Gordon E R, Goresky C A
Can J Biochem. 1982 Nov;60(11):1050-7. doi: 10.1139/o82-135.
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 IX alpha 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 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 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.
已开发出一种快速定量的高效液相色谱法，用于测定胆汁中的胆红素及其结合物。该方法基于离子对反相色谱，在醋酸盐缓冲液中使用庚烷磺酸，并采用乙腈梯度逐渐增加的方式。该方法可分离胆汁中的以下胆红素IXα结合物：胆红素二葡萄糖醛酸酯、胆红素单葡萄糖苷单葡萄糖醛酸酯二酯的两种异构体形式、胆红素单葡萄糖醛酸酯的两种异构体形式以及胆红素二糖苷。然后通过增加流速从柱上洗脱未结合的胆红素。该方法还可分离胆红素及其结合物的胆红素XIIIα和IIIα异构体。在每种情况下，胆红素XIIIα先于胆红素IXα成分洗脱，胆红素IIIα跟随其后。高效色谱运行在35分钟内完成。结合物的身份通过使用薄层色谱分离的参考化合物以及通过回忆色谱峰并鉴定其重氮衍生物来确定。结果表明该方法具有足够的灵敏度，可用于探索体外结合系统。回收和重新进样表明在分析过程中没有发生异构体混淆。
