An anion-exchange chromatographic method for measuring bilirubin covalently bound to albumin

After alkalinizing the diazo products of serum bilirubins, we apply them to a column of anion-exchange resin. The azodipyrroles derived from unconjugated and sugar-conjugated bilirubins, as well as from one half of the tetrapyrrole covalently linked to albumin ("Bil-Alb"), are anionic and protein-free and adsorb to the resin. The other half of the azo product of Bil-Alb, with absorptivity similar to that of the protein-free azodipyrroles, remains attached to albumin (Clin Chem 28:629-637, 1982) and passes through the resin unadsorbed. The color of the eluate correlates directly with the original amount of Bil-Alb present. Estimates of authentic Bil-Alb by the method agree with those by a Jendrassik-Gróf total-bilirubin method (in mg/L: intercept = -0.8, slope = 1.005, r = 0.999) and by liquid chromatography (Clin Chem 29:800-805, 1983) (n = 52, intercept = 5.2, slope = 0.724, r = 0.965). The CV of the method for serum with Bil-Alb of 99.4 mg/L was 3.8% (n = 8).