FLUORESCENT WHITENING AGENTS IN COSMETICS AND LIQUID DETERGENT 281 of ordinary HPLC which use DAD or FLD solely. Qualifi cation with both the ultraviolet spectral comparison (210–400 nm) obtained by DAD and fl uorescence of analytes ob tained by FLD would confi rm the presence of FWAs more convincingly. The method is suitable for laboratories that do not have access to HPLC-tandem MS. The sensitivity and selectivity of the developed method was demonstrated to be suitable for this application. EXPERIMENTAL INSTRUMENTATION AND REAGENTS HPLC method was carried out using a Waters 2695 system with DAD in tandem with FLD (Waters, Milford, MA). FWAs ( 95%) was received from Anpel Laboratory Technologies (Shanghai) Inc (Anpel, Shanghai, China). HPLC-grade solvents (acetonitrile) were purchased from Merck (Merck, Darmstadt, Germany). Reagent-grade ion-pair reagent di-n-hexylammonium acetate (DHAA) was got from Tokyo Chemical Industry (Tokyo Chemical Industry, Tokyo, Japan). Other reagents were purchased from Sinopharm Chemical Reagent (Sinopharm, Shanghai, China). Samples were obtained from local markets. PREPARATION OF STANDARDS AND QUALITY CONTROL SAMPLES The fi ve standard FWAs (50.0 mg) were separately suspended in 90 ml water–acetonitrile solution (2:1, v/v) with ultrasonication in the dark. The solution was cooled to room Figure 1 . Name and structure of fi ve distyryl-type fl uorescent whitening agents (FWA).

JOURNAL OF COSMETIC SCIENCE 282 temperature, diluted to 100 ml with 2:1 water–acetonitrile, and passed through a 0.22-μm fi lter. The stock solution was mixed by equal amount of these fi ve standard solutions. Working standard solutions were obtained by diluting the stock solution to 0.10, 0.50, 2.00, 10.0, 50.0, and 100 μg·ml-1. All standard solutions were stored in the refrigerator in darkness to prevent the light-induced conversion of the trans isomers of FWAs to the cis isomers. Quality control samples were prepared containing each FWAs at low (5.0 mg·kg-1), medium (50.0 mg·kg-1), and high (500.0 mg·kg-1) concentrations to evaluate the accuracy, precision, and stability. EXTRACTION OF FWAS Cosmetics: a 1.00 g of sample was accurately weighed, treated with 8 ml of 2:1 water– acetonitrile (a small amount of quartz sand were added to emulsion, cream, lotion, and mask samples), and shaken vigorously for 1 min. The samples were sonicated for 10 min, cooled to room temperature, diluted to 10 ml with 2:1 water–acetonitrile, centrifuged at 10,000 revolutions·min-1 for 5 min, and passed through a 0.22-μm membrane fi lter. Liquid detergent: A 0.20 g of sample was accurately weighed, treated with 8 ml of 2:1 water–acetonitrile, and shaken vigorously for 1 min. The samples were sonicated for 10 min, cooled to room temperature, diluted to 10 ml with 2:1 water–acetonitrile, and passed through a 0.22 μm membrane fi lter. HPLC HPLC was carried out using a Waters 2695 system and Shiseido MG C18 column (250 × 4.6 mm, 5 μm). The target analytes were separated by a gradient elution program (Table I) with the mobile phase of a mixture of acetonitrile (A) and 2 mmol·l-1 DHAA in 10 mmol·l-1 ammonium acetate (B). The column temperature was maintained at 40°C, the fl ow rate was 1 ml·min-1, and the injection volume was 10 μl. DAD detector was set at a compro- mised optimal absorption wavelength of 210–400 nm for all target FWAs. The fl uorescence detection was monitored at an excitation wavelength of 350 nm and an emission wave- length of 432 nm. The specifi city of the HPLC protocol was evaluated by the analysis of a variety of cosmetics and detergent. These samples included makeup water, lotion, cream, emulsion, mask, and liquid detergent. The suppliers confi rmed that the samples did not contain FWAs. Table I Gradien t Elution Program Time (min) Mobile phase A: acetonitrile (%) Mobile phase B: 2 mmol·l-1 DHAA in 10 mmol·l-1 ammonium acetate (%) 0.0 35 65 12.0 35 65 24.0 45 55 25.0 35 65 35.0 35 65