Specificity of hemoglobin A1c measurement by cation exchange liquid chromatography. Evaluation of a Mono S column...

A method was developed for evaluating the specificity of the ion exchange chromatographic HbA1c method by Pharmacia which uses a Mono S column. To investigate hemoglobin fractions potentially interfering in HbA1c analysis, the chromatographic resolution was enhanced and a peak integration program was used which enables the quantitation of overlapping and even shoulder-like peaks. Hemoglobin was incubated with glucose in vitro and the chromatograms were analyzed before and after incubation. Five minor peaks were detected, close to hemoglobin A1c, which could not be conclusively identified as hemoglobins previously described. Four of these peaks, the stable ones, were included in the fraction of the chromatogram to be measured as HbA1c by the routine method. In practice, these peaks comprise 20%-35% of the routine HbA1c result in diabetic patients and 30%-45% in non-diabetic subjects. Three of these minor peaks were non-dependent on glucose. The chromatography and the peak integration method described can also be used to study hemoglobin adducts other than glycohemoglobin.