Abstract
A high performance, reversed-phase liquid chromatographic (HPLC) procedure has been developed for the separation of bilirubin fractions using a column packed with octadecyl silane-bonded wide pore silica and a new elution system which consists of acidic methanol, dimethylsulfoxide and tetra-n-butylammonium hydrogen sulfate. This procedure does not need any prior treatment of serum unlike the method of Lauff et al., in which the deproteinization of globulin with sodium sulfate was required before chromatography. Human serum bilirubin was resolved into four fractions by linear gradient elution, in which the concentration of methanol was raised. The elution profile of serum bilirubin showed the diglucuronide bilirubin as the earliest eluting peak, followed in order by the monoglucuronide, serum albumin-bound bilirubin (delta fraction of bilirubin) and finally, unconjugated bilirubin. A single column has been used for more than 300 injections of serum samples without serious degradation of resolution or efficiency.
