Fluorescence Methods For The Rapid Determination Of Metabolic Porphyrins In Blood

The determination of the amount of porphyrins in human blood is important for the diagnosis of porphyrias and other porphyrin related diseases, and also for the monitoring of environmental contamination. The molecular structure of the dominant porphyrins in blood are similar with each other, therefore the spectra of these porphyrins overlap mutually. It is difficult to determine the porphyrins simultaneously with conventional spectrofluorometric methods. The characteristics of synchronous fluorescence spectrometry are spectral simplification, light scattering reduction, and selectivity improvement. Synchronous fluorescence spectrometry is suitable for the analysis of multicomponent mixtures. In this dissertation, synchronous fluorescence methods that are suitable for the simply and rapidly simultaneous determination of porphyrins in blood were established. The background interference could be eliminated by derivative technique. The dissertation is composed of following parts:In the first chapter, the literature was summarized. First, the development and application of synchronous fluorescence spectrometry, the theory foundation and analytical application of variable-angle synchronous fluorescence spectrometry and matrix isopotential synchronous fluorescence spectrometry, the development and characteristics of derivative technique were described in detail. Second, the investigation of porphyrins as biomarker and the analytical methods of porphyrins were introduced. At last, the plans for the whole dissertation were put forward.In the second chapter, the derivative variable-angle synchronous fluorescence spectrometry, a simple and rapid method for the simultaneous determination of protoporphyrin and zinc protoporphyrin in whole blood was proposed. Protopoprhyrin and zinc protoporphyrin can be determined simultaneously in one scan, avoiding the spectral compensation factor for protoporphyrin and chromatographic separation. The detection limit of protoporphyrin and zinc protoporphyrin was 0.098 nmol/L and 0.088 nmol/L, respectively. The within-run imprecision (RSD, n=5) for protoporphyrin was 4.1%, and for zinc protoporphyrin was 4.2%. Mean recoveries of protoporphyrin and zinc protoporphyrin added to blood sample were 86.4 % and 72.9%, respectively. The proposed derivative variable-angle synchronous fluorescence method was applied to the determination of 30 blood samples and the total amount of protoporphyrin obtained from this method was compared with the results from the conventional fluorescence method. The results from these two methods correlate well with each other. The Bland-Altman analysis indicates that the results of total protoporphyrin from these two methods are consistent with each other, and no significant difference between these two methods.In the third chapter, the combination of derivative variable-angle synchronous fluorescence spectrometry and matrix isopotential synchronous fluorescence spectrometry was proposed. The feasibility of the proposed method for the simultaneous analysis of uroporphyrin, protoporphyrin and zinc protoporphyrin in blood was investigated. The spectra of uroporphyrin and protoporphyrin, which were overlapped each other seriously, were interfered by the spectrum of zinc protoporphyrin. Based on the selection of optimal scanning path, uroporphyrin, protoporphyrin and zinc protoporphyrin were distinguished in a single scan by derivative variable-angle matrix isopotential synchronous fluorimetric method. The spectral interference between porphyrins was eliminated. Derivative variable-angle matrix isopotential synchronous fluorescence spectrometry would hopefully become a new method for the determination of porphyrins in blood.