🔬 1. Direct Bilirubin (Conjugated) — Diazo Method

Principle

Direct (conjugated) bilirubin reacts directly with diazotized sulfanilic acid to form azobilirubin, a reddish-purple complex.

• Absorbance: ~540–560 nm

• Method: Endpoint or timed endpoint

• No accelerator needed (only conjugated bilirubin reacts)

Reagent Components

• Sulfanilic acid

• Sodium nitrite

• Hydrochloric acid (for diazo formation)

• Buffer (acetate)

• Preservatives

Some manufacturers supply:

• R1: Buffer

• R2: Diazo reagent

🧪 2. Total Bilirubin — Diazo Method (Accelerated)

Principle

Unconjugated bilirubin requires an accelerator to react with the diazo reagent.

Common accelerators:

• Caffeine–benzoate

• Surfactants

• Methanol or DMSO (older methods)

Total bilirubin reacts with diazo reagent to form azobilirubin.

• Absorbance: ~540–560 nm

• Method: Endpoint or timed endpoint

Reagent Components

• Caffeine-benzoate accelerator

• Sulfanilic acid

• Sodium nitrite

• Acid buffer

• Stabilizers and preservatives

Formats:

• 1-reagent systems (common today)

• 2-reagent systems (R1 accelerator + R2 diazo reagent)