A rapid and quantitative high performance liquid chromatographic method for assaying bilirubin and its conjugates in bile has been developed. It is based on the use of ion-pair reverse-phase chromatography, utilizing heptanesulfonic acid in an acetate buffer, with a progressively increasing gradient of acetonitrile. The method resolves the following bilirubin IXα conjugates from bile: bilirubin diglucuronide, the two isomeric forms of bilirubin monoglucoside monoglucuronide diester, the two isomeric forms of bilirubin monoglucuronide, and bilirubin diglucoside. Unconjugated bilirubin is then eluted from the column by increasing the flow rate. The method also resolves the bilirubin XIIIα and IIIα isomers of both bilirubin and the conjugates. In each case, the bilirubin XIIIα precedes and the bilirubin IIIα follows the bilirubin IXα component. The high performance chromatographic run is completed in 35 min. The identity of the conjugates was ascertained by use of reference compounds isolated by thin-layer chromatography and by recollection of chromatographic peaks with identification of their diazo derivatives. The method was shown to have sufficient sensitivity that in vitro conjugating systems can be explored. Recollection and reinjection indicated that no isomeric scrambling occurs during the analytical procedure.
