Determination of Allantoin in Urine

In the course of studies involving allantoin, difficulty was experienced in finding a suitable method for the determination of this substance in urine. The method recently introduced by Larson, 1 while including such favorable features as rapidity and the avoidance of the hydrolysis of allantoin, was not found satisfactory under varying experimental conditions. Since the Folin ammoniacal copper reagent employed in this method is not specific for allantoin, further removal of interfering substances from the urine prior to its addition to the copper reagent seemed desirable. A method involving this modification is the basis of the present report.

Following the precipitation of interfering substances with phosphotungstic acid and lead acetate, the removal of excess of the heavy metals is effected with hydrogen sulphide. The allantoin is then precipitated with mercuric acetate, the precipitate washed and redissolved with hydrochloric acid. The solution is then freed of mercury by means of hydrogen sulphide, and the allantoin in a relatively pure solution is determined colorimetrically with the Folin ammoniacal reagent adopted for this purpose by Larson.

The amount of phosphotungstic acid to be added to the urine is determined by preliminary tests as follows: To a series of 6 tubes add 2 cc. of neutral urine. To the first add one cc. of 10% phosphotungstic acid and to the others increasing amounts, namely 1.5, 2.0, 2.5, 3.0, and 3.5 cc. The tubes are shaken, allowed to stand for 5 minutes, and then filtered. To each of the clear filtrates, a few drops of 10% phosphotungstic acid are added. The least amount of phosphotungstic acid that yields a filtrate remaining clear despite the further addition of the acid determines the amount necessary for precipitation of interfering substances from the urine sample. A large excess of phosphotungstic acid must be avoided since it leads to bulky precipitates, which cause a loss of solution volume. If no one of the filtrates remains clear after the addition of the acid, the urine is too concentrated and must be diluted.