In-capillary derivatization approach applied to the analysis of insulin by capillary electrophoresis with laser-induced fluorescence detection

An in-capillary derivatization procedure of insulin for its subsequent capillary electrophoretic analysis (with laser-induced fluorescence detection) was developed. The in-capillary derivatization performed using the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in a borate buffer pH 8.9, was achieved by successive introduction of plugs of sample and AQC reagent followed by application of a voltage (30 kV). Derivatization reaction results from the differential transport velocities that permit the distinct zones to penetrate each other under the applied field. Reagent/sample molar ratio (Rm) and plug lengths ratio were shown to have an influence on the efficiency of the derivatization reaction. A single peak could be obtained with a high reagent/sample molar ratio (Rm ≥ 68). The tagged derivative peak intensity and efficiency were improved when reagent solution time injection was at least twice higher than that of insulin sample. The validation of the method showed a good linearity between the corrected area of the derivative peak and insulin concentrations. The relative standard deviations of the migration times and the corrected areas obtained for the tagged derivative were 2.3 and 4.6%, respectively. An efficient derivatization and separation of a mixture of insulin and two glycated forms of insulin was obtained using the technique.