Application of HPLC-ELSD for the Quantification of 5-Aminolevulinic Acid after Penetration into Human Skin Ex Vivo

摘要

5-Aminolevulinic acid (5-ALA) has been used for treatment of different skin diseases (e.g., skin cancer, actinic keratosis (AK), psoriasis, and acne). The quality of the treatment is directly associated to the amount of 5-ALA that penetrates into skin. 5-ALA was extracted from skin samples after performing the experiment with Bronaugh cells for 4 hours. Two methods were developed by applying different column systems and mobile phase compositions: YMC-Pack Hydrosphere C18 column (250 × 4 mm, 5 μm) in series with Hichrom Hypersil H5ODS (150 × 4 mm, 5 μm), mobile phase consisted of 0.1% TFA in water with 2% of ACN (method A); ACE 3AQ column (150 × 4 mm, 3 μm) eluted with 0.05% TFA in water (method B). Exploratory fluorimetric analysis requiring pre-column or post-column derivatization did not warrant the expectations of simple and effective analysis; therefore, ELS detection for 5-ALA was considered. Parallel chromatography was applied for double-validation of the methods in order to correctly evaluate the possibility of ELSD application for 5-ALA detection and the quantification amount in skin extracts. The retention time and adequate lowest limit of quantification (LLOQ) of 5-ALA were determined in both methods: 5.5 min and 20 μg/ml (method A); 6.2 min and 8.4 μg/ml (method B). Both HPLC-ELSD methods proved to be simple, accurate, precise, reliable, and showed reproducible values in the concentration range of 20-200 μg/ml in human skin extracts.