CE-HPLC Testing of Hemoglobin A1c for Assessment of Long Term Glycemic Control in Patients with Diabetes Mellitus

Glycated hemoglobin A1c (HbA1c) is routinely used in the diagnosis of diabetes mellitus (DM) and for monitoring the long-term glycemic control in patients already diagnosed with DM. HbA1c is measured in the clinical laboratory based either on the charge of the glycated molecule by cation exchange high performance liquid chromatography (CE-HPLC) or electrophoresis, or on the physical structure of the glycated hemoglobin via immunoassays, boronate affinity chromatography or mass spectrometry. The addition of glucose to HbA triggers a decrease in the negative charge of the molecule making chromatographic separation of HbA1c from HbA using CE-HPLC possible. Estimation of HbA1c by CE-HPLC depends upon the separation of each of the patient’s hemoglobin species from HbA1c. In this context, the estimation of HbA1c using CE-HPLC is affected by Hb mutations (Hb variants) and posttranslational modifications (e.g. carbamylation and acetylation) that also alter the charge of hemoglobin molecule. Due to the clinical implications of diagnostic of DM and maintaining glucose control, accurate quantification of HbA1c is essential. A thorough understanding of the analytical and clinical limitations of the methods used (e.g. CE-HPLC) for HbA1c evaluation is extremely important. This review discusses the interferences of Hb variants and other factors in the evaluation of HbA1c using CEHPLC methodology.