Application of solid-phase extraction in the method development for determination of SEPA, an acronym for soft enhancement of percutaneous absorption, in human, rat, and rabbit serum using GC-FID method.
A new nonaqueous topical minoxidil formulation containing SEPA (2-n-nonyl-1,3-dioxolane) for enhancement of percutaneous absorption was under evaluation. SEPA does not have chromophore for either ultraviolet or fluorescence detection using liquid chromatography and has no functional groups for derivatization. Therefore, a direct gas-chromatographic method with flame-ionization detection (GC-FID) was developed. Owing to the limited detection response of the FID detection, it needs a selective and concentrated extract for GC-FID analysis to improve the assay sensitivity to meet the requirement for pharmacokinetic evaluation after topical application. In addition, SEPA is a very volatile compound. Any extraction procedures involving evaporation will result in a poor recovery. The application of solid-phase extraction (SPE) makes it possible to achieve a selective and a 10-fold concentrated extract with an absolute extraction recovery of approximately 90%, which greatly improved the assay sensitivity. This method involved the extraction of SEPA and the internal standard (2-n-heptyl-1,3-dioxolane) from serum (0.1-1 ml) with 100 microliter of hexane-chloroform (1:1, v:v) using a 50 mg 1.0 ml-1 phenyl SPE column (Varian, Harbor City, CA, USA), followed by direct GC-FID analysis on a fused-silica column chemically bonded with cross-linked methyl silicone gum phase (Hewlett Packard Ultra-1, 12 m x 0.2 mm x 0.33 micron, Avondale, PA, USA). The assay demonstrated a lower limit of quantitation of 2.5 ng ml-1 and a linear range of 2.5 to 250 ng ml-1 with intra- and inter-assay precision and accuracy of < or = 10%.