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Gomisin D, a lignan compound isolated from <i>Fructus Schisandra</i>, is a potential antidiabetic and anti-Alzheimer&#8217;s agent. Recently, gomisin D was used as a quality marker of some traditional Chinese medicine (TCM) formulas. In this study, a rapid ultra-performance liquid chromatography/tandem mass spectrometry method (UPLC-MS/MS) was developed and validated to quantify gomisin D in rat plasma for a pharmacokinetic and bioavailability study. Acetonitrile was used to precipitate plasma proteins. Separations were performed on a BEH C18 column with a gradient mobile phase comprising of acetonitrile and water (0.1% formic acid). An electrospray ionization source was applied and operated in the positive ion mode. The multiple reaction monitoring mode (MRM) was utilized to quantify gomisin D and nomilin (internal standard, IS) using the transitions of <i>m</i>/<i>z</i> 531.2 &#8594; 383.1 and <i>m</i>/<i>z</i> 515.3 &#8594; 161.0, respectively. The calibration curve was linear over the working range from 1 to 4000 ng/mL (<i>R²</i> = 0.993). The intra- and interday precision ranged from 1.9% to 12.9%. The extraction recovery of gomisin D was in the range of 79.2&#8211;86.3%. The validated UPLC-MS/MS method was then used to obtain the pharmacokinetic characteristics of gomisin D after intravenous (5 mg/kg) and intragastric (50 mg/kg) administration to rats. The bioavailability of gomisin D was 107.6%, indicating that this compound may become a promising intragastrical medication. Our results provided useful information for further preclinical studies on gomisin D.