Comparison of Spectrofluorometric and HPLC Methods for the Characterization of Fecal Porphyrins in River Otters

Abstract A spectrofluorometric method (B. Grandchamp et al. , 1980, Biochem. Biophys. Acta 629, 577–586) developed for the determination of amounts of uroporphyrin I (Uro I), coproporphyrin III (Copro III), and protoporphyrin IX (Proto IX) in skin fibroblasts was compared with a high-performance liquid chromatography (HPLC) method for the analysis of porphyrins in fecal samples of river otters ( Lutra canadensis ). Heptacarboxylate porphyrin I and coproporphyrin I, two porphyrins determined to be critical in defining the porphyrin profile in fecal samples of river otters with the HPLC method, contributed substantially to the calculation of the concentrations of Uro I and Copro III, respectively, in standard solutions of porphyrins with the spectrofluorometric method. Fluorescent components of the fecal matrix complicated the determination of the concentrations of Uro I, Copro III, and Proto IX with the spectrofluorometric method and resulted in erroneous values for the concentrations of these porphyrins compared with values determined with the HPLC method. These results indicate that the complexity of the sample, particularly with regard to the potential presence of interfering fluorescent compounds, as well as porphyrins additional to Uro I, Copro III, and Proto IX, should be considered prior to the application of the spectrofluorometric method. An alternative HPLC method developed for the rapid characterization of porphyrin profiles in fecal samples of river otters is described.