High-performance liquid chromatographic separation of bilirubin conjugates: the effects of change in molarity and pH
Goresky, C.A.; Gordon, E.R.
Journal of Chromatography 528(1): 123-141
1990
ISSN/ISBN: 0021-9673 PMID: 2384548 Document Number: 358728
A fast, sensitive high-performance liquid chromatographic method has been developed for the separation and quantitation of biliary bile pigments; this utilizes a C18 reversed-phase column with two solvents, a buffer and an organic solvent, which were changed in a linear gradient from a polar to a less polar combination. Nine glycosidic conjugates of bilirubin as well as unconjugated bilirubin and a suitable internal standard, in conjugated mesobiliribun IX.alpha., were all separated to baseline by gradient elution; the species eluted in a polar to less polar fashion. Increasing the molarity of the solvent decreased the binding of non-glucuronide pigments to the column, with a decrease in their retention times, whereas for bilirubin monoglucuronide they increased. Decrease in pH, similarly, preferentially increased bilirubin monoglucuronide retention times.