ALPHA-TOCOPHEROL ACETATE PERMEATION 97 sampler, a Hitachi 1-7400 UV detector, and a Hitachi 1-7100 pump. The chromatog­ raphy was performed on a reverse-phase C 18 (µBondapak™, 3.9 x 300 mm, Milford, MA). The detection wavelength was 285 nm. The mobile phase consisted of acetoni­ trile:water (96:4). The isocratic flow rate was changed to 2.0 ml/min to reduce the retention time. (The retention times were 10. 3 and 12 .4 minutes for al pha-tocopherol and for alpha-tocopherol acetate, respectively, compared to 13 and 16 minutes in the source paper.) For both analytes, the peak areas vs concentration (µg/ml) curves were linear in the range of 10-1000 µg/ml. The injection volume was 10 µl. DIFFUSION STUDIES USING CELLULOSE MEMBRANES In vitro diffusion studies were carried out using a modified Franz diffusion cell apparatus (Crown Glass Company, Somerville, NJ) with a diameter of 15 mm and a diffusional area of 1.76 cm2 . A Spectra/Por®7 regenerated cellulose membrane (Spectrum, Laguna Hills, CA) was inserted between the donor and the receiving compartment and secured in place by means of a pinch clamp. The membrane had a thickness of 60-65 µm and a molecular weight cutoff point of 1,000. According to the manufacturer's direction, the cellulose membrane was rinsed with distilled water in order to remove traces of the preservative sodium azide before use. The receiving compartment (volume 13.1 ml) was filled with degassed ethanol USP and it was maintained at 37°C by means of a water bath circulator and a jacket surrounding the cell, resulting in a membrane surface temperature of 32°C (18). The receiving medium was continuously stirred by a Teflon™-coated magnetic stirrer, to avoid diffusion layer effects. A 1.00-ml sample of each ATA formulation was accurately measured and placed in the donor compartment and sealed with aluminum foil and parafilm. Aliquots of 0.5 ml were withdrawn from the receiving compartment at 15, 30, and 45 minutes and 1, 2, 3, and 4 hours using a microsyringe, and replaced immediately with an equal volume of degassed ethanol USP. All samples were trans­ ferred to 1.5-ml vials and diluted with degassed ethanol USP up to 1.0 ml before analysis by HPLC. The experiment was carried out in triplicate for each formulation. This method was applied to the ethanol solution, 1 % Klucel® gel, and 3% Klucel® gel. Where appropriate, the cumulative amount released of ATA vs time data was analyzed and equation 5 was used to calculate the diffusion coefficients of AT A in the formula­ tion. In cases where the slope of the M-vs-t plots was nearly constant, the diffusion coefficient of AT A in the formulation was not calculated exactly, but lower limits for the value were calculated instead using equation 6 with t = 4 hours. RELATIVE VISCOSITY ESTIMATES The viscosity of ethanol USP, isopropyl myristate, and light mineral oil solvents was determined at 32°C by using the technique of falling ball viscometer. For each solvent measured, 105 ml was poured into a 100-ml graduated cylinder, which was then kept in a water bath for at least 30 minutes before starting the experiment, to ensure that the solvent temperature was 32°C. The plastic sphere (p = 1.0038 g/cm3) was carefully placed into the cylinder and allowed to fall through the liquid without touching the wall. The time of descent between the 100-ml and 10-ml marks was timed by means of a stopwatch. The experiments were repeated ten times for each solvent, and the average values were used for further calculation.

98 JOURNAL OF COSMETIC SCIENCE IN VITRO CADA VER SKIN PERMEABILITY STUDIES The same apparatus and experimental procedure described in the previous section were used to perform cadaver skin permeability studies. The sampling times were 2, 4, 6, 8, 12, 24, 30, 36, and 48 hours. The experiment was carried out in triplicate for each formulation. Human cadaver skin from the back of Caucasian subjects was kindly donated by Novartis (Somerville, NJ). Cadaver skin was prepared by hydrating it in an isotonic phosphate-buffered solution for one hour at room temperature before it was placed between the donor and receiving compartments. The integrity of the skin was checked by visual inspection of the cumulative amount-vs-time plots. Experiments in which the amount of permeated ATA suddenly reached a plateau or jumped by large amounts were disregarded and repeated. STATISTICAL DATA ANALYSIS Permeability calculations were done by nonlinear regression, using the Solver function in Microsoft Excel™. Other statistical analyses were performed with the SPSS 10.1 for Windows 2000 version using one-way analysis of variance (ANOVA). Analyses were performed primarily to determine whether there were any significant differences in alpha-tocopherol acetate release among the different concentrations of Klucel® in the formulations and between each solution formulation. RESULTS DIFFUSION COEFFICIENTS OF ATA IN THE DONOR MEDIA The release profiles of ATA from the ethanol solution, 1 % Klucel® gel, and 3% Klucel® gel through regenerated cellulose membrane were analyzed using equations 5 and 6. The diffusion coefficients of ATA in the formulation were 2.5 x 10- 2 cm2/h for the ethanol solution, and greater than 1 x 10- 2 cm2/h for the 1 % and 3% Klucel® gels, respectively. The ratios of solvent viscosity were used to calculate diffusion coefficients of ATA for the isopropyl myristate solution and the light mineral oil solution. The ethanol/isopropyl myristate viscosity ratio was 0.91 and the ethanol/light mineral oil viscosity ratio was 0.70, and the estimated diffusion coefficients of ATA in isopropyl myristate and light mineral oil were 2.3 x 10- 2 cm2/h and 1.8 x 10- 2 cm2/h, respectively. CADA VER SKIN PERMEABILITY The permeability coefficient of ATA though human cadaver skin was calculated using equation 1, which applies when the concentration profile in the membrane develops relatively slowly and diffusion through the membrane is the rate-controlling step. Figure 1 shows the experimental data for the cumulative amount released as a function of time for the five formulations, and the model fits the data. The calculated permeabilities through human cadaver skin membrane were 1.0 x 10-4 ± 1.5 x 10- 5 cm/h for the ethanol solution, 1.1 x 10- 2 ± 1.6 x 10- 3 cm/h for the isopropyl myristate solution, 1.4 x 10-4 ± 3.8 x 10- 6 cm/h for the light mineral oil solution, 2.1 x 10- 4 ± 2.6 x 10- 5 cm/h for 1 % Klucel® gel, and 4.7 x 10-4 ± 4.6 x 10- 5 cm/h for 3% Klucel® gel.