Studies on the determination of bile pigments. III. Standardization of the determination of urobilinogen as urobilinogen-aldehyde.

Several preparations of urobilin and stercobilin were investigated with regard to their value as standards for the determination of urobilinogen and urobilin in biologic specimens. Products of commercial origin were found to be impure and therefore unsuitable for this purpose. Absorptivities were measured for solutions of the pigments in 3% FICI in methanol and for the chromogens, obtained by ferrous hydroxide reduction, in the “quantitative” and “semiquantitative” methods for urobilinogen. Ascorbic acid was shown to be effective as a protective agent in filtrates from reduction mixtures. Spectra of urobilinogen-aldehyde, phenolsulfonphthalein, Pontacyl dye mixture and phenolphthalein were compared. Phenolsulfonphthalein is recommended as the artificial standard of choice. The independence of this procedurefrom instrumentalconsiderations, within limits, is demonstratedwith the useof this dye.