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Rothera’s Test for Ketone Bodies in Urine

Rothera’s Test for ketone bodies in urine post we briefly summarise about: principle of rothera test reaction, reagents requirements, Rothera test reaction  procedure, result, application and limitations of Rothera’s Test.

Rothera’s Test for Ketone Bodies in Urine

Rothera’s test is a sort of laboratory test that is used to detect ketone bodies in urine qualitatively. Three ketones bodies or acetone bodies are present in the urine during “ketosis” and are the results of fat metabolism: acetone (2%), acetoacetic acid (20%), and beta-hydroxybutyrate (78 %).Ketone bodies are generated in the liver to re-utilize energy during hunger and uncontrolled diabetes. These are acids that can cause metabolic acidosis in people with uncontrolled diabetes.

When the rate of ketone body generation exceeds the rate of excretion, the excess ketone bodies are excreted in the urine, resulting in ketonuria. Acetone is a volatile substance that is also exhaled. Ketosis can be caused by a high-fat, low-carbohydrate diet, or it can be caused by starvation, recurrent vomiting, or a high-fat, low-carbohydrate diet. Rothera test reaction, Gerhardt’s test, Lang’s test, Lindeman’s test, Han’s test, and Tablet test are some of the current methods for detecting ketones in urine. The premise of rothera test reaction is employed in all of the tests used to identify ketonuria.


The main aim of rothera test reaction is to detect the presence of Ketone bodies within the supplied urine sample.


An alkaline solution of sodium nitroprusside reacts with acetoacetic acid and acetone to generate a purple-coloured complex. Rothera test reaction can detect acetoacetic acid concentrations of 1-5 mg/dl and acetone concentrations of 10-20 mg/dl. There is no beta-hydroxybutyrate found.



1. Urine

2. Sodium nitroprusside = 0.75 gm

3. Ammonium sulphate = 20gm.

4. Liquor Ammonia


1. Glassware

2. Test tubes

3. Pipette

4. Test tube stand


1. Take a clean test tube and add 5 ml of urine to it. Transfers  1 gm of Rothera’s powder mixture within the test tube and mix well. 

2. Add 1-2 ml of concentrated ammonium hydroxide to the urine sample within the test tube. It will create a thin layer over the urine sample. Observe the pink-purple ring at the interface.


Ketone (acetone) bodies include acetone, acetoacetic acid and β-hydroxybutyric acid. To detect the latter a modified test has to be done. (oxidize the β-hydroxybutyric acid with hydrogen peroxide to form acetoacetic acid.  Add a few drops of acetic acid to 2 ml of 1:1 diluted urine with distilled water. Boil for few minutes to discard the acetone and the acetic acid present in the urine. 

Then add 1 ml of hydrogen peroxide warm gently and carry out Rothera’s test.  It will give a positive response if β-hydroxybutyric acid is present in the urine.) Normal urine contains approximately 20 mg per 24 hours only. Ketone bodies are produced excessively in the body in starvation and in uncontrolled diabetes mellitus and starvation.


Rothera’s test

Rothera Test Reaction

Positive Test: Ketone bodies are present if a purple permanganate-coloured ring forms quickly at the contact in rothera test reaction.

Negative Test: If no permanganate coloured ring forms at the contact in rothera test reaction, the test sample does not contain ketone bodies.

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