Nitroprusside Test: Principle, Reaction, Procedure And Results Interpretation

What Is Nitroprusside Test?

Nitroprusside test is a biochemical test used for the detection of the free-SH groups in amino acids or the cysteine amino acid in a protein. This test uses sodium Nitroprusside reagents and ammonia solution. Nitroprusside is a complex anion consisting of an octahedral ferrous center which is surrounded by five tightly bound cyanide groups.

In this test, the thiol groups (cysteine) react with sodium Nitroprusside in the presence of excess ammonia to give a red or purple color. Nitroprusside test is commonly used to detect the presence of ketones in the urine. The test can also be used for the differentiation between cysteine and cystine as cysteine gives a positive result to this test but cystine does not.

Objective of Nitroprusside Test

  • To detect the presence of cysteine in the urine sample and diagnose cystinuria.
  • To detect the presence of amino acid cysteine in a protein solution.
  • To differentiate between the amino acids cysteine and cystine.

Principle of Nitroprusside Test

The test is based on the detection of free sulfur atoms in a solution as a result of the degradation of the –SH group. In other words, the Nitroprusside test is specific for cysteine, the only amino acid containing sulfhydryl group (-SH). The testing principle is that the free-SH group called thiol or mercapto, which is contained in the amino acid cysteine will react with Nitroprusside in the case of excess ammonia to form red compounds. The S-S-group in cysteine will give positive results in Nitroprusside testing if it is reduced first.



Reagent And Material Required


  • 2% freshly prepared Sodium Nitroprusside
  • Concentrated Sodium hydroxide
  • Sample (1% cysteine)


  • Test tubes
  • Test tube stand
  • Pipettes
  • Water bath

Procedure For Nitroprusside Test

  1. Add 2ml of amino acid solution or sample to a test tube.
  2. To the test tube, add 0.5ml of freshly prepared sodium Nitroprusside and mix thoroughly.
  3. Then add 0.5 ml of the concentrated sodium hydroxide. If a positive result is not observed immediately, the test tube can be heated in a water bath for 2 minutes.
  4. Observed the test tube for any color change or the formation of a colored complex.

Nitroprusside Test Result Interpretation

  • Positive Result: A positive results is confirmed by the presence or appearance of a red-colored solution. The color indicates presence of cysteine.
  • Negative Result: A negative result is confirmed by the absence of a red-colored solution. This confirms the absence of cysteine.

Uses of Nitroprusside Test

  1. Nitroprusside test is used to detect presence of cysteine or cysteine –containing proteins in a sample.
  2. It is also very accurate in detection of compounds like ketoacids and ketones in blood and urine that are important in the determination of the degree of ketonuria and ketonemia.
  3. It is used in the diagnosis of cystinuria as the presence of cysteine in urine is a pathological feature of the disease.

Limitations of Nitroprusside Test

  • When used on methionine, the test does not show positive result because the –SH group in methionine is not free as it is involved in the thioester linkages.
  • Some proteins may require heat coagulation for the liberation of the –SH group.