terms: D000596:Amino Acids / Q000032:analysis; D002853:Chromatography, Liquid / Q000379:methods; D006442:Glycated Hemoglobin / Q000032:analysis*; D006801:Humans; D007201:Indicator Dilution Techniques; D007553:Isotope Labeling / Q000379:methods; D057230:Limit of Detection; D008958:Models, Molecular; D059748:Proteolysis; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000596:Amino Acids; D006442:Glycated Hemoglobin
keywords: Hemoglobin A1c; diabetes; enzymatic proteolysis; isotope-dilution mass spectrometry (IDMS); peptide hydrolysis; reference measurement method