Validated LC–MS/MS assay for quantitative determination of deoxypodophyllotoxin in rat plasma and its application in pharmacokinetic study

Abstract

A rapid and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of deoxypodophyllotoxin (DPT) concentration in rat plasma with diazepam as internal standard (IS). DPT and IS were extracted with ethyl acetate, and the chromatographic separation was accomplished by using a Waters Symmetry C18 analytical column (2.1mm×150mm, 5μm) with a mobile phase consisting of acetonitrile and deionized water (70:30, v:v) containing 0.1% formic acid at a flow rate of 0.2mL/min. Multiple Reaction Monitoring (MRM), using electrospray ionization in positive ion mode, was employed to quantitatively detect DPT and IS. The monitored transitions were set at m/z 399.05–231.00 and m/z 285.00–154.00 for DPT and IS, respectively. The calibration curve was linear over the concentration range of 7.8–1000ng/mL (R2≥0.9999). The intra- and inter-day precision values were less than 7%. Similarly, the mean intra- and inter-day accuracy were found to be within −2.8% to 1.9% of the interval, with all samples locating within general assay acceptability criteria for QC samples according to FDA guidelines. This method was further and successfully applied in the pharmacokinetics study of DPT in rat.