DETERMINATION OF ALLANTOIN AND THE AL ALLANTOINATES 307 ml. with hot distilled water. Add 1 g. of activated carbon and filter while hot. The clear colorless flitrate is transferred to the separatory funnel, and the allantoin content is determined by either of the above methods. 5. Cream or Ointment Containing/tllantoin or/tluminum Chlorhydroxy- allantoinate /tlone or in Combination with/tntiseptics, /tntibiotics, Sulfur, Resorcin, etc. Thoroughly mix 25.0 g. of the cream with 50 ml. of dis- tilled water. Dissolve 5 g. of ammonium sulfate in the mixture. Bring to a volume of 75 ml. and boil for 2-5 minutes. Add 3 g. of activated carbon, mix thoroughly and filter immediately. Transfer the flitrate to the separatory funnel and determine the allantoin content of the sample by either of the above methods. 6. Cream or Ointment with/tluminum Dihydroxyallantoinate /tlone or in Combination with/tntiseptics,/tntibiotics, etc. Thoroughly mix 25.0 g. of the cream with 50 ml. of hot distilled water. Add $ g. of ammonium sulfate and 5 ml. of concentrated hydrochloric acid. Add sufficient hot distilled water to bring the volume of the mixture to 75 ml. and place in a boiling water bath for 5-7 minutes. Add 2 g. of activated carbon and filter while hot. Transfer the flitrate to the separatory funnel and deter- mine the allantoin content of the sample by either of the above methods. 7. Shampoo with /tllantoin /tlone or in Combination with /tntiseptics, Sulfur, etc. A 25.0 g. sample of the shampoo is treated by the same pro- cedure as the cream or ointment in (5) above. 8. Lotions with /tllantoin or /tluminum Chlorhydroxyallantoinate. A 25.0 g. sample of the lotion is treated by the same procedure as the cream or ointment in (5) above. 9. Lotion with/tluminum Dihydroxyallantoinate. A 25.0 g. sample of the lotion is treated by the same procedure as the cream with aluminum dihydroxyallantoinate in (6) above. 10. /tfter-shave Lotion with /tllantoin or /tluminum Chlorhydroxy- allantoinate. A 25.0 g. sample of the lotion is treated by the same pro- cedure as the cream or ointment in (5) above. 11. Talcums and Compressed Powders with /tllantoin or /tluminum Chlorhydroxyallantoinate. Thoroughly mix 25.0 g. of the talc or powder with 50 ml. of hot distilled water. Dissolve $ g. of ammonium sulfate in the mixture. Add sufficient hot distilled water to give a total volume of 75 ml. Boil for several minutes. Add 2 g. of activated carbon and mix thoroughly. Boil for 1 minute and filter immediately. Transfer the flitrate to the separatory funnel and determine the allantoin content by either of the methods described above. 12. Talcums and Compressed Powders with ztluminum Dihydroxyal- lantoinate ztlone or in Combination wtth /tntiseptics, Sulfur, etc. Thoroughly mix 25.0 g. of the talcum or powder with 50 ml. of hot distilled water and

308 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 10 mi. of concentrated hydrochloric acid. Dissolve 5 g. of ammonium sulfate in the mixture and place the mixture in a boiling water bath for 10 minutes with constant stirring. Add 2 g. of activated carbon, mix well and filter while still hot. Transfer the tiltrate to the separatory funnel and determine the allantoin content by either of the above procedures. E. Determination of the ztluminum ztllantoinates In formulations containing either aluminum chlorhydroxyallantoinate or aluminum dihydroxyallantoinate the aluminum content of the complex can be determined by alkalinizing the extract with ammonia water (28%) to precipitate the aluminum hydroxide which is then filtered, washed, dried, ignited and weighed. Extraction of aluminum dihydroxyallantoinate is accomplished by using 10 to 15% hydrochloric acid as the extraction medium. F. Calculations The allantoin content of the material under investigation is readily cal- culated from the following formula: % Allantoin = aliquot factor X 107 X mg. allantoin found/mi. sample weight in g. RESULTS Calibration curves were prepared for both the phenylhydrazine ferri- cyanide method and the copper tartrate-Folin acid molybdate method. In both cases, absorbance was found to be linear with concentration over the 0.1 to 1.0 mg./ml. concentration range. Several commercial cosmetic preparations were analyzed for allantoin by both methods, using appropriate sample preparation methods. The results of these analyses are presented in Table I. To demonstrate that the recorded results are specific allantoin assays, and that there were no interfering substances, the tests were repeated on controls i.e., preparations of the same formulation but without allantoin were also extracted by the same means and subjected to the same determi- nations. The results were negative for the controls in all cases. It was also noted that by the methods described herein it can be determined whether allantoin is intact or decomposed. Thus, if an aliquot of the extract was treated with the hydrochloric acid, phenylhydrazine HC1 and potassium ferricyanide without boiling, the results were negative, indicating the absence of allantoin degradation products. But when aliquots were subjected to boiling, the results were positive, indicating the presence of intact allantoin.