High-performance liquid chromatographic separation of bilirubin conjugates: the effects of change in molarity and pH.
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, unconjugated mesobilirubin 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.
Goresky
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CA
McGill University Medical Clinic, Montreal, Quebec, Canada.
Gordon
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ER
D000818:Animals; D001646:Bile / Q000032:analysis*; D001663:Bilirubin / Q000031:analogs & derivatives* / Q000302:isolation & purification* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002855:Chromatography, Thin Layer / Q000379:methods; D004285:Dogs; D006027:Glycosides / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008861:Microsomes / Q000378:metabolism; D009994:Osmolar Concentration
D006027:Glycosides; D001663:Bilirubin