Chromatographic assay and identification of mono- and diester conjugates of bilirubin in normal serum.

This liquid-chromatographic procedure for determining bilirubin mono- and diester conjugates in normal serum is based on pre-analysis conversion of bilirubin monosugar and disugar conjugates to the corresponding methyl esters by alkaline methanolysis. Here, extracted unconjugated bilirubin, bilirubin monomethyl esters, bilirubin dimethyl ester, and internal standard are separated on a reversed-phase column within 15 min, detected in the effluent at 436 nm, and quantified from their peak areas. Carotenoids do not interfere. Within-day and day-to-day CVs range from 5 to 13%. The smallest concentrations of monoconjugated and diconjugated pigments that are detectable and measurable are about 10 and 20 nmol/L, respectively. Such data are given for sera from 43 healthy adults. Total bilirubin concentrations in serum tended to be lower in women than in men, but the relative amounts of the various bilirubin fractions in sera from men and women were comparable. Analysis of ethyl anthranilate azoderivatives from serum permitted identification of the bilirubin ester conjugates in normal serum as bilirubin 1-O-acyl glucuronides.