Rothera`s test for urine

Principle of Rothera’s Test
Acetoacetic acid and acetone react with alkaline solution of sodium nitroprusside to form a purple colored complex. This method can detect above 1-5 mg/dl of acetoacetic acid and 10-20 mg/dl of acetone. Beta-hydroxybutyrate is not detected.
Requirements
Urine specimen
Test tubes
Rothera’s powder:
Sodium nitroprusside = 0.75 gm
Ammonium sulphate = 20gm
Mix and pulverize.
Liquor ammonia (Ammonium hydroxide)
Procedure of Rothera’s Test
Transfer about 5 ml of urine to a test tube.
Add 1 gm of Rothera’s powder mixture and mix well.
Layer over the urine 1-2 ml of concentrated ammonium hydroxide.
Observe the pink-purple ring at the interface.
Rothera's test for ketone bodies
Observations and Results
Immediate formation of purple permanganate colored ring at the interface: Ketone bodies present (Positive)
No formation of purple permanganate colored ring at the interface: ketone bodies absent (Negative)

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