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

Abstract

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.

Keywords:

• 5-aminolevulinic acid
• HPLC-ELSD
• Parallel validation
• Penetration into human skin

Acknowledgments

The authors are grateful to Prof. Dr. R. Rimdeika from the Department of Plastic and Reconstructive Surgery, Hospital of LUHS, Lithuania for supplying the human skin for this research.

Notes

R², coefficient of determination; RSS, sum of squares; RSD(E), line relative standard deviation.