The diazo reaction used for bilirubin quantification includes which of the following components?

• A. Contains sulfanilic acid, hydrochloric acid, and sodium nitrite
• B. Contains only sulfanilic acid
• C. Contains only sodium nitrite
• D. Contains sodium hydroxide and salt

Answer: (A)

The essential idea is that bilirubin quantification by the diazo method relies on forming a diazonium compound from an aromatic amine (sulfanilic acid) under strongly acidic conditions, using nitrous acid generated from nitrite. The diazonium then couples with bilirubin to produce a colored azobilirubin complex that can be measured.

All three components are needed: sulfanilic acid provides the amine that becomes diazonium; sodium nitrite supplies the nitrous acid that converts the amine to the diazonium ion; and a strong acid like hydrochloric acid creates the acidic environment required for diazotization and stabilizes the diazonium. Without any one of these, the diazo reaction won’t proceed properly. For example, without nitrite there’s no nitrous acid to form the diazonium; without acid the diazonium wouldn’t form or would decompose; and using a base would inhibit diazotization.

Therefore, containing sulfanilic acid, hydrochloric acid, and sodium nitrite is the correct combination.