A rapid and quantitative high performance liquid chromatographic method for assaying bilirubin and its conjugates in bile

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Abstract
A rapid and quantitative high performance liquid chromatographic method for assaying bilirubin and its conjugates in [human, dog and rat] bile was 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 IX.alpha. conjugates from bile: bilirubin diglucuronide, the 2 isomeric forms of bilirubin monoglucoside monoglucuronide diester, the 2 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 XIII.alpha. and III.alpha. isomers of both bilirubin and the conjugates. In each case, the bilirubin XIII.alpha. precedes and the bilirubin III.alpha. follows the bilirubin IX.alpha. 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 TLC and by recollection of chromatographic peaks with identification of their diazo derivatives. The method has sufficient sensitivity that in vitro conjugating systems can be explored. Recollection and reinjection indicated that no isomeric scrambling occurs during the analytical procedure.

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