The structures of some azopigments obtained by diazotization of conjugated and unconjugated bile pigments with diazotized ethyl anthranilate were studied by mass spectrometry. The α0-azopigments derived from rat bile and human bile were shown to be identical (t.l.c. and mass spectra) with azobilirubin derived from unconjugated bilirubin. The presence of two methyl vinyl isomers (Ia) and (Ib) in equal amounts was shown by t.l.c. and mass spectrometry. The structure of the δ-azopigment derived from rat bile was studied by two methods: (a) ammonolysis gave rise to an amide having a CH2·CH2·CO·NH2 side chain as shown by its mass spectrum; (b) the mass spectrum of a trimethylsilyl derivative of the δ-azopigment methyl ester confirmed the ester to be a β-d-monoglucuronide ester of azobilirubin I.
