ALL-TRANS-RETINOL IN CREAM 193 DETERMINATION OF ALL-TRANS-RETINOL AND 13-CIS-RETINOL We weighed, to the nearest 0.1 mg, approximately 0.3 g of sample into a 25-ml volumetric flask, added methanol/ethyl acetate solvent (1:1) including 0.5% (w/v) BHT, and dissolved using ultrasonics. The sample solution was obtained by filtering through a 0.45-micron filter. The sample solution was analyzed by HPLC under the following conditions: ß Detection: Ultraviolet spectrophotometer (325 nm) (U-best 55, Japan Spectroscopic Co.) ß Column: Vydac 201 TP 104-C18 (25 x 0.46 cm) (Vydac Co., U.S.A.) ß Flow Rate: 1.0 ml/min ß Oven Temperature: 35øC ß Mobile phase: 0. 136% (w/v) potassium phosphate monobasic and 0.034% (w/v) potassium phosphate dibasic solution/acetonitrile (3:7) The ratio of the value (peak response of all-trans-retinol and 13-cis-retinol in the sample solution obtained from the chromatogram/sample weight in grams) after the storage test to that before the test is evaluated as % remaining. Standard 13-cis-retinol (Sigma Chemical Co, St. Louis, U.S.A.) was used for identification of 13-cis-retinol. Since retinol is light-sensitive to photoisomerization, sampling was done in a room having pure yellow-colored fluorescent light and using brown-colored volumetric flasks and autosampler. RESULTS INFLUENCE OF OILS IN CREAM ON RETINOL Figure 1 shows an HPLC chromatogram (325 nm) of water-in-oil cream with 400 ppm of retinol after six months storage at 40øC. The cream storage test result shows decrease of all-trans-retinol and increase of 13-cis-retinol, a main isomer of all-trans-retinol. This conversion from all-trans-retinol to 13-cis-retinol is thermal isomerization (6), which can 13-cis-retinol BHT , /X, all-tras-retinoi Retention Time (min.) Figure 1. Chrornatograrn of HPLC (325 nrn) of water-in-oil cream with 400 pprn of retinol after six months storage at 40øC.

194 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS be observed when all-trans-retinol in oil is preserved at higher storage temperatures. Figure 2 shows the stability of all-tram-retinol in squalane during ten days storage at 0øC, 25øC, 50øC, and 80øC. Thermal isomerization was accelerated due to increase of temperature. This isomerization was observed in other oils, including liquid petrola- turn, jojoba oil, macadamia nut oil, and silicone oils. Figure 3 shows the relation between the weight percentage of total oils in cream and percent increase of the original amount of 13-cis-retinol after two months storage at 50øC. The larger the percent of oils in the cream formula became, from 15% to 38%, the more thermal isomerization from all-trans-retinol to 13-cis-retinol occurred proportionally. This result shows that the quantity of the oil phase had influence on the degree of thermal isomerization. INFLUENCE OF WATER IN CREAM ON RETINOL In order to investigate the influence of water in the cream on all-tram-retinol, the relation between the weight percentage of water in cream and the percent remaining of the original amount of all-trans-retinol after two months storage at 50øC was examined (Figure 4). As a result, it was clarified that the larger the percent of water in the cream formula became, from 42.03% to 65.03%, the lower was the percent remaining of all-tram-retinol. Apparently the result of thermal isomerization seems to contradict this result. But we can infer that the influence of water is stronger than that of thermal isomerization under this condition. Therefore, it is considered important for the stabi- lizing of all-trans-retinol in cream that the appropriate balance of oil and water be identified. lOO 80 70 •0 50 40 30 2O lO o o 5 lO Time(days) Figure 2. Percent remaining of all-trans-retinol in 500 ppm of retinol in squalane during ten days storage at 0øC O, 25øC ', 50øC &, and 80øC ß (with argon gas blanket).