In the Malloy and Evelyn method for the determination of bilirubin, the reagent that is reacted with bilirubin to form a purple azobilirubin is:

• A. Dilute sulfuric acid
• B. Diazonium sulfate
• C. Sulfobromophthalein
• D. Diazotized sulfanilic acid

Answer: (D)

In this assay, the color that indicates bilirubin formation comes from azo coupling with a diazonium ion. Sulfanilic acid is converted in acid to a diazonium ion, making diazotized sulfanilic acid. This electrophilic diazonium species reacts with bilirubin to form azobilirubin, a purple-colored dye. The intensity of this purple color is proportional to the bilirubin concentration in the sample, which is why this reagent is used. The other options don’t generate the azo dye with bilirubin in this method: dilute sulfuric acid is just the acid medium, not the reactive coupling partner; a general diazonium salt like diazonium sulfate isn’t specifically formed from sulfanilic acid in this context; and sulfobromophthalein acts differently and isn’t the reagent that couples with bilirubin to yield azobilirubin.