Rearranged glucuronic acid conjugates of bilirubin‐IXα in post‐obstructive bile. Structure elucidation of azopigments β and γ as ethyl anthranilate N ‐glycosides derived from 2‐, 3‐ and 4‐ O ‐acyl glucuronides

Azopigment analysis was performed on conjugates of bilirubin‐IXα in bile of man and rats obtained after obstruction of the bile duct or in bile incubated under N 2 . The azopigments β and γ, formed by applying a pH 2.7 diazonium reagent containing an excess of ethyl anthranilate, correspond to rearranged ethyl anthranilate N ‐glucuronides having the azodipyrrole acyl group on positions 2, 3 and 4 of the sugar. These assignments were verified, first by conversion of the structurally known 2‐, 3‐ and 4‐ O ‐acyl glucuronide azopigments, unsubstituted at C‐1, into ethyl anthranilate N ‐glucuronide reference compounds, and second, by mass spectrometry of trimethylsilyl ether methyl ester derivatives of unknown and reference compounds. The C‐1 ethyl anthranilate group of the N ‐glucuronides triggers characteristic fragmentation reactions of the carbohydrate moiety revealing the position of the azodipyrrole O ‐acyl group.