A novel rapid method for simultaneous determination of three diagnostically important porphyrins in erythrocytes using hyphenated synchronous fluorescence techniques

The species and concentrations of porphyrins in erythrocytes are of great importance in the clinical screening and diagnosis of porphyrias. However, it is difficult to analyze them simultaneously by conventional spectrofluorimetry. In this paper, we proposed a novel, simple and rapid method for the simultaneous determination of three diagnostically important porphyrins in human erythrocytes, protoporphyrin IX (PP), coproporphyrin III (CP) and zinc protoporphyrin IX (ZnPP), using hyphenated techniques based on derivative matrix isopotential synchronous fluorescence spectrometry and nonlinear variable-angle synchronous fluorescence spectrometry (DMI-NLVASFS). The spectral overlapping problems were well resolved and these three components were determined in one scanning without spectral compensation factors and chromatographic separation. The detection limits were 0.58, 0.21, 0.05 nmol L(-1) for PP, CP and ZnPP, respectively. Only 30s was needed for a single scanning and the recoveries were from 73% to 105% in erythrocytes. The Bland-Altman analysis indicates no significant difference between the proposed DMI-NLVASFS method and conventional fluorimetry method. The PP level of the erythropoietic protoporphyria (EPP) patients was significantly higher than that of healthy volunteers. This method can determine PP, CP and ZnPP simultaneously in a single scanning, thus providing a potential tool for the clinical analysis of porphyrins in human erythrocytes and the differential diagnosis of porphyrias.