Novel method for high-performance liquid chromatography of azo derivatives of conjugated and unconjugated bilirubin.

A method for the separation and quantitation of ethyl anthranilate or p-iodoaniline azo derivatives of bile pigments was developed using reversed-phase high-performance liquid chromatography. A convenient separation was achieved in 15 min, permitting the quantitation of the unconjugated azo-dipyrrole (alpha o) and its glucuronide (delta), xyloside (alpha 2) and glucoside (alpha 3) conjugates. The pathological beta- and gamma-azo pigments, derived from bilirubin glucuronide isomers that occur in cholestatic bile or plasma, are also detected in this system. The results of this method as applied to bile from 25 healthy dogs were in excellent agreement with the values obtained by reversed-phase chromatography of bilirubin and its mono- and dimethyl esters produced from the corresponding conjugates by alkaline methanolysis. This system permits the sensitive and convenient determination of bilirubin and its conjugation pattern in biological fluids.