Quantitative determination of hemoglobin A1c by thin-layer isoelectric focusing

A method for the quantitation of hemoglobin A1c using isoelectric focusing is reported. Hemolysates were prepared and stabilised with carbon monoxide and with potassium cyanide before the quantitation. The two preparations gave identical results. The potassium cyanide method is simple and adequate for routine purposes, but the cyanohemiglobin compound remains stable for one week only. The carbon monoxide method is more laborious, but the carboxyhemoglobin remains stable for up to one year. Quantitations of the separated fractions emerging after isoelectric focusing were made with spectrophotometry or with densitometry. No significant difference in the results could be shown. Reproducibility tests were improved by introducing transferrin as an internal standard. The specificity of the method was checked by the in vitro addition of oral hypoglycemic drugs and of insulin.