Determination of Porphyrins by High Performance Liquid Chromatography with Chemiluminescence Detection

Abstract

The peroxyoxalate chemiluminescence reaction, based on the oxidation of bis (2, 4 dinitrophenyl) oxalate with hydrogen peroxide, was developed as a new detection method for the determination of porphyrins after separation by reverse phase high performance liquid chromatography. To optimize signal to noise ratio, experimental parameters such as design of the detection flow-cell and the effects of pH and organic solvents were investigated. Significant enhancements in detectability, selectivity and linearity were demonstrated for the determination of various hydrophilic and hydrophobic porphyrins when compared to absorbance and fluorescence methods (e.g., the detection limits (S/N = 3) for uroporphyrin and coprophorphyrin were about 40 and 5 fmol, respectively). Application of this method for the sensitive and selective determination of urinary porphyrins was also demonstrated.