j. Cosmet. Sci., 54, 119-131 (March/April 2003) Penetration studies of vitamin E acetate applied from cosmetic formulations to the stratum corneum of an in vitro model using quantification by tape stripping, UV spectroscopy, and HPLC P. LAMPEN, W. PITTERMANN, H. M. HEISE, M. SCHMITT, H. JUNGMANN, and M. KIETZMANN, Institute of Spectrochemistry and Applied Spectroscopy, D-44013 Dortmund (P.L., H.M.H.), Henkel KGaA, D-40191 Diisseldorf (W.P., M.S.), MBR Messtechnik GmbH, D-45896 Gehenkirchen (H.J.), and Institute for Pharmacology, Toxicology and Pharmacy, Veterinary School Hannover, D-30559 Hannover (M.K.), Germany. Accepted for publication September 6, 2002. Presented in part at the ConjSrence on Stratum Corneum III, Basel, Switzerland, September 12-14, 2001, and in Proceedings of the Conference Stratum Corneum, R. Marks, J.-C. Lgvb'que, and R. Voegeli, Eds. (Martin Dunitz Ltd., London, 2002). Synopsis The skin activation and penetration capability of vitamin E acetate as an ingredient in a basic o/w cream (lameliar type), in liposomes (Rovisome ©) and microparticles (Roviparts©), was investigated under in vitro conditions (BUS model) by the adhesive stripping method. The aim of the study was to compare the analytical results obtained by UV spectroscopy (transmission) and the conventional HPLC method. For the quantitative spectrometric assay, a classical least-squares evaluation of the spectra between 265 and 350 nm, based on the constituent spectra, was used. UV spectroscopy is an economic analytical method for evaluating a large population of samples of the horny layer taken by the adhesive tape stripping method, which is an established tool for depth profiling of substances within the stratum corneum. With regard to the irritation test, no cytotoxicity was recorded for all formulations tested. However, the Roviparts © and Rovisome © cream formulations induced a considerable activation of the epidermal cells that may contribute to the penetration efficiency of Rovisome©-formulated vitamin E acetate. The Rovisome ©- formulated cream delivered a maximum amount of vitamin E acetate into the horny layer compared to the other formulations tested. The difference can be explained by an alteration of the plasticity of the horny layer inducing a strong reservoir capacity and an activation of upper epidermal cells. Moreover, the opening of the potential pathway for a follicular penetration may be part of the increased reservoir capacity. INTRODUCTION Today's cosmetic emulsions usually contain several ingredients whose function is to ameliorate the condition of the skin with respect, for example, to skin hydration or Address all correspondence to W. Pittermann. 119

120 JOURNAL OF COSMETIC SCIENCE barrier properties of the stratum corneum. Well known is the importance of vitamins, such as vitamin E or D-panthenol, as bioactive substances in cosmetic formulations. However, the release of vitamins from cosmetics into the stratum corneum requires certain application conditions and exposure times (1,2). After application, the formu- lations undergo a dramatic change due to evaporation processes, which may influence the penetration of ingredients into the horny layer, to be demonstrated by analytical meth- ods. The use of liposome- and microparticle-based formulations as transport vehicles may also increase the bioavailability of the compounds within the surface skin layer as well as diminish the exposure period for penetrating the skin surface (3-5). Depth profiling for substances penetrated can be carried out by repeated adhesive tape stripping and subsequent tape analysis (3). Experimental dermatological in vivo studies for cosmetics on animals are prohibited nowadays due to ethical and legal restrictions. Therefore, the lateral follicular skin of the isolated perfused bovine udder skin (BUS model) was used here as a viable in vitro substitute for the penetration efficiency studies carried out (6). UV spectroscopy is well suited for fast quantitative determination of trace amounts of vitamin E acetate due to the strongly absorbing chromophore. Multivariate calibration models have come into wide use when implementing quantitative spectroscopic assays based on the Beer-Lambert law in the case of overlapping spectral bands from complex samples. Classical least-squares (CLS) modeling has been used for the quantitative analy- sis of spectra under the premises that the linear additive model is valid and all compo- nent spectra contributing to the sample spectrum are known (7). The applicability of such a model for vitamin E acetate-loaded adhesive tape strips was tested in this study. The results spectrometrically obtained are compared with those from a more complex and time-consuming HPLC method, which is mainly used for such dermatological studies. MATERIALS AND METHODS LIPOSOMES/MICROPARTICLES Liposomes and microparticles are vesicles composed of phospholipids, phophatidylcho- line in particular, surrounding an inner fluid compartment. The liposomes consist of an aqueous inner compartment, and therefore the lipophilic vitamin is found in the lipid bilayer, in contrast to the microparticles that have an oily compartment surrounded by only one layer of lipids. For this study, the tocopheryl acetate was dissolved in the natural oil phase. Liposomes and microparticles were prepared by dissolving all lipo- philic components in ethanol and than carefully adding the buffer while stirring. These crude carrier suspensions were then homogenized and extruded several times through a microporous filter of 0.2 pm diameter. The particles described in this study have a size of 150 nm, measured by laser light scattering. Both types of vesicles are used for cosmetics due to their ability to transport active ingredients into the skin and to stabilize sensitive active components (4). FORMULATIONS Several formulations (Table I) that include liposomes (Rovisome ©, RS) or microparticles (Roviparts ©, RP) (ROVI GmbH & Co., Kosmetische Rohstoffe KG, Schluechtern,