Development of a sensitive and rapid UPLC-MS/MS method for the determination of koumine in rat plasma: application to...

A rapid, selective and sensitive method using UPLC-MS/MS was first developed and validated for quantitative analysis of koumine in rat plasma. A one-step protein precipitation with methanol was employed as a sample preparation technique. Plasma samples were separated on an Acquity UPLC BEH C18 column (50 × 2.1 mm, i.d. 1.7 µm) with a gradient mobile phase consisting of methanol with 0.1% (v/v) formic acid and water containing 0.1% (v/v) formic acid at a flow rate of 0.3 mL/min. Detection and quantification were performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via positive eletrospray ionization. Good linearity (r > 0.9997) was achieved using weighted (1/x(2) ) least squares linear regression over a concentration range of 0.025-15 µg/mL with a lower limit of quantification of 0.025 µg/mL for koumine. The intra- and inter- precisions (relative standard deviation) of the assay at all three quality control samples were 5.6-14.1% with an accuracy (relative error) of 5.0-14.0%, which meets the requirements of the US Food and Drug Administration guidance. This developed method was successfully applied to an in vivo pharmacokinetic study in rats after a single intravenous dose of 20 mg/kg koumine.