Peroxyoxalate chemiluminescence in aqueous solutions: Coupling of immobilized enzyme reactors and 1,1′-oxalyldiimidazole chemiluminescence reaction to flow-injection analysis and liquid chromatographic systems

A highly sensitive method for the determination of enzymatically generated hydrogen peroxide in flow-injection analysis (FIA) and liquid chromatography (LC) has been developed. A dual-line flow system is used, one carrier (or eluent) delivering the analyte and the other one the chemiluminescent reagent 1,1′-oxalyldiimidazole (ODI). The results show that the composition of the analyte flow line is not critical for the chemiluminescence detection step; even purely aqueous buffers, as generally applied if immobilized enzyme reactors (IMERs) are involved in FIA and LC, can be used without loss of sensitivity. IMERs containing either glucose oxidase or acetylcholine esterase/choline oxidase were incorporated in this flow line and favourable detection limits (S/N = 3) were obtained, i.e. 3 nM for glucose and 50 nM for acetylcholine and choline. The performance of the approach in real-sample analysis was tested by determining glucose and choline in urine samples.