454 PHYSIOLOGICAL CHEMISTRY 



2. Ferric Chloride Test (Gerhardt). To 5 c.c. of urine in a test-tube add 

 ferric chloride solution, drop by drop, until no more precipitate forms. In the 

 presence of acetoacetic acid a Bordeaux-red color is produced ; this color may be 

 somewhat masked by the precipitate of ferric phosphate, in which case the fluid 

 should be filtered. 



A positive result from the above manipulation simply indicates the possible 

 presence of acetoacetic acid. Before making a final decision regarding the pres- 

 ence of this body make the two following control experiments : 



(a) Place 5 c.c. of urine in a test-tube, small beaker, or Erlenmeyer flask 

 and boil it vigorously for 3-5 minutes. Cool the vessel and, with the boiled 

 urine, make the test as given above. As has been already stated, acetoacetic 

 acid yields acetone upon decomposition and acetone does not give a Bordeaux- 

 red color with ferric chloride. By boiling as indicated above, therefore, any 

 acetoacetic acid present would be decomposed into acetone and carbon dioxide 

 and the test upon the resulting fluid would be negative. If positive, the color 

 is due to the presence of bodies other than acetoacetic acid. 



(b) Place 5 c.c. of urine in a test-tube, acidify with H 2 SO 4 , to free aceto- 

 acetic acid from its salts, and carefully extract the mixture with ether by shaking. 

 If acetoacetic acid is present it will be extracted by the ether. Now remove 

 the ethereal solution, evaporate it to dryness, dissolve the residue hi 1-2 c.c. 

 of water and add 3-5 drops of 3 per cent ferric chloride. Acetoacetic acid is 

 indicated by the production of the characteristic Bordeaux-red color. 



This color disappears spontaneously in 24-48 hours. Such sub- 

 stances as antipyrin, kairin, phenacetin, salicylic acid, salicylates, 

 sodium acetate, thiocyanates, and thallin yield a similar red color 

 under these conditions, but when due to the presence of any of these 

 substances the color does not disappear spontaneously but may re- 

 main permanent for days. Many of these disturbing substances are 

 soluble in benzene or chloroform and may be removed from the urine 

 by this means before extracting with ether as above. Acetoacetic 

 acid is insoluble in benzene or chloroform. 



Maxwell 1 points out possible error in use of ferric chloride test for 

 acetoacetic acid in urine of patients taking sodium bicarbonate. 



3. Sodium Nitrite Ferrous Sulphate Reaction (Hurtley). Place 10 c.c. 

 of urine in a large test-tube, add 2.5 c.c. of concentrated hydrochloric acid 

 and i c.c. of fresh i per cent sodium nitrite. Shake the tube and permit it to 

 stand for two minutes. Add 15 c.c. of concentrated ammonium hydroxide and 

 5 c.c. of 10 per cent ferrous sulphate. Shake the tube and permit it to stand. 

 Note the slow development of a violet or purple color hi the presence of aceto- 

 acetic acid. 



This test serves to detect acetoacetic acid when present in a dilu- 

 tion of i to 50,000. The concentration of the acetoacetic acid regulates 

 the speed at which the color develops. If the concentration be very 



1 Maxwell: Med. Jour. Australia, i, 458, 1920. 



