PERMEATION AND METABOLISM OF o•-T and o•-TAc 39 termined to be 5 pl, corresponding to a weight of about 4 mg. After application the preparation was uniformly spread on the stratum corneum (SC) side of the skin with the help of a glass rod, and the tip of the rod was washed into a vial containing 2 ml acetonitrile in order to account for the material lost on spreading. With this technique the exact amount of material applied on the skin surface was determined. IN VITRO SKIN PERMEATION/METABOLISM METHODOLOGY A flow-through system was used for conducting in vitro permeation experiments. The total system consisted of a receptor fluid reservoir, a variable flow rate peristaltic pump, Cassette © (Manostat, New York, NY), a circulating water bath, Lauda © (Brickman Instrument Co., Westbury, NY), two cell-holding heating blocks, 14 Teflon © flow- through diffusion cells, and a Retriever IV fraction collector (ISCO Inc., Lincoln, NE) to collect effluent fractions over the adjusted time period. Each diffusion cell had an inner diameter of 9 mm and a surface area of 0.636 cm 2 exposed to the receptor fluid. The receptor fluid was pumped at a flow rate of 1.5 ml/h from the reservoir to the diffusion cells placed in the holding blocks. The skin surface temperature was maintained at 32øC by adjusting the circulating water bath temperature to 39øC (24). Effluent from diffu- sion cells was collected directly into glass scintillation vials. The experiments (four replicates for each formulation) were stopped at time intervals of 2, 6, 12, and 24 hours. SKIN TREATMENT At the conclusion of the experiment at each time point in the kinetic study, the donor compartment was washed thrice with 1 ml of acetonitrile. Washes were collected and analyzed by HPLC for the amount of drug remaining on the surface. Washed skin samples were removed from the cells. The tape-stripping technique was used to separate the stratum corneum (SC) from the rest of the epidermis to get an estimate of material remaining in the barrier layer of the skin. In this technique, seventeen strips of the drug-treated side of the skin, using a 3M Scotch TM tape, were taken as two + 15 strips. The first two strips represented the drug superficially adhering to the surface (and so included in the wash), and the next 15 strips represented the drug recovered from the SC. To each of the two strips and 15 strips about 15 ml of n-hexane was added exactly. Both these strips were shaken in a wrist-action shaker for 45 minutes at the end of which the mixture was filtered and injected into the HPLC. The remainder of the skin was placed in plastic culture tubes, 5 ml of ice cold DMPBS was added, and the skin was homogenized (Polytron homogenizer, Switzerland) for five minutes until a buff-colored suspension was obtained. Absence of chunks of skin was ensured. This suspension was extracted three times each with 5 ml of n-hexane. Each extraction involved shaking the mixture on a wrist action shaker for 45 minutes. After extraction a 45-minute centrifugation process helped to separate the n-hexane and DMPBS layers, and the upper n-hexane layer was carefully removed with a pipette and pooled together into 30-ml glass tubes. This procedure was repeated three times, each time shaking for 45 minutes and centrifuging for 45 minutes for each skin sample. The pooled hexane layer was evaporated under vacuum. Acetonitrile (2 ml) was added to this mixture, vortexed to ensure complete mixing, and the solution was filtered and injected into the HPLC column. The whole extraction procedure had been validated previously

40 JOURNAL OF COSMETIC SCIENCE to confirm the absence of any degradation during these analytical manipulations and to ensure complete extraction of a-TAc and a-T. RECEPTOR TREATMENT Receptor solutions were collected in glass scintillation vials every eight hours, and the BSA was precipitated using 4 ml of ACN. The drug was extracted into the organic n-hexane layer (5 ml) by shaking the above mixture for 45 minutes on a wrist-action shaker followed by a 45-minute centrifugation process. This procedure was repeated two times. Centrifugation was followed by careful removal of the n-hexane layer into 30-ml glass tubes and evaporation of the organic phase. This procedure had been validated previously to ensure complete extraction of the •-TAc and •-T from the receptor. Acetonitrile (1.5 ml) was added and vortexed to ensure good mixing. Sufficient quantity of this mixture was filtered and injected into the HPLC. Thus, the amount of ot-TAc and its metabolite was estimated in the following four locations in each in vitro permeation experiment: (a) receptor fluid, (b) washes, (c) stratum corneum (from strippings), and (d) viable tissues of the skin. •-TAC AND •-T ANALYSIS For the quantitative determination of •-TAc and •-T alone and in the presence of each other, HPLC analysis was used. By means of a Waters 717 autosampler, samples were taken from glass vials and injected into an HPLC apparatus. The latter consisted of a Waters 600 controller connected to a column (3.9 x 300 ram, p Bondapack RP (Waters Corporation) and a variable wavelength detector (Waters 486 tunable absor- bance detector) set at 285 nm. Chromatographic data were processed by a Waters 746 data module integrator. With acetonitrile (ACN)-water (96:4) as the mobile phase (flow rate 1.4 ml/min), the retention times were 16 minutes for •-TAc and 13 minutes for •-T. The detection limits were 0.5 pg/ml (ot-TAc) and 0.25 pg/ml (a-T). The HPLC method was validated prior to use, using USP standards. Limit of quantitation, interday variability, and system suitability were performed on the system previously described. The peak areas were converted into concentrations (pg/ml) using a standard curve developed under conditions of Beer-Lambert's law. This was further converted into micrograms of compound using the dilution factor for each sample. The final results were expressed as percentage of applied dose. STATISTICAL ANALYSES Microsoft Excel (1997) was used to compile and statistically analyze the data. Student's t-test was performed to find significant differences at the o• = 0.05 level. Values are given as means _+ SEM. RESULTS In order to delineate the kinetics of permeation and metabolism of ot-TAc across pig skin, a series of in vitro permeation experiments terminating at the end of 2, 6, 12, and