DIFFUSION OF PRESERVATIVES 241 EXPERIMENTAL MATERIALS Methylparaben (MP), ethylparaben (EP), and propylparaben (PP) were purchased from Fluka Chemie AG (Buchs, Switzerland). Pemulen © TR2 (acrylates/C10-30 alkyl acrylate crosspolymer) and Carbopol © 940 (Carbomer 940) were a generous gift of Biochim (Milan, Italy). All other materials and solvents of high purity grade were from Sigma Chemical Co. (St. Louis, MO). PRODUCTION OF TOPICAL DOSAGE FORMS Four different topical formulations were produced, namely a water-in-oil emulsion, an oil-in-water emulsion, and two hydrophilic gels (Pemulen gel and Carbopol gel), whose compositions are reported in Table I. After production, all the dosage forms here described were stored at 4øC until use, to minimize possible degradations. W/O emulsions. Briefly, for the preparation of the water phase, MP, EP, and PP were solubilized in boiling water. The oil-soluble components of the formulation were fused and heated to about 70øC. Production of the W/O emulsion was performed by slow addition of the aqueous phase to the oil phase under vigorous stirring by a turbine mixer. The emulsion obtained was then cooled down at room temperature. O/W emulsions. As for the preparation of the W/O emulsion, MP, EP, and PP were solubilized in boiling water. The oil-soluble components of the formulation were fused and heated to about 70øC. Production of the O/W emulsion was performed, slowly adding the oil phase to the aqueous phase under vigorous stirring by a turbine mixer. The emulsion was then cooled at room temperature. Hydrophilic gels. The production procedure was the same for both gels. MP, EP, and PP were solubilized in boiling water. For the preparation of the hydrophilic gel, the acry- lates/C10-30 alkyl acrylate crosspolymer (Pemulen © TR2) or the carboxyvinyl polymer carbomer (Carbopol © 940) was added to the solution obtained and left to swell at room temperature to obtain a homogenous and liquified mixture. After an overnight incuba- tion, triethanolamine was added to neutralize the solution. QUANTITATIVE DETERMINATION OF PRESERVATIVES Preservatives extraction from formulations. A liquid procedure was performed in order to extract parabens from formulations. Briefly, 20 ml of a mixture constituted of tetrahy- drofurane (THF)/water 90:10 v/v was added to 1 g of topical formulation and stirred for 30 min at room temperature. The solution obtained was vortexed and sonicated at 25øC for 2 min in a bath-type sonicator, Branson 2200 (Branson Ultrasonics Co., Danbury, CT) and centrifuged (Centrifuge Hareus Sepatech GmbH, Germany) at 6000 rpm for 10 min. Extracted samples (5 ial) were injected onto an HPLC column for the quantitative analysis. HPLC analysis. A high-performance liquid chromatographic method was employed for the quali-quantitative analysis of preservatives. The analyses were performed with a Bruker apparatus (Bremer, FRG) consisting of three plungers, an alternative pump, a

242 JOURNAL OF COSMETIC SCIENCE Table I Composition of Utilized Topical Formulations Component % (w/w) Component % (w/w) A. W/O emulsion Oil phase Cetearyl alcohol White petrolatum Mineral oil Ceteth-20 B. O/W emulsion Oil phase Cetyl alcohol Cetearyl glucoside Jojoba oil Tocopherol Lecithin Ascorbyl palmirate Capric/caprilic triglyceride C. PemMen gel Acrylates/C 10-30 alkyl acrylate Crosspolymer Methylparaben Ethylparaben Propylparaben Triethanolamine Water D. Carbopol gel Carbomer 940 Isopropyl alcohol Mineral oil Methylparaben Ethylparaben Propylparaben Triethanolamine Tetrasodium EDTA Water q.s. q.s. 10.0 7.50 4.00 2.00 2.50 2.00 3.00 0.05 6.00 5.00 2.00 1.00 0.05 0.05 0.05 1.00 to 100 0.80 4.50 2.50 0.05 0.05 0.05 0.80 0.10 to 100 Water phase Glycerine 3.00 Methylparaben 0.05 Ethylparaben 0.05 Propylparaben 0.05 Water q.s. to 100 Water phase Glycerine 4.00 Methylparaben 0.05 Ethylparaben 0.05 Propylparaben 0.05 Citric acid 0.05 Water q.s. to 100 variable-wavelength UV detector, a Rheodine Inc. injection valve, and a Shimadzu integrator. For the analysis of parabens, a Hypersil C18 stainless steel column (25 x 0.46 cm) packed with 5-1nm particles and equipped with a precolumn was eluted with an isocratic mobile phase consisting of acetonitrile/water (40:60 v/v), the flow rate being 1.0 ml/ min, at room temperature, with detection at 260 nm. Extracted samples were quanti- tated by a calibration curve constructed from standard preservative solutions. Quantitative analysis of preservatives was, in addition, performed by UV spectroscopic analysis. The analyses were performed by an NIR Lambda 19 (Perkin-Elmer) spectro- photometer equipped with a double ray and a double monochromator UV-VIS-NIR. DIFFUSION EXPERIMENTS The experiments were carried out using a standard glass Franz diffusion cell (12,13) with