Development of a targeted method for quantification of gypenoside XLIX in rat plasma, using SPE and LC-MS/MS.

A sensitive, selective and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of gypenoside XLIX, a naturally occurring gypenoside of Gynostemma pentaphyllum in rat plasma and then validated according to the US Food and Drug Administration's Guidance for Industry: Bioanalytical Method Validation. Plasma samples were prepared by a simple solid-phase extraction. Separation was performed on a Waters XBridgeTM BEH C18 chromatography column (4.6 × 50 mm, 2.5 μm) using a mobile phase of acetonitrile and water (62.5:37.5, v/v). Gypenoside XLIX and the internal standard gypenoside A were detected in the negative ion mode using selection reaction monitoring of the transitions at m/z 1045.6 → 913.5 and 897.5 → 765.4, respectively. The calibration curve was linear (R2  > 0.990) over a concentration range of 10-7500 ng/mL with the lower quantification limit of 10 ng/mL. Intra- and inter-day precision was within 8.6% and accuracy was ≤10.2%. Stability results proved that gypenoside XLIX and the IS remained stable throughout the analytical procedure. The validated LC-MS/MS method was then applied to analyze the pharmacokinetics of gypenoside XLIX after intravenous administration to rats (1.0, 2.0 and 4.0 mg/kg).