New less temperature-sensitive microchromatographic method for the separation and quantitation of glycosylated...

This paper describes a new microchromatographic method on Bio-Rex 70 ion-exchanger, which enables the isolation and quantitation of the minor components hemoglobin AIa+b and hemoglobin AIc. The method relies on the equilibration of the resin in a polyphosphate buffer with a pH closer to the pKa of the carboxylic group of the resin, and on the adjustment of the sample pH at 5. This induces linear pH and ionic strength gradient during the elution of the hemoglobin components. The method is little affected by temperature between 20 and 30 degrees C, by the presence of the aldimine Schiff base, and is not expected to be greatly influenced by moderate fluctuations in pH and ionic strength of the buffers used. There was good correlation between the values obtained by the new micromethod and four procedures currently used, namely high-performance liquid chromatography (r = 0.96), and Trivelli Bio-Rex 70 macromethod (r = 0.99), bioaffinity chromatography (r = 0.98), and Isolab hemoglobin AI kit (r = 0.91). The method is reproducible, the interassay and the intra-assay correlation coefficients did not exceed 4.2%. The mean value for hemoglobin AIc was 4.6 +/- 0.35% for eleven non-diabetics and 8.9 +/- 2.6% for twenty-one diabetics.