Azodipyrroles of unconjugated and conjugated bilirubin using diazotized ethyl anthranilate in dimethyl sulfoxide

We have developed a diazotization technique in which both conjugated and unconjugated bilirubin react completely. The method represents a crucial modification of the ethyl anthranilate diazo reaction originally described by K. P. M. Heirwegh, J. Fevery, J. A. T. P. Meuwissen, and J. de Groote (1974, Methods Biochem. Anal. 22, 205-250). In the presence of dimethyl sulfoxide (2 ml/ml of sample and diazo reagent), conjugated and unconjugated bilirubin in human serum and human, rat, and mouse bile reacted rapidly and completely. The azopigments were stable for at least 4 h. Addition of human serum to unconjugated bilirubin, bilirubin monoglucuronide, and human bile did not influence azopigment formation. Because the reaction solution was optically clear, total azopigments could be measured by spectrophotometry or separated and quantitated by high-performance liquid chromatography without prior extraction into nonpolar solvents. Alternatively, the pigments could also be extracted into 2-pentanone for analysis by thin-layer or high-performance liquid chromatography. This method allows the quantitation of total bilirubin and analysis of individual ethyl anthranilate azopigments after a single diazotization step.