Assay of tolnaftate in human skin samples after in vitro penetration studines

Abstract

Purpose: To optimize and validate a simple, rapid, accurate and reproducible procedure for tolnaftate extraction and determination in human skin layers after in vitro skin penetration studies. Methods: High performance liquid chromatography (HPLC) method with UV detection was used to validate tolnaftate assay for linearity, specificity, accuracy, precision, limit of quantitation, limit of detection, drug extraction recovery and stability in skin extracts. In vitro tolnaftate penetration studies were carried out using flow-through diffusion cells, mounted with human skin. Epidermis and dermis, separated by heat-separation method, were extracted using ultrasonication in methanol. Results: Linear range of the analytical procedure was within 0.6-100 µg/mL. The assay was specific, accurate (intra-day and inter-day recovery values were 98.2-104.2% and 98.7-101.4%, respectively) and precise (intra-day and inter-day imprecision was <3.8%). Mean extraction recoveries of tolnaftate from epidermis and dermis were satisfactory and exceeding 90%. In vitro skin penetration studies revealed that after application of 1% (w/w) tolnaftate solution in PEG400 for 24 hours, the mean amount of tolnaftate penetrating into the epidermis and dermis was 2.60±0.28 µg/cm2 and 0.92±0.12 µg/cm2, respectively. Conclusion: A validated reliable HPLC method is suitable for biopharmaceutical evaluation of tolnaftate pharmacokinetics in human skin after in vitro penetration studies