Simultaneous Determination of Deoxypodophyllotoxin and Its Major Metabolites in Rat Plasma by a Sensitive LC–MS/MS Method and Its Application in a Pharmacokinetic Study

Abstract

The purpose of this study is to develop and validate a rapid and sensitive liquid chromatography-tandem mass spectrometry method for the quantification of deoxypodophyllotoxin (DPT) and its major metabolites (M1, M2, M7) in rat plasma. Diazepam was used as the internal standard. The method involves a simple liquid–liquid extraction with ethyl acetate. Chromatographic separation was accomplished on a Shim-pack VP-ODS analytical column (2.0 mm × 150 mm, 5 μm) with gradient elution using a mobile phase consisting of acetonitrile and water (containing 0.1 % formic acid) at a flow rate of 0.2 mL min−1. The detection was performed by multiple reaction monitoring mode via a positive electrospray ionization interface. The calibration curves were linear for all analytes over investigated range. The lower limits of quantification were 7.734 ng mL−1 for DPT, 1.973 ng mL−1 for M1, 7.969 ng mL−1 for M2, and 1.973 ng mL−1 for M7, respectively. The intra- and inter-day precision values were less than 10.59 %, and the accuracy ranged from −6.787 to 12.74 %. The extraction recoveries of the analytes were higher than 90 % and in this method no apparent matrix effect was observed. The validated method was successfully applied to pharmacokinetic study of DPT and its main metabolites in rat plasma.