Quantification of glycohemoglobin in blood by mass spectrometry applying multiple-reaction monitoring.
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Chromatography, High Pressure Liquid↗
Biomedical subjects
W Hoelzel
Publications and source records attributed to W Hoelzel.
Preparation of a candidate primary reference material for the international standardisation of HbA1c determinations.
We prepared a candidate primary reference material for the forthcoming international standardisation of beta-N-terminal glycated hemoglobin A measurements. It consists of well-defined mixtures of purified beta-N-terminal glycated hemoglobin A and non-glycated hemoglobin A. First, beta-N-terminal glycated hemoglobin A and non-glycated hemoglobin A were isolated, purified to homogeneity, and characterised. The techniques used were cation exchange and affinity chromatography for the purification, and high performance liquid chromatography, capillary isoelectric focusing, electrospray ionisation mass spectrometry, and peptide mapping for the characterisation. Hemoglobins from blood of healthy, non-diabetic volunteers were obtained with a purity of > 99.5% for non-glycated hemoglobin A and of > 98.5% for beta-N-terminal glycated hemoglobin A. However, results from peptide mapping indicate that the beta-N-terminal glycated hemoglobin A preparations still contain some non-beta-N-terminal glycated hemoglobins, co-eluting with beta-N-terminal glycated hemoglobin A. The exact content of beta-N-terminal glycated hemoglobin A in these preparations could be determined by a procedure consisting of standard addition, enzymatic cleavage and quantification of the resulting beta-N-terminal peptides to be in the range from 95-97.5%. Since the beta-N-terminal glycated hemoglobin A and non-glycated hemoglobin A content could be exactly determined in the materials prepared, mixtures of both components could be successfully used to calibrate the candidate reference methods.
Chromatography, Affinity↗
Candidate reference methods for hemoglobin A1c based on peptide mapping.
A reference method that specifically measures hemoglobin (Hb) A1c is an essential part of the reference system for the international standardization of Hb A1c/glycohemoglobin. We have developed a new method for quantification, based on the specific N-terminal residue of the hemoglobin beta-chains. Enzymatic cleavage of the intact hemoglobin molecule with endoproteinase Glu-C has been optimized to obtain the beta-N-terminal hexapeptides of Hb A1c and Hb A0. These peptides have been separated by reversed-phase HPLC and quantitated by electrospray ionization-mass spectrometry (method A) or by capillary electrophoresis (method B). With these peptides and hyphenated separation techniques, it has been possible to overcome the insufficient resolution of currently used protein separation systems for Hb A1c.
Chromatography, High Pressure Liquid↗
Development of a reference system for the international standardization of HbA1c/glycohemoglobin determinations.
The essential role of glycohemoglobin measurements in diabetes care and the currently poor comparability of results between different methods used in clinical laboratories makes an international standardization absolutely necessary. The IFCC working group on HbA1c standardization is going to develop a reference system for the international standardization which is based on HbA1c as the biochemically well-defined major glycohemoglobin component, and consisting of primary and secondary reference materials, a reference method and adjusted reference and target values. The system will serve for the standardization of all glycohemoglobin assays.
Glycated Hemoglobin↗