**2.2.3 Determination of glycated hemoglobin HbA1c**

HbA1c was determined from EDTA capillary blood immediately after obtained by the lowpressure liquid chromatography (LPLC) (DiaSTAT, USA) in conjunction with gradient elution. Before testing hemolysate is heated at 62-68°C to eliminate unstable fractions and after 5 minutes is introduced into the column. Hemoglobin species elute from the cationexchange column at different times, depending on their charge, with the application of buffers of increasing ionic strength. The concentration of hemoglobins is measured after elution from the column, which is then used to quantify HbA1c by calculating the area under each peak. Instrument calibration is always carried out when introducing a new column set procedure (Bio-RAD, Inc., 2003).
