Usc1200 thd

Epidermis was separated from dermis by applying the dry heat separation method. DHQ was extracted from separated skin layers with methanol and sonication for 30 minutes in sonication bath (USC1200 THD, VWR). DHQ content in skin extracts was quantified at 289 nm using Acquity UPLC H-Class chromatography system (Waters, Milford, MA, USA). Separation of DHQ from endogenous skin matrix compounds was performed on Acquity UPLC BEH C18 (130 Å, 1.7 µm, 2.1 mm × 50 mm, Waters, Milford, MA, USA) column. The mobile phase was delivered in a linear elution gradient from 20 to 58% of solvent A (acetonitrile) in B (0.1% (v/v) trifluoracetic acid in ultrapure water) for 3 min; the injection volume was 1 µL, flow rate was 0.5 mL/min, and the column temperature was 40 °C. A standard calibration curve was built up by using standard solutions (0.35–28.35 µg/mL). Calibration graphs were plotted according to the linear regression analysis, which gave a correlation coefficient (R²) of 0.9999. The method was tested and validated for DHQ analysis in methanolic extracts of human skin layers.

The evaluation of physical enhancers was performed by using 800 µm human nail clippings as a nail model system. Fractional CO₂ laser and ultrasound were applied as efficient physical enhancement methods.
Candela CO₂RE laser (Syneron Candela, Wayland, MA, USA) was used to disrupt the nail barrier to enhance transungual permeation of naftifine hydrochloride. Nail clippings were treated by the laser at different energy levels: “fusion” (50–70 mJ energy) and “deep” (60–80 mJ energy) before the application of nail lacquers.
To determine the possible effect of ultrasound on transungual permeation of naftifine, nail clippings were placed in ultrasonic bath USC 1200 THD (VWR, Penang, Malaysia) for 15 min, 30 min, and 45 min. Ultrasound was used before or after nail lacquer application.
After using both methods separately, ultrasound and “fusion” mode energy level laser were concomitantly applied on nails before nail lacquer application. Nail clippings were treated by laser and placed in an ultrasonic bath for 30 min. After these steps, nail lacquer was applied and left until the “dry-to-touch” condition was achieved.
Structural changes of the nail were visualized by optical microscope Optika B-353FL (Optika, Ponteranica, Italy).