JOURNAL OF COSMETIC SCIENCE 286 the retention and improving the separation of FWAs. Thus, DHAA was selected in the present work. Because the ion-pairing reagent caused damage to the chromatographic column, concen- tration of acetonitrile in the mobile phase was decreased to reduce the use of DHAA. Various concentrations of DHAA (1–5 mmol·l-1, in the ammonium acetate solution) and acetonitrile (30–40%, in the mobile phase) were evaluated to determine the mobile phase composition that offers the highest signal-to-noise (S/N) ratio and suffi cient resolution for the FWAs. The highest S/N ratio and good separation of these fi ve analytes were achieved with 2 mmol·l-1 DHAA as the ion-pairing reagent to form neutral ion pairs, which promote the hydrophobic interaction between the analytes and the C18 stationary phase. Under this condition, the best concentration of acetonitrile in the mobile phase was 35%. Moreover, the ratio of ammonium acetate, which was commonly used in the HPLC anal- ysis for improving separation, was optimized. Lower concentrations of ammonium acetate produced excessively wide peaks and long retention times, whereas higher concentrations of ammonium acetate led to insuffi cient separation of target compounds and impurities. Hence, the best composition of ammonium acetate was 10 mmol·l-1. After optimization, the initial mobile phase was determined to be a mixture of 35% ace- tonitrile and 65% 2 mmol·l-1 DHAA in 10 mmol·l-1 ammonium acetate. However, the retention time of FWA351 was too long in this situation. Thus, a gradient elution pro- gram was adopted. The concentration of acetonitrile was linearly increased to 45% from 12 to 24 min, and then decreased back to 35% from 24 to 25 min, at last, maintained the concentration for 10 min. The effects of three C18 columns, including Shiseido MG C18 (Shiseido, Tokyo, Japan), Merck RP18 column (Merck, Darmstadt, Germany), and Agilent SB C18 (Agilent, Palo Alto, CA), on the separation of the fi ve target FWAs were investigated. Chromatographic peaks of some FWAs were tailed in Merck RP18 column and Agilent SB C18 column, whereas good shape of the peak was observed using Shiseido MG C18 column. Ionic surfactant, a commonly used component in liquid detergent, might interact with ion-pairing reagents in the mobile phase and led to the weak combination of the FWAs and ion-pairing reagents. Because of this, the retention time of some analytes in detergent samples became shorter than the retention time of the same FWAs in standard solutions. Figure 4. C hromatography of standard solutions which contained fi ve fl uorescent whitening agents (FWA) standards.

FLUORESCENT WHITENING AGENTS IN COSMETICS AND LIQUID DETERGENT 287 Table II Recovery Tests Sample FWAs Sample prepared with the added FWAs standard (mg·kg-1) Analysis results (mg·kg-1) Mean recovery (%) Liquid detergent 85 0.5 0.440 ± 0.025 88.0 ± 5.0 210 0.462 ± 0.020 92.4 ± 4.0 220 0.451 ± 0.024 90.2 ± 4.8 351 0.485 ± 0.017 97.0 ± 3.4 353 0.410 ± 0.015 82.0 ± 3.0 85 5 4.19 ± 0.23 83.7 ± 4.6 210 4.47 ± 0.19 89.3 ± 3.8 220 4.31 ± 0.16 86.1 ± 3.2 351 4.77 ± 0.34 95.4 ± 6.8 353 4.26 ± 0.20 85.2 ± 4.0 85 50 45.0 ± 2.8 89.9 ± 5.6 210 46.6 ± 2.3 93.1 ± 4.6 220 45.4 ± 1.4 90.8 ± 2.8 351 48.0 ± 1.1 96.0 ± 2.2 353 43.0 ± 1.9 85.9 ± 3.8 Makeup water 85 0.5 0.429 ± 0.033 85.8 ± 6.6 210 0.440 ± 0.029 88.0 ± 5.8 220 0.451 ± 0.030 90.2 ± 6.0 351 0.407 ± 0.024 81.4 ± 4.8 353 0.416 ± 0.020 83.2 ± 4.0 85 5 4.43 ± 0.25 88.6 ± 5.0 210 4.41 ± 0.18 88.1 ± 3.6 220 4.43 ± 0.21 88.6 ± 4.2 351 4.61 ± 0.32 92.1 ± 6.4 353 4.27 ± 0.29 85.4 ± 5.8 85 50 46.5 ± 2.7 92.9 ± 5.4 210 47.7 ± 2.4 95.3 ± 4.8 220 47.8 ± 1.5 95.5 ± 3.0 351 51.7 ± 1.2 103.3 ± 2.4 353 49.9 ± 1.8 99.7 ± 3.6 Mask 85 0.5 0.431 ± 0.029 86.2 ± 5.8 210 0.406 ± 0.025 81.2 ± 5.0 220 0.410 ± 0.028 82.0 ± 5.6 351 0.422 ± 0.017 84.4 ± 3.4 353 0.507 ± 0.030 101.4 ± 6.0 85 5 4.41 ± 0.25 88.2 ± 5.0 210 4.36 ± 0.21 87.2 ± 4.2 220 4.53 ± 0.23 90.6 ± 4.6 351 4.92 ± 0.12 98.3 ± 2.4 353 4.37 ± 0.20 87.4 ± 4.0 85 50 41.2 ± 2.1 82.3 ± 4.2 210 44.8 ± 1.0 89.5 ± 2.0 220 46.2 ± 1.3 92.3 ± 2.6 351 48.4 ± 1.5 96.8 ± 3 353 48.2 ± 2.1 96.3 ± 4.2