JOURNAL OF COSMETIC SCIENCE 118 Chromatographic separation was achieved by a linear gradient from 30% acetonitrile and 70% water to 80% acetonitrile and 20% water over 30 min. The fi nal ratio was held for 5 min before returning to the starting conditions. A re-equilibration time of 10 min was used prior to the next injection. A constant fl ow rate (1.0 ml/min) and temperature (25°C) were used throughout the analysis. Hormone concentrations were determined in sample extracts and standards by duplicate injections of 20 μL. The recovery of the estrogens and progesterone from two commercially available lotions which did not contain any estrogen or progesterone was determined by fortifying the lo- tions with 100, 1000 or 10,000 μg/g of each hormone followed by extraction and HPLC analysis as described above. RESULTS AND DISCUSSION The present study was initiated to develop a method for the extraction and HPLC separa- tion of the hormones estradiol, estriol, estrone, and progesterone. Each analyte peak was baseline separated and could be unambiguously quantifi ed. Each hormone was identifi ed by comparison of peak retention times and UV spectra with known standards. Chromato- gram A in Figure 4 shows the chromatographic separation and elution order of estriol, estradiol, estrone, and progesterone as standard solutions in methanol, with retention times of 5.4, 12.9, 15.7, and 21.8 min, respectively. As expected, the retention time was found to be inversely correlated with the number of alcohol groups present in the analyte as an indicator of the analyte’s polarity. All four hormone analytes were stable with no evidence of degradation, either during extraction or chromatographic separation. Chromatograms B through E show typical chromatographic separations of extracts of cosmetic products containing estriol, estradiol, estrone, and/or progesterone. As shown by the fi ve chromatograms in Figure 4, retention times observed for each hormone were very consistent and did not vary with sample matrix. The linearity ranges for estriol, estradiol, and estrone were from 0.60 to 600 μg/g, while the linearity range for progesterone was from 0.30 to 300 μg/g. The limit of quantifi ca- tion (10 times baseline noise) ranged from 0.46 to 1.0 μg/g. The limit of detection (3.3 times baseline noise) ranged from 0.15 to 0.30 μg/g. Regression correlation coeffi cients were better than 0.995. Recovery experiments were performed on two commercially available lotions (not among the products analyzed in the study) which did not contain estriol, estradiol, estrone, or progesterone. Table I shows specifi c recoveries for these sample matrices spiked with three different concentrations (100, 1000, 10000 μg/g) of estriol, estradiol, estrone, and progesterone. Recoveries of the four hormones ranged from 81.8% to 101%. A single liquid/solid extraction of 10 ml was chosen for simplicity and rapidity since increasing the volume of methanol extractant or repeat extractions was found not to signifi cantly increase the observed recovery. For lotion A, a second recovery experiment was completed on a different day (Table I). A total of 70 products marketed as cosmetics were selected and purchased from the Inter- net. The 70 products, as shown in Table II, were creams, lotions, moisturizers, oils, and extracts and had labels claiming a diverse range of product types, hormones present, and uses. Among the 35 products labeled as containing estrogen and/or progesterone, 22 (63%)

ESTRIOL, ESTRADIOL, ESTRONE, AND PROGESTERONE IN COSMETIC PRODUCTS 119 of the labels provided quantitative information. The labeled hormones were progesterone (23 out of 35 or 66%), estriol (16 out of 35 or 46%), estradiol (4 out of 35 or 11%), and estrone (2 out of 35 or 6%). For these 35 products, indicated uses fell into four broad categories: (a) relief for or treatment of premenstrual syndrome (PMS), menstruation, perimenopause, menopause, postmenopause (b) hormone or “women’s” balance (c) Figure 4. HPLC separation of a standard solution containing estriol, estradiol, estrone and progesterone in methanol (A). Typical chromatograms of sample extracts (B), (C), (D) and (E).