DETERMINATION OF ALLANTOIN AND THE AL ALLANTOINATES 309 TABLE I--REsULTS OF ALLANTOIN DETERMINATIONS IN VARIOUS COSMETIC PREPARATIONS Preparation Allantoin Found Allantoin Found (Copper Tartrate- (Phenylhydrazine- Folin Acid Ferricyanide Molybdate Method) Method) 2% allantoin in PPG* 0.2% allantoin in PPG* 0.1% allantoin in PPG* After shave lotion #1 After shave lotion #2 Cosmetic cream •1 Cosmetic cream #2 Lipstick Hand cream #1 Hand cream #2 1.88 1.79 ! .90 1.82 1.90 1.86 0.194 0.188 0.190 0.190 0.190 0.190 0.101 0. 098 0.100 0.100 0.099 0.100 0.178 0. 202 0.180 0.190 0.145 0.152 0.150 0.150 2.02 1.88 2.00 1.90 1.98 1.88 1.94 1.75 0.031 0.053 0.036 0. 055 0.186 0.190 0.188 0. 200 0.155 0.162 0.150 0.166 * PPG = polypropylene glycol, DISCUSSION The mechanism of the phenylhydrazine-ferricyanide method has been reported by Young and Conway (3). These authors propose hydrolysis of allantoin to urea and glyoxylic acid, condensation of the glyoxylic iacid with phenylhydrazine and subsequent reaction of this product with ferri- cyanide to give a cherry red chromophore: o NH--CHNH. CO. NH,,: NH2 C•H NH2 • C -- , (2)-- C C=:O q- 2H•O • I HC1 H=O I I NH--C•-O NH , CH----NH (allantoin) (allantoic acid) NH• I COOH 2CO +1 q-C•-tsNHNH2--• I CHO NH• (urea) (glyoxylic acid) (phenylhydrazine) CH: N. NH. C•H5 [ q- KaFe(CN)6--'- chromophore COOH (phenylhydrazone) (cherry-red -color)

310 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS The chemical changes involved in the ammoniacal copper tartrate-Folin acid-molybdate are, at present, undefined. In all probability, the reduc- tion of the molybdenum is involved in the formation of the chromophore. In the preparation of the samples from typical cosmetic products, several factors that hindered the determination of allantoin were observed. In the case of some suppositories containing aluminum dihydroxyallantoinate, it was found that a precipitate sometimes formed in the cooled tiltrate. If this occurs, the precipitate should be removed by filtration. Certain lotions containing aluminum chlorhydroxyallantoinate or allantoin were found to be difficult to decolorize. In these cases, more activated carbon was added, and the solutions were refiltered. This process was continued until a clear colorless tiltrate was obtained. Some after-shave lotions were found to give a pink to red coloration when treated with hydrochloric acid prior to the addition of phenylhydrazine in the phenylhydrazine-ferri- cyanide method. If this occurs, the color is removed by adding activated carbon and filtering. The colorless tiltrate is boiled for 2-3 minutes, and the phenylhydrazine and potassium ferricyanide are then added as directed in the procedure, CONCLUSIONS The determination of allantoin and its aluminum derivatives in cosmetic preparations is easily carried out by the procedures outlined above. Inter- fering substances are removed by chloroform extraction, and the allantoin content of the sample is determined colorimetrically. (Received December 16, 1963) REFERENCES (1) S. B. Mecca, Proc. Sci. Sect. Toilet Goods ztssoc. No. 23, 8 (1955). Idern, Ibid. No. 39, 7 (1963). (2) Toilet Goods ztssoc. Spec. JVo. 72. (3) E. Young and C. Conway, y. Biol. Chern., 142, 839 (1942). (4) M. E1 Ridi, A. Magd, and M. E1 Marsy, Proc. Pharrn. Soc. Egypt, Sci. Ed., 36, 71 (1954). (5) A. Domnas, y. Biol. Chern. (Tokyo), 50, 46 (1961). (6) J. Deshusses and P. Desbaumes, Parrurns Cosrn•t. Sar'ons, 4, 192 (1961).