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

Abstract

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.