Background: The aim of this study was to determine the concentrations of khasianine in
mouse whole blood sample and its application for the pharmacokinetics by a rapid, selective and sensitive
ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method.
Methods: The blood samples were preprocessed by one-step protein precipitation with acetonitrile. The
study was performed on an ACQUITY I-Class UPLC system with a UPLC BEH column. Lannaconitine
(internal standard, IS) and khasianine were gradient eluted by a mixture of acetonitrile and water with
0.1% formic acid at a flow rate of 0.4 mL/min. The mass spectrometer was equipped with an Electrospray
Ionization (ESI) source in positive mode. The quantitative detection was performed in a multiple
reaction monitoring modes at transitions m/z 722.4→70.7 for khasianine and m/z 585.3→119.9 for
the corresponding IS.
Results: The calibration curve was of good linearity ranging from 0.5 to 1000 ng/mL (r > 0.995). The
Lower Limit of Detection (LLOD) and Lower Limit of Quantitation (LLOQ) were 0.2 and 0.5 ng/mL,
respectively. The inter-day and intra-day precision (RSD%) were both less than 14%, and the accuracy
ranged from 86.6% to 108.3%. The matrix effects were between 98.0% and 103.7%, and the average
recovery was better than 67.4%.
Conclusion: This assay established a sensitive, rapid, selective UPLC-MS/MS method which was successfully
used for the pharmacokinetic study of khasianine in mouse blood, and the absolute availability
of khasianine was 0.78% which exhibited a poor oral absorption.