Highly sensitive analysis of four hemeproteins by dynamically-coated capillary electrophoresis with chemiluminescence detector using an off-column coaxial flow interface.

Dynamic coating of the surface in capillary electrophoresis with chemiluminescence detection (CE-CL) using an off-column coaxial flow interface for the determination of four hemeproteins was developed. This method is based on the luminol-hydrogen peroxide reaction catalyzed by metalloproteins in alkaline medium. The experimental setup of the CE-CL system with the proposed off-column coaxial interface was evaluated by separation and detection of dopamine and catechol based on inhibition of the luminol-potassium ferricyanide reaction. Highly efficient separation of the two model compounds with symmetrical peak shape and satisfactory reproducibility was achieved by using this interface. In addition, in order to obtain a good resolution for hemeproteins, polyvinylpyrrolidone (PVP) combined with sodium dodecyl sulfate (SDS) were introduced as dynamic modifiers to reduce the unwanted adsorption of non-specific protein. Several parameters affecting the CE separation and CL detection were investigated in detail. Under the optimized conditions, a mixture of the four hemeproteins (horseradish peroxidase (HRP), catalase (Cat), myoglobin (Mb) and cytochrome C (Cyt C)) could be well separated within 20 min. The linear ranges of the four proteins were 5.7 × 10(-8) to 1.1 × 10(-6) mol L(-1) for HRP, 4.0 × 10(-8) to 2.0 × 10(-6) mol L(-1) for Cat, 1.1 × 10(-10) to 5.6 × 10(-8) mol L(-1) for Mb, and 3.8 × 10(-7) to 7.7 × 10(-6) mol L(-1) for Cyt C. The limits of detection (LODs) (S/N = 3) for HRP, Cat, Mb and Cyt C were 2.2 × 10(-8) mol L(-1) (104.5 amol), 1.6 × 10(-8) mol L(-1) (74 amol), 5.6 × 10(-11) mol L(-1) (0.26 amol), and 1.95 × 10(-7) mol L(-1) (0.89 fmol), respectively. The proposed method has been successfully applied to the analysis of low-level Mb in a spiked human urine sample and the recoveries were above 97%. Our primary result demonstrated that the proposed CE-CL method has great potential for Mb determination in clinical diagnosis.