The content of the test compounds which penetrated into the acceptor fluid during the 24 h experiment and the amounts of extracted active ingredients which accumulated in the skin were determined using the high-performance liquid chromatography (HPLC) method, with the HPLC system from Knauer with a UV detector (Berlin, Germany).

The samples were separated on a 125 × 4 mm column containing Hyperisil ODS; particle size 5 µm. The flow rate of the mobile phase consisting of acetonitrile, water, and MeOH (28:64:8, by vol) was 1 mL/min. Twenty microliters of each analyzed sample were injected onto the column. The detection wavelength was 210 nm. Injections were repeated at least three times for each sample and the results were averaged. The concentration of betulin and its derivative was calculated based on the peak area measurements using the calibration curve method.

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