J. Cosmet. Sci., 70, 167–180 (July/August 2019) 167 Simultaneous Determination of Nine Sunscreen Agents by HPLC and Chemometric Analysis FATEMEH MORAFFAH, HANIEH ADLI, AKRAM ROSTAMI, MOHAMMAD R. KHOSHAYAND, MOHSEN AMINI, ZEINAB POURJABBAR, SAREH KARGAR, NAFICEH SADEGHI, and MANNAN HAJIMAHMOODI , Pharmaceutical Quality Assurance Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran (F.M., M.R.K., M.H.), Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran (F.M., H.A., A.R., M.R.K., Z.P., S.K., N.S., M.H.), Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran (M.A., M.H.), Food and Drug Administration, Tehran University of Medical Sciences, Tehran, Iran (M.A.) Accepted for publication March 13, 2019. Synopsis A worldwide outbreak of skin cancer, related to ultraviolet (UV) radiations, was reported. Therefore, primary prevention programs were initiated. Application of sunscreens is one of the most effi cient ways of protection however, their effi ciency and safety have remained a challenging issue. So, it seems necessary to consider the potential side effects for limiting the use and amount of sunscreens. In this study, an high performance liquid chromatography (HPLC) system equipped with a UV–visible detector has been used. For separation, an Agilent C18 column was used (Agilent Technologies, Santa Clara, CA). This method was applied for quantitative deter- mination of nine UV fi lters in commercial sunscreen products which were widely used in Iran. Fifty samples of Iranian and imported sunscreen products were analyzed. The detection limit was determined to be 0.439–1.481 μg/ml, and the quantization limit was determined to be 1.330–4.490 μg/ml. Also, in this study, chemometric methods were used to investigate the differences between Iranian and other countries’ sunscreen brands. It was observed that despite the amount of UV fi lters in Iranian sunscreens, which was in the allowed range, there were some differences between Iranian and other countries’ sunscreens. The proposed HPLC method allows effi cient and simultaneous analysis of UV fi lters and is suitable as a quality control assay for commercial sunscreen products. INT R ODUCTION Bec ause of the recent changes in lifestyle, people are being exposed to sunlight more and more. The harmful ultraviolet (UV) radiation, which consists of three parts (UVA, UVB, and UVC), is our main concern (1). The ozone layer screens UVC and does not let it reach the human body. UVA is the main cause of skin aging and UVB is the most hazardous part. It affects the skin quickly, causing skin burns (i.e., sunburn) and an increase in the risk of future skin cancer (2). Address all correspondence to Mannan Hajimahmoodi at hajimah@sina.tums.ac.ir.

JOURNAL OF COSMETIC SCIENCE 168 Use of sunscreens is one of the helpful recommendations to prevent the deleterious effects of UV rays. The active ingredients of sunscreens consist of physical (UV-scattering agents, such as zinc oxide and titanium dioxide) and chemical fi lters: UV absorbers such as avobenzone (BMDBM), oxybenzone, para-aminobenzoic acid (PABA), homosalate (HMS), octocrylene, 3-(4-methylbenzylidene) camphor (MBC), ethylhexyl salicylate (EHS), ethylhexyl methoxy- cinnamate (EHMC), and ethylhexyl triazone (EHT) (3). These organic compounds (chemical sunscreens) can penetrate the skin (4), where they act as a photosensitizer (5,6). They can cause an increase in the production of free radicals under illumination, which may contribute to enhanced malignant melanoma incidences (7) among consumers. Furthermore, some of the organic UV fi lters such as HMS and oxybenzone have been identifi ed as hormonal disruptors (8–10). As a result, types and amount of sunscreen agents need to be evaluated. Alt hough acid–base titration method is the current way to determine the type and amount of UV fi lters in British pharmacopeia (BP) and U.S. pharmacopeia (USP), manu- facturers cannot apply this simple method in their analysis because the substances and impurities used in their products interfere with each other. Man y high performance liquid chromatography (HPLC) methods have been developed by researchers over the years for the determination and photostability of UV fi lters in sunscreen and cosmetic products. Comparison of the photostability of fi ve UV fi lters in sunscreen agents was carried out by Vanquerp et al. (11) using a C8 column and a mobile phase consisting of methanol and water. Chi svert et al. (12) determined seven UV fi lters simultaneously, using a C18 stationary phase. This was combined with a mobile phase of ethanol, water, and acetic acid, with cyclodextrins as a mobile phase modifi er. Chi svert and Salvador (13) analyzed three most common water-soluble UV fi lters in sunscreen sprays. They used a C18 column and an isocratic mobile phase of ethanol and water with a 20 mM sodium acetate buffer. The analytical run time of this method was fi ve and a half minutes. Sch akel et al. (14) determined 16 UV fi lters, including benzophenone-3 (Benz-3), 4-tert-butyl- 4′-methoxydibenzoylmethane (BDM), octyl methoxycinnamate (OMC), and octocrylene (OCT), in sun care formulations. They used a C18 stationary phase and a gradient ethanol– aqueous acetate buffer mobile phase, containing 0.2 mM EDTA. EDTA is an agent added to the mobile phase to reduce tailing in the determination of BDM the analysis took 32 min. Smyrni otakis and Archontaki (15) used a 5-μm Hypersil BDS column for the determina- tion of OCT, OMC, Tinosorb M, and octyl salicylate using a mobile phase of methanol– acetonitrile (90:10, v/v). Salvado r and Chisvert developed an environmentally friendly method for the determina- tion of 18 UV fi lters in cosmetics. Their method used a C18 stationary phase and a mobile phase of ethanol and acetic acid, which was used for fat-soluble compounds. A mixture of ethanol and sodium acetate buffer was used for water-soluble compounds. The analysis run time was less than 30 min for the 12 fat-soluble fi lters, whereas the water-soluble fi lters took 10 min. They validated their method by the analysis of 27 samples with dif- ferent cosmetic forms, e.g., creams, lipsticks, makeup, and sunscreens (16). Kedor-H ackmann et al. (17) operated two C18 columns connected in series and a mobile phase containing acetonitrile and water to determine fi ve sunscreens in synthetic formulations. One of the most recent studies has developed and validated a HPLC method for the si- multaneous determination of 12 sunscreens in 30 minutes using a C18 column and a gradient mobile phase consisting of ethanol and acidifi ed water (18).