DETERMINATION OF ALLANTOIN AND THE AL ALLANTOINATES 305 volumetric flasks. Pipette 2.00 mi. of the ammoniacal copper tartrate reagent into each flask. Mix well and place the flasks in boiling water for 10 minutes. Remove the flasks from the water, cool and discharge any residual blue color by the dropwise addition of 10% sulfuric acid. Pipette 2.00 mi. of the Folin Acid Molybdate Reagent into each flask. Dilute to the mark with distilled water and mix well. Allow 15 minutes for full color development and then measure the absorbance at 685 m•. Plot absorbance against concentration of allantoin in mg./ml. for the cal- ibration curve. Procedure. The "prepared sample" is transferred to a 250 mi. separatory funnel and extracted with three 25 mi. portions of chloroform. The organic layers are discarded. If an emulsion forms, .5 g. of ammonium sulfate may be added to hasten separation of the phases. The aqueous layer is transferred to a 100 mi. volumetric flask and is treated as described above starting with "Pipette 2.00 mi. of ammoniacal copper tartrate reagent .... " The allantoin content of the sample is read directly from the calibration curve. C. Phenylhydrazine Ferricyanide Method (3) Preparation of Calibration Curve. Transfer 3.00, 6.00, 9.00, 12.00 and 15.00 ml. aliquots of the standard allantoin solution to individual 100 ml. volumetric flasks. Ten ml. of 10% hydrochloric acid is added to each flask and the contents are heated to boiling for 3-5 minutes. Ten ml. of 1% phenylhydrazine hydrochloride solution is added to each flask while still hot. Cool and add 0.5 ml. of 10% potassium ferricyanide solution to each flask. Mix well and add 1 ml. of concentrated hydro- chloric acid to each flask. Dilute to the mark with distilled water and mix well. Allow 1.5 minutes for development of the color, and then measure the absorbance of the solutions at 530 m•. Plot absorbance against concentration of allantoin in mg./ml. for the calibration curve. Procedure. The "prepared sample" is transferred to a 250 mi. separatory funnel and extracted with three 25 mi. portions of chloroform. The or- ganic layers are discarded. If an emulsion forms, .5 g. of ammonium sulfate may be added to hasten separation of the phases. The aqueous layer is transferred to a 100 mi. volumetric flask and treated as outlined above starting with "Ten mi. of 10% hydrochloric acid is added to the flask .... "The allantoin content of the sample is read directly from the calibration curve. D. Preparation of Samples from Typical Products The procedures for the preparation of the samples differ slightly for each of the cosmetic products. Therefore, sample preparation is best con-

306 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS sidered with respect to each sample type. In each case, the final colorless aqueous solution is the "Prepared Sample" in which the allantoin content is determined by either of the methods described above. The sample size is selected on the basis of the concentration of allantoin (or aluminum allantoinate) in the preparation. For example, in the case of lipsticks, generally 0.075 to 0.1% allantoin is used, and the sample size has to be such as to insure a sufficient amount of allantoin in the final extract. Also, in the case of lipsticks, suppositories and nonaqueous preparations, it is more difficult to extract allantoin from the sample, and the larger sample minimizes the chance of error in the final results. On the other hand, extraction of allantoin, etc., from lotions and hydro- alcoholic formulations is easier, and smaller samples may be employed. In other words, it is best to select that weight of sample which would yield enough allantoin (or aluminum allantoinate) in the final extract to attain reproducible results. 1. Lipstick with ztllantoin. Suspend 25.0 g. of the lipstick in 50 ml. of distilled water. Heat the solution to boiling and dissolve 5 g. of am- monium sulfate in the hot solution. Add 5 g. of activated carbon and mix thoroughly. Immediately filter the hot solution through a pre- moistened double fluted filter paper. If the flitrate is not colorless, refilter with additional activated carbon. The clear colorless tiltrate is then transferred to a separatory funnel and treated by either of the procedures outlined above. 2. Suppository with ztluminum Dihydroxyallantoinate. Suspend 25.0 g. of the suppository formula in 50 ml. of hot distilled water and add 5 ml. of concentrated hydrochloric acid. Place the solution in a boiling water bath. Dissolve 5 g. of ammonium sulfate in the hot solution and allow it to stand in the water bath 5-10 minutes. Add 2 g. of activated carbon and mix thoroughly. Filter through a premoistened double fluted filter paper. The clear colorless flitrate is transferred to the separatory funnel and treated by either of the procedures outlined above. 3. ,4erosol Shave Cream with ,4llantoin. Mix 15.0 g. of the shave cream with 40 ml. of distilled water and dissolve 5 g. of ammonium sulfate in the solution. With slow constant stirring, gradually heat the solution to boiling. Add sufficient boiling water to bring total volume to 50 ml. Boil 1-2 minutes and add 1 g. activated carbon. Filter while hot through a premoistened double fluted filter paper. Transfer the flitrate to the separatory funnel and determine the allantoin content of the sample by either of the above procedures. 4. zterosol Shave Cream with ztluminum Dihydroxyallantoinate. Mix 15.0 g. of the cream with 40 ml. of distilled water. Add 5 ml. of concen- trated hydrochloric acid and 5 g. of ammonium sulfate. Heat in a boiling water bath for 5 minutes with constant stirring. Bring to a volume of 50