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- E-ISSN 2093-8950
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Optimization of Enzyme Digestion Conditions for Quantification of Glycated Hemoglobin Using Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry
Mass Spectrometry Letters / Mass Spectrometry Letters, (P)2233-4203; (E)2093-8950
2014, v.5 no.2, pp.52-56
https://doi.org/10.5478/MSL.2014.5.2.52
(Korea Research Institute of Standard and Science)
Jeong,
J.
(2014). Optimization of Enzyme Digestion Conditions for Quantification of Glycated Hemoglobin Using Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry. Mass Spectrometry Letters, 5(2), 52-56, https://doi.org/10.5478/MSL.2014.5.2.52
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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.
- keywords
- glycated hemoglobin, isotope dilution LC-MS/MS, enzyme digestion, diabetes mellitus
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- Submission Date
- 2014-04-21
- Revised Date
- 2014-06-05
- Accepted Date
- 2014-06-05
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