DETERMINATION OF SUNSCREEN AGENTS BY UPLC AND HPLC 475 0 0 u � � � � � � � � � u � � .. Figure 2. Chromatogram of the standard solution of the six sunscreen agents by HPLC. Peaks (µg/ml): IMC = 26.3, OMC = 48.8, OS = 35.5, OT = 20.5, TM = 19.8, and TS = 41.0 (310.0 nm). mixture was chosen as a mobile phase for the simultaneous determination of the six sunscreen agents. OPTIMIZATION CONDITION OF UPLC (ULTRA-PERFORMANCE LIQUID CHROMATOGRAPHY) IN THE ANALYSIS OF THE SIX SUNSCREEN AGENTS As UPLC is a new technology for analysis, it has been developed to obtain increased efficiency together with a short time of analysis by the use of 1. 7 -um porous stationary phases, mobile phases at high linear velocities, and instrumentation that operates at high pressure (ca. 15.000 psi) (16). Analysis research of sunscreen agents in cosmetic products using UPLC has not been reported before. In this paper, we present a new analysis method using UPLC. At first, we performed a test of the six sunscreen agents with the default condition of UPLC in a 100-mm-long column with a flow rate of 0.4 ml/min and an ethanol-water gradient. Under this condition, the peaks of IMC and OS partially overlapped and the co-elution of OMC and IMC was observed. From the results, some modifications of the test condition were needed. In the gradient method, we tried to increase the water portion for satisfactory separation. Increasing the water portion, however, took additional time that led to poor productivity. Secondly, methanol was used as an organic solvent instead of ethanol. With methanol, we could make satisfactory determination simultaneously of the six sunscreen agents in 8 min (Figure 3). METHOD VALIDATION Specificity. Specificity was evaluated by comparing the chromatograms of a standard solution that contained six sunscreen agents and a placebo solution of the product that

476 JOURNAL OF COSMETIC SCIENCE ag Q ag ci I :i l/J I- I- 1 00 2.0[ 3CO l.OC 5 00 E. :X:• 700 9 00 ·c ro t.lrutK Figure 3. Chromatogram of the standard solution of the six sunscreen agents by UPLC. Peaks (µg/ml): IMC = 35.2, OMC = 67.8, OS = 60.1, OT = 31.0, TM = 36.2, and TS = 47.5 (310.0 nm). was mentioned in the validation studies above. Figure 4 shows HPLC and UPLC chromatograms of the standard solution and the placebo solution. As there was no significant interference in the chromatogram of the placebo solution, the specificity could be considered to be suitably validated for the proposed system. Linearity. Five standard solutions of different concentrations were prepared for the lin­ earity test. The solutions contained six sunscreen agents in which the concentration ranged from 5 µg/ml to 200 µg/ml, and the corresponding contents of active in the suncare products were from 0.25% to 10.0%. Each solution was injected three times and the calibration curve of each standard solution was analyzed by the least squares method. The test result was evaluated by the R 2 value of the regression line. Table III summarizes the observed R 2 values of HPLC and UPLC. The values were not less than 0.999, and therefore the linearity of the proposed method could be regarded as qualified. Repeatability. The sample solution from a cream A was injected to obtain the peak area and the retention time. Six injections were performed in a row to calculate the RSDs of the peak area and the retention time. Table III summarizes the observed RSDs of the HPLC and UPLC data. The RSDs were significantly smaller than 2% for the peak area and the retention time in the HPLC and UPLC system. System stability. The stability of the HPLC and UPLC system could be verified by measuring the RSDs of the retention time and the peak area of the standard solution in specific time frames, such as in the initial stage, 24 h, and 48 h. The RSDs of the peak area and the retention time were calculated. Table IV summarizes the observed RSDs of HPLC and UPLC. The RSDs of the peak area and the retention time were not more than 2% in both HPLC and UPLC. We could conclude that HPLC and UPLC constitute a stable system for the analysis of sunscreen agents.