Synchronous Fluorescence Technique Combined With Chemometrics Methods For The Determination Of Porphyrins In Blood

Synchronous fluorescence technique has the advantages of spectral simplification,light scatterring reduction,and selectivity improvement,which is suitable for the analysis of multicomponent mixture.Recently this technique was combined with chemometrics methods with the characteristic of "mathematical separation",and the problem of seriously overlapped spectra can be solved better with resorting to "spectral differentiation" united with "mathematical separation",so it is applied more and more widely in the resolution of complex system. In this dissertation,synchronous fluorescence methods that are suitable for the simple and rapidly simultaneous determination of porphyrins in blood are established.The background interference could be eliminated by derivative technique,or the multicollinearity problem could be solved by using chemometrics methods.The dissertation is composed of following parts:In the first chapter,the literature was summarized.First,the significance of porphyrins as biomarker and the analytical methods of porphyrins were introduced.Second,the theory foundation,the development and application of synchronous fluorescence spectrometry, andthe development of synchronous fluorescence technique combined with chemometrics methods were described in detail.At last,the plans for the whole dissertation were put forward.In the second chapter,we investigated fluorescene intensity,stability,feasibility of extraction of porphyrins in different solvents at room temperature and find that dimethylformamide was a new solvent, which was stable and suitable analysis of porphyrin in whole blood.Two fluorimetric methods were developed for the rapid and simultaneous determination of zinc protoporphyrinⅨ(ZnPP) and protoporphyrinⅨ(PP) extracted in whole blood with DMF,which were excitation-emission fluorescence spectra combined with alternating trilinear decomposition algorithm and derivative matrix isopotential synchronous fluorescence spectrometry.The detection limit,precision and recovery were investigated.The average recovery of the former was(82±9)%and the latter was(85±10)%in whole blood samples,and the correlation of measured results by these two methods for 20 samples was well.In the third chapter,non-linear variable angle synchronous fluorescence technique combined with partial least squares(NVASF/PLS) was developed for the simultaneous determination of protoporphyrinⅨ(PP),uroporphyrinⅨ(UP) and coproporphyrinⅢ(CP) with detection limits of 0.40,0.20 and 0.15nmol/L respectively.Only about 30s was needed in one scanning by this simple and quick method and the recoveries of real samples were(86.5±4.2)%for protoporphyrinⅨ,(89.6±2.3)%for uroporphyrinⅨand(89.8±3.9)%for coproporphyrinⅢrespectively.In the fourth chapter,derivative matrix isopotentia-non -variable angle synchronous fluorescence spectrometry(DMI-NVASF) was proposed for the simultaneous analysis of protoporphyrinⅨ,coproporphyrinⅢand zinc protoporphyrinⅨin ultramicro blood cells. ProtoporphyrinⅨ,coproporphyrinⅢand zinc protoporphyrinⅨ,the spectra of which overlapped with each other seriously,were distinguished in a single scan by derivative matrix isopotential-non -variable angle synchronous fluorimetric method with detection limits of 0.58,0.21 and 0.05nmol/L respectively.The recoveries in blood cells were(82.5±9.1)%for protoporphyrinⅨ,(83.5±7.8)%for coproporphyrinⅢand(89.2±13)%for zinc protoporphyrinⅨrespectively.When the proposed derivative matrix isopotential-non -variable angle synchronous fluorescence spectrometry was applied to the determination of 20 blood cells,the Correlation between the total amount of protoporphyrinⅨobtained from this method and the results from the conventional fluorescence method was well.