Application of a liquid chromatography–tandem mass spectrometry method to the pharmacokinetics, tissue distribution and excretion in the study of anemoside B4, a novel antiviral agent candidate, in rats

Abstract A simple, sensitive and reliable LC–MS/MS method was developed and validated for the quantification of anemoside B4, a potential antiviral constituent isolated from Pulsatilla chinensis in rat plasma, tissue, bile, urine and feces. All biological samples were prepared by protein precipitation method, and ginsenoside‐Rg1 was chosen as the internal standard (IS). The analyte and IS were separated using a C 18 column (2.1 × 50 mm, 1.8 μm) and a mobile phase consisting of 0.1% formic acid in water ( v /v) and acetonitrile running at a flow rate of 0.2 mL/min for 5 min. The multiple reaction monitoring transitions were monitored at m / z 1219.5–749.5 for anemoside B4 and 845.4–637.4 for ginsenoside‐Rg1 in electrospray ionization negative mode. The calibration curve was linear in the range of 10–2000 ng/mL for all biological matrices with a lower limit of quantification of 10 ng/mL. The validated method was successfully applied to a pharmacokinetics, tissue distribution and excretion study. These preclinical data will be beneficial for further development of anemoside B4 in future studies.