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Rothera’s test Principle, Procedure, and Uses

Ketone bodies in urine are hard to find with simple urine testing methods. The Rothera Test is a special test that can check for ketones in urine.

Rothera’s test Definition

Rothera test is a type of lab test that is used to find out if ketone bodies are present in urine. During “ketosis,” three byproducts of fat metabolism, called “ketone bodies” or “acetone bodies,” are found in the urine. These are acetone (2%), acetoacetic acid (20%), and beta-hydroxybutyrate (78%).

When a person is hungry or has diabetes that isn’t under control, the liver makes Ketone bodies to re-use energy. These are the kinds of acids that can cause metabolic acidosis in people with diabetes that is not under control. If the rate of making ketone bodies is too high, the body will get rid of the extra ketone bodies through the urine. This is called ketonuria. Acetone is volatile and also exhaled through the lungs.

Ketosis may be associated with diabetes, termed diabetic ketoacidosis, or it may be the result of malnutrition, recurrent vomiting, and a diet heavy in fat and low in carbohydrates.

Aim of the Rothera’s test

Rothera’s Test is utilised to determine the presence of ketone bodies in the urine sample provided.

Rothera’s test Principle

Acetoacetic acid and acetone combine with sodium nitroprusside in an alkaline solution to make a purple complex. Rothera test can find acetoacetic acid levels above 1–5 mg/dl and acetone levels between 10–20 mg/dl. Beta-hydroxybutyrate is not detected.

Rothera’s test Requirements

  1. Specimen: Urine
  2. Glassware: Test tubes, pipette.
  3. Rothera’s powder
  4. Liquid Ammonia

Rothera’s test Procedure

  1. To perform the Rothera’s test, start with a clean test tube. Then, add 5 ml of urine to the test tube. 
  2. Next, add 1 g of Rothera’s powder mixture to the test tube and mix well. 
  3. Finally, add 1-2 ml of concentrated ammonium hydroxide to the test tube. 
  4. This will create a thin layer over the urine sample. 
  5. Observe for the formation of a pink-purple ring at the interface between the layers.

Rothera’s test Results

Positive Result: If a purple permanganate-colored ring forms immediately at the interface upon performing the test, this indicates the presence of ketone bodies in the sample.

Negative Result: On the other hand, if no permanganate-colored ring is formed at the interface, this suggests that ketone bodies are absent in the sample.

Rothera’s test

Figure 1: Rothera’s test Results


Rothera test is used for testing urine for the presence of acetone or acetoacetic acid. To ensure the accuracy and safety of the experiment, be sure to follow these guidelines:

Wash the apparatus before and after the experiment. Handle all chemicals in the laboratory with care. Do not touch the urine sample during the experiment. Use test tube holders to hold the test tubes. Make sure the test tubes are clean, as contamination may affect the results. After completing the experiment, put all the apparatus back in their designated place.


FAQs on Rothera’s test

Ammonium sulphate is used to concentrate the ketone bodies to the center of the solution.