NEW PHOTOTOXICITY METHODS 323 Each of these systems is useful, depending on the type of application and specific needs. The MatTek EPI-100 system is the most sophisticated system tested, as it is con- structed to be similar to human skin, including a well-defined stratum corneum and barrier function. Because the MatTek system is constructed to be similar to human skin, it is considered to most closely mimic in viv0 test conditions. As the monolayer system is limited by the fact that (a) materials cannot easily be tested neat, (b) there is no pro- tective stratum corneum, and (c) the variability of monolayer systems is inherently high, it may be most useful as a screening tool for phototoxic effects. Finally, the yeast method is a useful and inexpensive means to assess phototoxicity on a large number of materials prior to more expensive testing. The yeast method, however, does not measure the phototoxic potential of test material metabolites in mammalian cells and tests on a substrate dissimilar from the conditions of use. With a good understanding of the limitations and advantages of the different systems, any one of these assays may be used effectively to screen materials for phototoxic effects. Since statistical measurements are more meaningful, the MatTek EPI-100 shows the most promise in this research as a final test for phototoxicity. The data presented here with the monolayer and MatTek EPI-100 systems indicate a good degree of correlation to data obtained using the published yeast assays. Any lack of correlation may be due to differences in the systems as described previously. Finally, it should be noted that the use of the monolayer and MatTek EPI-100 systems for evaluating the phototoxic potential of metabolites from topical preparations is promising. Evaluating metabolites from oral products, however, is more difficult, due to the increased complexity associated with the metabolism of oral products. REFERENCES (1) J. W. Gould, M. S. Mercurio, and C. A. Elmers, Cutaneous photosensitivity diseases induced by ex- ogenous agents,J. Am. Acad. Dermatol., 33(4), 551-573 (1995). (2) E Daniels, A simple microbiological method for demonstrating phototoxic compunts,J. Invest. Der- matol., 44, 259-267 (1965). (3) E. H. Weinbert and S. T. Spring, The evaluation in vitro of fragrance materials for phototoxic activity, J. Soc. Cosmet. Chem., 32, 303-315 (1981). (4) J. D. DiNardo, B. A. Wolf, W. E. Morris, S. Tenenbaum, and R. W. Schnetzinger, A quantitative in vitro assay for the evaluation of phototoxic potential of topically applied materials: Modification and assessment of low-level activity, J. Soc Cosmet. Chem., 36, 423-433 (1985). (5) P. A. Duffy, A. Bennett, M. Roberts, and O. P. Flint, The prediction ofphototoxic potential using hu- man A431 cells and mouse 3T3 cells, In Vitro Toxico/. New Directions, 7, 327-335 (1989).

j. Soc. Cosmet. Chem., 47, 325-336 (September/October 1996) Tretinoin assay in cosmetics and pharmaceuticals by carbon phase extraction G. RAGNO, M. VERONICO, R. MADDALENA, and C. VETUSCHI, Pharmaco Chemistry Department of University, Via E. Orabona 4, 70126 Bari, Italy. Accepted for publication September 30, 1996. Presented at IV CRSAAE Bologna, Italy, October 1991. Synopsis A tretinoin assay, useful to verify its absence in cosmetics, where the drug is forbidden, was proposed. The method was based on a carbon phase extraction that allows one to obtain the analyte free from interfering matrix components and in an enriched concentration in such a way as to attain a higher sensitivity. When the analyte was present, a quantitation was performed by UV-derivative spectrophotometry. In the presence of substances with a high absorptivity, like sun filters, besides the carbon extraction step, a further separa- tion by HPTLC was required. The drug absence was validated over 0.1 mg/100g. Since the TRT amount in pharmaceuticals for topical use is 10 to 100 mg/100g, this revelation limit assures a sufficient warranty. A simplified procedure, by direct derivative spectrophotometry, may be applied for tretinoin determination in pharmaceuticals. INTRODUCTION Tretinoin (TRT), all-trans retinoic acid, is used for the treatment of severe cystic acne (1-2) and other skin diseases (3), for its positive action on cell proliferation and kera- tinization of the skin, as well as for decreasing sebum secretion and inflammation (4). Research with retinoids and clinical observations in humans sometimes gives apparently contradictory results: while stimulating the proliferation of normal epidermal cells, retinoids may check the growth of psoriatic cells and neoplasms, but the side effects of these compounds are fairly unpleasant (3). Sun exposure during treatment must be stringently avoided. Teratogenicity for isotretinoin is well documented (5,6), and it is suspected as a potential effect of tretinoin. For these reasons, the use of tretinoin in pregnancy is forbidden. On 27 July 1976 the European Community promulgated Law 76/768, actuated in Italy with Law 11 October 1986 no. 713, which forbids the use of TRT and several other chemicals in the cosmetic field. Furthermore, in Italy the Ministry of Health, with Cir- cular 18 October 1990 no. 27, has committed the public laboratories to check for the absence of TRT in cosmetics. Analytical methods for TRT have been developed by using HPLC on biological samples (7) and on anti-aging cosmetics (8,9) the U.S. Pharmacopeia 23rd Rev. (10) reports a 325