Abstract

We prepared a candidate primary reference material for the forthcoming international standardisation of β-N-terminal glycated hemoglobin A measurements. It consists of well-defined mixtures of purified β-N-terminal glycated hemoglobin A and non-glycated hemoglobin A.

First, β-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 β-N-terminal glycated hemoglobin A. However, results from peptide mapping indicate that the β-N-terminal glycated hemoglobin A preparations still contain some non-β-N-terminal glycated hemoglobins, co-eluting with β-N-terminal glycated hemoglobin A. The exact content of β-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 β-Nterminal peptides to be in the range from 95–97.5%.

Since the β-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.