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The aim of this study was to explore the transdermal delivery potential of a new compound W/O microemulsion system.Permeation of different drugs from microemulsions was enhanced significantly compared with the extraction.Optimized batches were selected through construction of phase diagrams following stability studies.Globule size analyzed by zeta sizer of selected batches were found within the desired range (<200 nm).Selected microemulsion batches with acceptable viscosities, contained 5%, 8% and 10% of water content, for in-vitro skin penetration study using Franz-cells and excised rat skin.A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated in transdermal research.The drug permeation flux and the cumulative permeation amount of HSYA, Icariin and Gallic acid in the microemulsion batch ME-4 after 24 h increased significantly compared with extracting solution.Therefore, the microemulsion batch ME-4 was selected for future in vivo pharmacodynamics studies.