In the Jendrassik-Grof reaction method for the determination of serum bilirubin concentration, quantitation is obtained by measuring the green color of:

• A. Azobilirubin
• B. Bilirubin glucuronide
• C. Urobilin
• D. Urobilinogen

Answer: (A)

In this method, bilirubin is converted to a green azo dye called azobilirubin by reacting with diazotized sulfanilic acid. The intensity of this green color, when measured spectrophotometrically, is proportional to the amount of bilirubin present in the serum. The green chromophore azobilirubin is specifically formed from bilirubin in the diazo reaction, so the color observed directly reflects bilirubin concentration. Urobilin or urobilinogen do not form the same green azo dye under these conditions, which is why the quantified color corresponds to bilirubin. The procedure often includes solvents or additives to keep bilirubin soluble and to maximize complete dye formation, but the key point for quantitation is the green azobilirubin color being measured.