J. Cosmet. Sci., 66, 113–128 (March/April 2015) 113 Determination of estriol, estradiol, estrone, and progesterone in cosmetic products JEAN C. HUBINGER, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, MD 20740-3835. Accepted for publication March 1, 2015. Synopsis This report describes the development and validation of a reverse phase high-performance liquid chro- matography (HPLC) method with UV detection for the determination of the hormones estriol, estra- diol, estrone, and progesterone in topically applied products. The developed method was then used to conduct a postmarket survey of consumer products for these hormones. Each product was first mixed with Celite and then extracted with methanol. Extracts were cleaned on a Waters Oasis HLB solid phase extraction cartridge, and then analyzed using reversed phase HPLC. The analytes were separated using an Agilent Zorbax Eclipse XDB C8 (5 μm, 250 mm by 4.6 mm) analytical column and detected by their absorbance at 230 nm. Chromatographic separation was achieved by a 1.0-ml/min linear gradi- ent from 30% acetonitrile and 70% water to 80% acetonitrile and 20% water over 30 min. A final 5 min hold time and a re-equilibration time of 10 min were used to prepare the column for subsequent analysis. Recovery from two different brand lotions spiked with three different levels of estriol, estra- diol, estrone, and progesterone ranged from 81.8% to 101%. In this study, a total of 70 cosmetic prod- ucts were surveyed. Twenty two (63%) of the 35 products were labeled as containing an estrogen and/ or progesterone and also provided quantitative label information about the hormone ingredient. The most frequently labeled hormones were progesterone (66%), estriol (46%), estradiol (11%), and estrone (6%). Six products labeled as containing estriol were found to contain estradiol. An estrogen and/or progesterone were found in 34 products at concentrations ranging from 86.0 to 26,800 μg/g. Proges- terone was not found in one product labeled as containing this hormone. An additional 35 products, which did not list hormones on their labels, were analyzed and estrogen or progesterone was not de- tected in these products. INTRODUCTION The three naturally occurring estrogens in humans are the steroids estriol, 17β-estradiol, and estrone, with estriol being the predominant estrogen during pregnancy, 17β-estradiol being the major estrogen in nonpregnant women of child-bearing age, and estrone being the primary estrogen in postmenopausal women (1,2) (Figure 1). Another important class of hormones is progestagens (or progestogens), with progester- one being the naturally occurring pr ogestagen in humans. The term “progestagen” Address all correspondence to Jean C. Hubinger at Jean.Hubinger@fda.hhs.gov.

JOURNAL OF COSMETIC SCIENCE 114 derives from its function of maintaining pregnancy (progestational) and includes all ste- roids with a pregnane skeleton. However, synthetic progestagens are usually referred to as progestins (3) (Figure 2). Many plants contain compounds with chemical structures similar to that of estrogens and progesterone. For example, the plant Dioscorea mexicana, a part of the yam family native to Mexico, contains a steroid, sapogenin called diosgenin, which can be converted in the laboratory to progesterone and other steroids. Some manufacturers have incorporated plant compounds, such as diosgenin, into their cosmetic products (Figure 3). Studies of postmenopausal women show that low systemic estrogen levels are associated with dry skin, fi ne wrinkling, and declining levels of dermal collagen (4,5). Additional studies have indicated that estrogens and progesterone may have a benefi cial effect on skin functions such as elasticity and water-holding ability and on dermal collagen levels (6–11). Beginning in 2002, large-scale epidemiological studies under the Women’s Health Initiative have shown that hormone therapy poses risks as well as benefi ts. In particular, an increased risk of breast cancer and cardiovascular disease (12,13) as well as endometrial cancer, stroke, blood clots, ovarian cancer, and gallbladder disease have been associated with hormone therapies. In addition, an explicit relationship has been proposed between excessive exposure to exog- enous female sex hormones (including estriol, estradiol, estrone, and progesterone) and the risk of breast cancer in women (14,15). Because of the reported relationship between estro- gen levels and skin attributes such as dryness and fi ne wrinkling (4) and the fact that creams sold for cosmetic uses are capable of producing signifi cant drug exposures compared to oral drug products (16), there is a concern that topical application of products containing hor- mones could result in signifi cant drug exposure. There are also reports in the literature of Figure 1. Naturally occurring estrogens. Figure 2. Progestagens.