Development and validation of a bioanalytical method to quantify povorcitinib in human skin with clinical application

Quantification of drug concentrations in human skin is essential for understanding mechanisms of action in dermatologic therapeutics but remains analytically challenging. The study aimed to develop and validate a standardized tissue homogenization and liquid chromatography – tandem mass spectrometry workflow for quantifying povorcitinib in human skin biopsies. Key skin homogenization parameters, including solvent composition, processing volume, and homogenate density, were systematically evaluated. Wet and dry homogenization approaches were compared. Following protein precipitation, samples were analyzed using liquid chromatography – tandem mass spectrometry. Eighteen solvent systems and various processing volumes were evaluated, with three solvent mixtures producing homogeneous preparations. Limiting homogenization volumes to less than half tube capacity and using 20 mg/mL improved sample consistency. Povorcitinib stability under acidic and thermal conditions was confirmed. Comparable performance was observed between wet and dry homogenization methods, and a dry workflow was validated. This study provides the first systemic evaluation of human skin homogenization parameters and a direct comparison of wet and dry approaches. The validated dry homogenization method enabled accurate and reproducible quantification of povorcitinib in skin biopsies from a phase I clinical study and offers a robust framework for quantifying a broad range of analytes in human skin tissues. Clinical trial registration:https://www.clinicaltrials.gov/study/NCT06505265 [clinicaltrials.gov] identifier is NCT06505265. Knowing how much a medicine taken by mouth reaches the skin helps scientists understand how skin treatments work. However, measuring this can be difficult. In this study, we figured out a dependable way to measure the amount of a medicine called povorcitinib in very small skin samples collected from people. We tested different ways to break down skin samples to make sure the medicine could be measured reliably. This included trying different types and amounts of liquid and comparing two ways of crushing the skin. In the wet method, skin samples were placed in liquid right away. In the dry method, the skin was first frozen and then crushed into very tiny pieces before liquid was added. We also checked that povorcitinib did not change during these steps. We found that using several types of liquids could produce consistent skin samples and that carefully controlling how much liquid to use helped with sample consistency. Both the wet and dry methods worked equally well, and the dry method was tested further to make sure it worked every time. Overall, this study provides a reliable way to measure how much povorcitinib reaches the skin in people taking part in a research study. This same approach can also be used to measure other medicines in skin samples and help scientists develop better treatments for skin diseases in the future.