Determination of haptoglobin in human serum by high-performance gel-permeation chromatography

Summary A high-performance gel-permeation chromatographic (GPC) method is described for the determination of serum haptoglobin (Hp), based on the estimation of the haptoglobin-hemoglobin (Hp−Hb) complex. Hemoglobin (Hb) is added to the serum and the resulting Hp−Hb complex is separated by GPC and detected at 415 nm. The Hp−Hb complex separated chromatographically from serum migrated electrophoretically with the globulin fraction. It was found to be stable over a period of one week at 4°C. The total analysis time for a serum sample is approximately 20 min. The minimum detection limit is 30mg/L. Relative standard deviation values were below 1% (peak area). No interferences from bilirubin or from turbidity of samples were observed. The results of the GPC analysis correlate well with those obtained by nephelometric assay (r=0.996).