Analysis of HbA1c on Ion Exchange HPLC

When glucose binds at the β N-terminal to form HbA1c the hemoglobin molecule undergoes a conformational change that results in the molecule presenting with an extra negative charge. This means that on ion-exchange chromatography systems it will separate out from normal hemoglobin (HbA0) and from hemoglobin glycated at lysine side chains which often co-elute with HbA0.

With refinement of chromatography methods and instrumentation (HPLC), hemoglobin is routinely separated into a number of peaks such as HbA1a, HbA1b, HbF (foetal Hemoglobin), HbA1c, HbA0, HbA2 and a number of hemoglobin products resulting from reaction with other molecules (glutathione, urea, aspirin etc.).

There are also a number of hemoglobin degradation products which show up with samples that have aged. On some HPLC systems these may co-elute with, or be incompletely separated from, HbA1c. In these cases, the HbA1c value obtained may be reported as higher than it actually is. Fresh blood samples contain a significant amount of labile HbA1c which in some of the earlier analysers is removed by a pre-incubation step. Newer analysers separate the labile form from the stable HbA1c and do not need pre-incubation of the sample.

Platforms that use ion exchange HPLC include BioRad D-10, Diastat, Variant, Variant II and Variant Turbo; TOSOH 2.2 Plus, G7, G8, Menarini Arkray Adams HA 8160. The integrity of a HbA1c control is more critical with these methods because any degradation can result in the increase of interfering peaks that may co-elute (and co-integrate) with HbA1c. For this reason, the useable reconstituted or open vial times for these methods may be less than for an Immuno Assay procedure.