Study of bilirubin binding in human serum by high-performance liquid chromatography

We describe a high-performance single-run liquid chromatographic method for the separation of protein-bound and "free" (loosely bound) bilirubin in human serum samples. The procedure is based on both the size-exclusion and the adsorptive properties of the column packing material, LiChrosorb Diol. The elution of all sample constituents is accomplished by means of an aqueous mobile phase containing a phosphate-buffered solution of human serum albumin or adult serum, supplemented by a small amount of bilirubin. The efficacy of the developed method is exemplified by assaying the bilirubin binding to human adult and infant sera.