Quantitative fractionation of serum bilirubin species by reversed-phase high-performance liquid chromatography

A rapid high-performance liquid chromatographic method is described for separating and quantifying four bilirubin species present in serum: bilirubin, bilirubin monoglucuronide, bilirubin diglucuronide, and bilialbumin. Sample preparation consists of dilution with ascorbic acid and dimethylsulfoxide and filtration to remove solid materials. The diluted serum was injected directly onto a wide-pore (300 A) reversed-phase column and the bilirubin species eluted with a water-isopropanol gradient. Excellent agreement was found between the total bilirubin concentration measured by the high-performance liquid chromatographic method and a diazotized sulfanilic acid procedure.