Optimization of Enzyme Digestion Conditions for Quantification of Glycated Hemoglobin Using Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Glycated hemoglobin (HbA1c) is used as an index of mean glycemia over prolonged periods. This study describes anoptimization of enzyme digestion conditions for quantification of non-glycated hemoglobin (HbA0) and HbA1c as diagnosticmarkers of diabetes mellitus. Both HbA0 and HbA1c were quantitatively determined followed by enzyme digestion using isotopedilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) with synthesized N-terminal hexapeptides asstandards and synthesized isotope labeled hexapeptides as internal standards. Prior to quantification, each peptide was additionallyquantified by amino acid composition analysis using ID-LC-MS/MS via acid hydrolysis. Each parameter was consideredstrictly as a means to improve digestion efficiency and repeatability. Digestion of hemoglobin was optimized when using100 mM ammonium acetate (pH 4.2) and a Glu-C-to-HbA1c ratio of 1:50 at 37oC for 20 h. Quantification was satisfactorilyreproducible with a 2.6% relative standard deviation. These conditions were recommended for a primary reference method ofHbA1c quantification and for the certification of HbA1c reference material.