Diazo coupling reaction for the determination of biliverdin

Abstract

We studied the diazo coupling reaction of biliverdin using twelve kinds of diazonium salts to determine serum bilirubin concentration accurately. It was found that biliverdin reacted with various diazonium salts to form red-violet colored products with the absorption maximum at about 510 to 550 nm, and the rate constant of the reaction ranged from 1.80 × 10^-4 to 1.22× 10^-5 s^-1 at 25°C under acidic condition. The rate of reaction with the diazonium salt of 3-, 4-nitroaniline and 4-nitroaniline-2-sulfonic acid was comparatively fast, while with 4-bromoaniline and sulfanilic acid was extremely slow. By increasing pH of the reaction mixture by addition of sodium hydroxide, the reaction rate increased significantly. In this reaction, it seemed that one mole of biliverdin formed one mole of azobilirubin unlike the diazo coupling reaction of bilirubin. It was considered that serum bilirubin concentration can be determined more accurately by use of the diazonium salt which reacts readily with biliverdin.