Liquid-chromatographic determination of bilirubin and its conjugates in rat serum and human amniotic fluid.

Abstract

Abstract This newly developed and highly specific and sensitive procedure was applied to the determination of unconjugated bilirubin and its ester conjugates in rat serum and human amniotic fluid. Bilirubin conjugates in biological samples are converted to methyl esters by alkaline methanolysis, extracted into chloroform, and the unconjugated bilirubin and esterified pigment derivatives are fractionated by "high-performance" liquid chromatography. The separated pigments are measured spectrophotometrically. Bilirubin and its mono- and di-conjugates are readily quantitated, even in previously undetectable concentrations. Linearity was established from 0.07 to 121.2 mumol/L for unconjugated bilirubin, 0.07 to 34.6 mumol/L for the C-8 monoconjugate, 0.06 to 69.3 mumol/L for the C-12 monoconjugate, and 0.17 to 43.7 mumol/L for the di-conjugate fraction. The detection limit was 0.03 mumol/L for unconjugated bilirubin and for each monoconjugate, and 0.1 mumol/L for the di-conjugated pigment.