DETERMINATION OF RETINOIDS IN COSMETICS 487 not obtained from direct chemical analysis but from formulators of cosmetics who re- ported the number of their products falling within several ranges of retinoid concentra- tion. In 2005, the CIR Expert Panel re-examined the use of retinoids in cosmetics and found that the concentrations of retinol and retinyl palmitate used to formulate cosmetics had not substantially changed (8). Although there are currently uncertainties in the information on the frequency and level of use, it is clear that the use of retinoids in cosmetics is widespread. After reviewing ani- mal and clinical data available in 1987 and 2005, the CIR Expert Panel concluded that retinyl palmitate and retinol were safe as cosmetic ingredients in the then-current prac- tices of use and concentration. However, questions have persisted concerning the safety of exposure to retinoids in cosmetic products. Because topically applied retinoids such as retinol and retinyl palmitate readily penetrate the skin, systemic increases in retinoid levels could result from exposure to retinoid-containing cosmetics (9). Clinical studies have shown that no signifi cant increase in serum levels of retinol is observed following multiple topical applications of retinol or retinyl palmitate (10,11). However, since serum levels of retinol are in tight homeostatic control under most physiological conditions, serum concentrations of retinol may be insensitive indicators of vitamin A status under exposure conditions leading to vitamin A toxicity (12). Therefore, additional information is needed about the effects of topically applied retinol and retinyl palmitate on retinoid levels in tissues, such as tissues in the developing fetus, which are sensitive targets for retinoid toxicity (9). In addition, animal studies have shown that topically applied reti- noic acid can be photocarcinogenic (13). While these results are controversial and are found to vary with experimental conditions (13), concerns about the effects of topically applied retinoids such as retinyl palmitate on photocarcinogenesis have been raised and are being addressed by a study funded by the National Toxicology Program (14). Chemical identifi cation and quantitation of retinoids in cosmetics are needed to address the uncertainties in currently available information on the levels of retinoids in cosmetics and to provide data on exposure levels for use in risk assessments. Several reports of analysis of cosmetic products for retinoids have appeared. Scalia et al. have reported the use of supercritical fl uid extraction and isocratic, reversed-phase high-performance liquid chromatography (HPLC) for analysis of tocopheryl acetate and retinyl palmitate in cos- metic products (15). Using this method, they successfully analyzed one day cream con- taining 0.1% retinyl palmitate. Ceugniet et al. have described the use of an isocratic, reversed-phase HPLC method suitable for analysis of cosmetic skin creams (16). These investigators were able to chromatographically resolve and identify, through use of chro- matographic standards, retinaldehyde and its isomers, all-trans-retinol, 5,8-peroxyretinal, and 5,6-epoxyretinal. The method was used to analyze a commercial cosmetic formula- tion containing 0.05% retinalaldehyde. Similarly, an isocratic, reversed-phase HPLC method, optimized for separation of retinol, retinyl acetate, and retinyl palmitate, has been used for analysis of a body lotion containing 0.075% retinyl palmitate (17). The fat-soluble vitamins A, D, E, and K were also chromatographically separated using this method. Electrodeposition of retinal, retinol, and isomers of retinoic acid has also been investigated as an approach for determining the levels of retinoids in cosmetics (18). Fail- loux et al. have reported the use of Raman spectroscopy for the analysis of vitamin A degradation products in cosmetic product type matrices (19). In this work, specifi c condi- tions under which retinol degrades were examined. Retinol decomposition products were then identifi ed and quantifi ed by Raman spectroscopy. Flores-Perez et al. report the use of

JOURNAL OF COSMETIC SCIENCE 488 cantilever-based sensors for the detection of the three retinoids, 9-cis retinol, 13-cis retinol, and all-trans retinol, in solution (20). This method utilized changes in the resonance fre- quency of the micro-cantilever sensor, due to the attachment of the analyte molecule to the micro-cantilever’s surface, to detect and quantify retinoid molecules. While these studies demonstrate utility for the analysis of retinoids in cosmetics, none of the methods involved the simultaneous analysis of a wide variety of cosmetic matrices, using real cosmetic samples, for all three retinoids—from the highly polar retinoic acid, to the less polar retinol, to the non-polar retinyl palmitate. Nor did these methods in- volve solid-phase extraction of the retinoid from the cosmetic matrix prior to analysis. Given the diversity of cosmetic products, the complexity of their formulations, and the many potential interfering compounds contained in them, purifi cation prior to HPLC analysis is critical. Therefore, we sought to develop and validate a new procedure suitable for use in a survey of consumer cosmetics for retinoic acid, retinol, and retinyl palmitate. In addition, a limited survey of cosmetic products labeled to contain retinoids was per- formed to demonstrate the applicability of this procedure. MATERIALS AND METHODS REAGENTS AND MATERIALS The following HPLC grade reagents were used: hexane and ethyl acetate (Burdick and Jackson, Muskegon, MI), isopropanol and ammonium acetate (Fisher Scientifi c Corpora- tion, Fair Lawn, NJ), acetic acid ( J. T. Baker, Phillipsburg, PA), and methanol and dichlo- romethane (Mallikrodt, Phillipsburg, N.J.). Butylated hydroxytoluene (BHT) was obtained from Sigma-Aldrich (St. Louis, MO). Retinoids for preparation of standards were obtained from Sigma-Aldrich and were stored at −20°C. Purities reported by Sigma- Aldrich were as follows: 13-cis-retinoic acid (98%), 9-cis-retinoic acid (98%), all-trans- retinoic acid (98%), 13-cis-retinol (90%), all-trans-retinol (95%), all-trans-retinaldehyde (98%), and all-trans-retinyl palmitate (99%). De-ionized water was prepared with a Milli-Q purifi cation system from Millipore (Billerica, MA). The extraction tube and adaptors were obtained from Supelco (Bellefonte, PA). The Symmetry C18 analytical column (250 mm × 4.6 mm with a 5-μm particle size) was obtained from Waters Corporation (Milford, MA). Celite 545 was obtained from Fisher Scientifi c (Fairlawn, NJ). SAMPLES OF COSMETIC PRODUCTS Twenty-nine samples were purchased via the Internet or from local stores and included a wide range of different types of skin care products with retinol or retinyl palmitate listed as ingredients. Samples were stored in their original containers and packaging at room temperature until opened for analysis. CALIBRATION STANDARDS Actinic glassware was used for preparation and storage of all solutions of standards and samples. Seven stock solutions were prepared in a solvent comprised of 1/3 (v/v) hexane,