304 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Deshusses and Desbaumes (6) is applicable to the determination of al- lantoin in these preparations, but the procedure is somewhat time consum- ing. The procedures described in this report offer the advantages of speed, simplicity, and wide scope. EXPERIMENTAL vt. ztpparatus and Reagents A Beckman Model DU spectrophotometer with 1 cm. cells was used for all spectrophotometric measurements. Atmmoniacal Copper Tartrate Reagent. Dissolve 100 g. of ammonium sulfate in approximately 400 ml. of distilled water and filter into a 1 1. volumetric flask. Add 100 ml. of 10% sodium hydroxide and 12 g. of sodium tartrate. Dissolve 5 g. of cuptic sulfate in distilled water and add this solution to the flask. Dilute the contents of the flask to the mark with distilled water and mix well. This reagent will not give a blank for months if it is stored in a well-filled, tightly stoppered, amber glass bottle. Folin ztcid Molybdate Reagent. Prepare a stock solution of 30% bro- minated sodium molybdate by dissolving 3C0 g. of sodium molybdate in distilled water and diluting to 1 1. Add 2-3 drops of liquid bromine and allow the solution to stand overnight. Transfer 500 ml. of the clear supernatant liquid to a 1 1. volumetric flask and add 225 ml. of 85% phosphoric acid while stirring. Add 150 ml. of cold 25% v/v sulfuric acid while stirring, and place the flask on a hot water bath in a hood for 15 min- utes to remove excess bromine, as indicated by the disappearance of the yellow color. Cool the solution, add 75 ml. of glacial acetic acid with stirring, dilute to the mark with distilled water and mix well. Phenylhydrazine. One % aqueous solution of phenylhydrazine hydro- chloride. Potassium Ferricyanide. Ten % aqueous solution of potassium fer- ricyanide prepared fresh weekly. Standard ztllantoin Solution. Accurately weigh out 1.000 g. of al- lantoin and dissolve it in approximately 50 ml. of distilled water with the aid of heat. Transfer this solution to a 100 ml. volumetric flask and dilute to the mark with distilled water. The solution contains 10.0 rag. of allantoin/ml. This solution should be prepared fresh weekly. B. Copper Tartrate Folin ztcid Molybdate Method (¾) Preparation of Calibration Curve. Transfer 2.00, 4.00, 6.00, 8.00 and 10.00 ml. aliquots of the standard allantoin solution to individual 100 ml.

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-