Quantitative determination of arenobufagin in rat plasma by ultra fast liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study

• A UFLC–MS method was developed to determination of arenobufagin in rat plasma.
• A post-column switching valve was applied to reduce the matrix effect.
• The method successfully applied to pharmacokinetic study of arenobufagin in rat plasma.

A rapid, sensitive, and selective ultra fast liquid chromatography–tandem mass spectrometry method was developed for quantitative determination of arenobufagin in rat plasma. Sample pretreatment involved a one-step protein precipitation with methanol using 0.1 mL rat plasma. The separation was carried out on a Shim-pack XR-ODS II (75 mm × 2.0 mm, i.d. 2.1 μm) column with gradient elution at a flow rate of 0.30 mL min−1. The mobile phase was acetonitrile and 0.1% formic acid in water. A post-column switching valve was applied to reduce the matrix effect. The detection was performed on a triple-quadruple tandem mass spectrometer in the multiple reaction monitoring mode after electrospray ionization. Linear calibration curves for arenobufagin were obtained over the concentration range 1.056–1056 ng mL−1, with a lower limit of quantification of 1.056 ng mL−1. The intra-day and inter-day precision values were lower than 15% and the accuracy ranged from 5.4% to 9.8% at all quality control levels. The method was successfully applied to the determination and pharmacokinetic study of arenobufagin in rat plasma following intraperitoneal administration.