VITAMIN E METABOLISM 87 Table I Assay Condition of Vitamin E with HPLC in Line With a Liquid Scintillation Counter Flow rate Mobile phase (ml/min) Detection wavelength Column type MeOH:water = 95:5 1.0 280 nm •-bondapak C18(RP), 4 X 30 mm HPLC system: pump Waters 510 injector WatersWISP 710B detector Kratos Spectroflow 773 integrator Waters 730 data module. LSC system: B-Ram Micropump (CA) elution buffer Bioflour (1 ml/min). Time was calculated from the corrected values of dpm and the specific activity (dpm/mg) of radiolabeled vitamin E in the donor solution at time zero and the concentration of vitamin E in the receptor solution at each sampling. Permeation study. A freshly excised full thickness of abdominal skin of a female hairless mouse (5-7 weeks old, Jackson Lab. HRS/J Strain) was mounted between the half cells of the in vitro skin permeation system, as shown in Figure 1. Vitamin E (13 mg/ml) or [3H]2-dl-tx-tocopherol with vitamin E in silicone fluid was employed as a donor solu- tion. An aqueous solution of Tween-80 (5 mM) was used as a receptor solution. The solubility of vitamin E in silicone fluid was 39 mg/ml. To increase the solubility of vitamin E in receptor solution, maintaining a sink condition during the permeation experiment, Tween-80 was used. Tween-80 can increase the solubility of the lipophilic compound by incorporating vitamin E into a micelle. Imai et al. (11) reported that the solubility of vitamin E in water increased linearly with increasing concentration of ......•j.•...- Glass stopper Sampling port .•,• •. • • to other cells 37øC water in • '-"•:•i- (in series) skin Receptor compartment (3.5 ml ) Donor compartment • (:•5 ml) Water- (37 ø ) Star- head magnet (dia.,8.5 mm height,6 ram) Stirring platform (dia., 10 ram) (height, 4mm) Revolving magnet (6oo rpm) •ynchronous motors -- • Connecting • on-off button tubing SKIN PERMEATION SYSTEM by VALIA & CHIEN Figure 1. Schematic diagram of a Valia-Chien diffusion cell for skin permeation study.

88 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Tween-80. For the receptor solution, 5 mM Tween-80 was chosen after considering the critical micelie concentration of Tween-80 in water (0.4 mM) and the possible skin damage by the surfactant at high concentration. The donor and receptor solutions were then charged in each cell compartment. At predetermined time intervals, 30 I•l of receptor solution was withdrawn and assayed for the drug concentration with HPLC or liquid scintillation counting. The total amount of drug that permeated through the skin was plotted as a function of time. Metabolism of vitamin E in the skin during the permeation study. In vitro skin permeation experiments of radiolabeled vitamin E were performed as mentioned above. A 70-1•1 sample was withdrawn from the receptor cell at predetermined time intervals and analyzed with HPLC in line with the liquid scintillation counter. Table I summarizes the assay conditions and Figure 2 shows the schematic diagram of HPLC in line with the liquid scintillation counter. Vitamin E degradation without contacting the skin in receptor cell. Fifty I•l of vitamin E in methanol solution (10 mg/ml) and 20 I•l of 2 mCi [•H]2-3,4-ot-tocopherol were added to 3.5 ml of 5 mM Tween-80 aqueous solution. The resulting vitamin E solution (140 I•g/ml) was charged in the diffusion cells. The donor and receptor cells were separated with impermeable teflon film. The stability study was carried out at 37øC. The diffusion cell was thoroughly covered with aluminum foil to prevent photo-oxidation of vitamin E. At times, 70-1•1 samples withdrawn from the diffusion cell were stored in nitrogen gas-saturated inserts at -20øC until analysis. Valla-Chlen cell Donor IReceptor HPLC/InJector Sample i C18 RP i Column I i Computer Printer UV detector i•.•.•.•.•.----a Liquid Scintillation J Counter Flow of solvent ........ Flow of Information Figure 2. Schematic diagram of HPLC in line with a liquid scintillation counter.