Novel approach for the analysis of glycated hemoglobin using capillary isoelectric focusing with chemical mobilization

In this work, a novel CIEF methodology for the analysis of the glycated hemoglobin, HbA1c, in dimethylpolysiloxane coated fused-silica capillaries (DB-1, 50 μm I.D., 27 cm, 0.20 μm coating thickness), using a narrow pH ampholyte mixture (4% pH 6–8:pH 3–10, 10:1, v/v) in 0.30% methylcellulose, was developed. In the focusing procedure, a 0.100-mol l−1 phosphoric acid solution was used as anolyte and a 0.040-mol l−1 NaOH solution was used as catholyte. During method development, two types of mobilization of the focused hemoglobins were tested: pressure and chemical mobilization. Chemical mobilization performed better, allowing the complete baseline resolution of the hemoglobin of interest, HbA1c, from its adjacent peak, HbA, in less than 8 min. In the chemical mobilization procedure, the catholyte was replaced by a 0.040-mol l−1 NaOH solution containing 0.080 mol l−1 NaCl. The proposed methodology was applied to the analysis of 31 hemolysate samples and validated with respect to the selectivity, inter-assay and intra-assay precision (both migration time and hemoglobin percentage concentration). In addition, HbA1c determinations were compared for the CIEF method and a chromatographic standardized procedure using cation-exchanger columns (Variant™, Bio-Rad), adopted in a local clinical laboratory, showing excellent correlation (r2=0.872, n=31). The slope was found to be statistically equal to one but the intercept differed from zero. Also the Bland-Altman plot indicates bias, implying that the CIEF method yields HbA1c concentration higher than the reference method. The separation of the hemoglobins HbA, HbA2, HbF and HbA1c and the variants HbS and HbC was also demonstrated (8 min run). The resolving power of the proposed CIEF method allowed baseline resolution of hemoglobins with a pI difference as small as ca. 0.03, as it is the case for the pairs HbC/HbA2 and HbA/HbA1c.