Mass Spectrometry Letters

Korean Society for Mass Spectrometry (한국질량분석학회)
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Optimization of Enzyme Digestion Conditions for Quantification of Glycated Hemoglobin Using Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry

  • Jeong, Ji-Seon (Center for Bioanalysis, Department of Metrology for Quality of Life, Korea Research Institute of Standards and Science)
  • Received : 2014.04.21
  • Accepted : 2014.06.05
  • Published : 2014.06.30
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Abstract

Glycated hemoglobin (HbA1c) is used as an index of mean glycemia over prolonged periods. This study describes an optimization of enzyme digestion conditions for quantification of non-glycated hemoglobin (HbA0) and HbA1c as diagnostic markers of diabetes mellitus. Both HbA0 and HbA1c were quantitatively determined followed by enzyme digestion using isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) with synthesized N-terminal hexapeptides as standards and synthesized isotope labeled hexapeptides as internal standards. Prior to quantification, each peptide was additionally quantified by amino acid composition analysis using ID-LC-MS/MS via acid hydrolysis. Each parameter was considered strictly as a means to improve digestion efficiency and repeatability. Digestion of hemoglobin was optimized when using 100 mM ammonium acetate (pH 4.2) and a Glu-C-to-HbA1c ratio of 1:50 at $37^{\circ}C$ for 20 h. Quantification was satisfactorily reproducible with a 2.6% relative standard deviation. These conditions were recommended for a primary reference method of HbA1c quantification and for the certification of HbA1c reference material.

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