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Azopigments derived from bilirubin tetrapyrroles in bile by coupling with the diazonium salts of ethyl anthranilate and p-iodoaniline were analyzed by thin layer chromatography. From the difference between the values of alpha O-fraction in both diazo reactions, percentage of unconjugated azopigment from unconjugated bilirubin (alpha OF) was determined. The recovery rate of unconjugated bilirubin added to bile sample was 98.68 +/- 2.97% with this method. alpha OF in calcium bilirubinate gallstone group was 3.19 +/- 3.88% (mean +/- S.D., n = 14) and that of control group was 0.20 +/- 1.23% (mean +/- S.D., n = 6). There was no significant difference. But only in calcium bilirubinate gallstone group, high values of alpha OF were seen in 29% of bile samples. Although this method has been critically evaluated, it is useful to study the factors affecting the deconjugation of conjugated bilirubin in bile, such as long time incubation, changes in pH, addition of chemical agents, etc.

Quantitative determination of conjugated and unconjugated bilirubin in bile by thin layer chromatography of their azoderivatives.