Quantitative liquid-chromatographic estimation of bilirubin species in pathological serum

Earlier we described a "high-performance" liquid-chromatographic procedure for separating the four distinct fractions of bilirubin (unconjugated, monoconjugated, diconjugated, and protein-bound) in pathological human serum (J. Chromatogr. 226: 391-402, 1981). We have modified the prechromatography precipitation of the serum globulins required in that method and have measured the bilirubin content of the precipitate spectrophotometrically. On average, the precipitate contained less than 10% of the total bilirubin in the serum samples. Adding the value obtained for the precipitate to that obtained by chromatography for the individual bilirubin fractions gave an estimate of the concentration of the total bilirubin in the sample. For 357 samples from 132 patients, this total value correlated well with that obtained by the Jendrassik-Gróf diazo procedure (slope = 1.00; r = 0.995, linear least-squares fit). The CV for the total and fractional bilirubin measurements was, on average, less than or equal to 5% for pathological sera. Serum sampled at different times from the same patient showed significant changes in the distribution of bilirubin among the four fractions.