DETERMINATION OF SUNSCREEN AGENTS BY UPLC AND HPLC 471 Ethylhexyl Methoxycinnamate (OMC) OH � C-OCH2 I H(CH2hCH3 CH2CH3 # Ethylhexyl Salicylate (OS) lsoamyl p-Methoxycinnamate (Th1C) � �NOH I N-N Methylene Bis-Benzotriazolyl -Tetramethylbutylphenol(TM) Ethylhexyl Triazone(OT) Bis-Ethylhexyloxyphenol -Methoxyphenyl Triazine(TS) Figure 1. Chemical structures of OMC, IMC, OS, OT, TM, and TS. which enabled temperature control of the analytical column. Analytical data was col­ lected and processed by the system's own chromatographic software, Empower II. The column used was a Waters Symmetry ClS column (4.6 mm x 75 mm, 3.5 µm).

472 JOURNAL OF COSMETIC SCIENCE The UPLC used in the test was the Waters Acquity Ultra Performance Liquid Chro­ matographic system (Waters, Milford, MA). The system consisted of an Acquity 996 PDA detector, a binary pump, an autosampler, and a column oven. The data was collected and processed by Empower II. The column used was a Waters UPLC BHS Cl8 column (2.1 mm x 50 mm, 1.7 µm). REAGENTS Ethylhexyl methoxycinnamate (Parsol MCX, OMC) was purchased from TCI (Tokyo, Japan), isoamyl p-methoxycinnamate (Neoheliopan ElO00, IMC) was purchased from Symrise (Holzminden, Germany), ethylhexyl salicylate (Escalol 587, OS) was purchased from Sigma (St. Louis, MO), and ethylhexyl triazone (Uvinul Tl 50, OT) was purchased from BASF (New Jersey, USA). Methylene bis-benzotriazolyl tetramethyl butylphenol (Tinosorb M, TM) and bis-ethyl­ hexyloxy phenol methoxyphenyl triazine (Tinosorb S, TS) were purchased from Ciba (North Carolina, USA). Methanol, ethanol, acetonitrile, and dimethyl formamide of HPLC grade were purchased from Merck. All of the other chemicals were analytical grade (Sigma). SOLUTION PREPARATION Standard solutions. Stock standard solutions of the six sunscreen agents were prepared daily. Into a 100-ml volumetric flask were accurately weighed 19.5, 10.5, 14.2, 8.2, 16.4, and 7 .9 mg of OMC, IMC, OS, OT, TS, and TM, respectively. The solutions were dispersed with 100 ml of dimethyl formamide/methanol (50:50, v/v) by ultrasonication for 10 min. In order to build the corresponding calibration curves and evaluate the precision of the proposed method, working standard solutions of OMC, IMC, OS OT, TS, and TM were prepared as shown in Table I. Sample solution. Two hundred milligrams of the suncare product was accurately weighed into a 100-ml volumetric flask and dispersed with 100 ml of dimethyl formamide/ methanol (50:50, v/v) by ultrasonication for 10 min. The sample was filtered through 0.2-µm membrane filters (Whatman, NJ) and then analyzed by HPLC or UPLC. Method of analysis. The samples were analyzed through HPLC and UPLC. The operation conditions are summarized in Table II. Table I Preparation of Working Standard Solutions Cone. (g/ml) Flask size Flask ID Volume of Flask ID IMC OMC OS OT TM TS (ml) (source) source (ml) A 105 195 142 82 79 164 100 B 52.5 97.5 71 41 39.5 82 50 A 25 C 26.3 48.8 35.5 20.5 19.8 41 50 B 25 D 13.1 24.4 17.8 10.3 9.9 20.5 50 C 25 E 6.6 12.2 8.9 5.11 4.9 10.3 50 D 25