Simultaneous determination of 20(S)-protopanaxadiol and its three metabolites in rat plasma by LC-MS/MS: application to their pharmacokinetic studies

The aim of this study was to develop an LC-MS/MS method for simultaneous determination of 20(S) protopanaxadiol (PPD) and its three metabolites, PPD-glucuronide (M1), (20S,24S)-epoxy-dammarane-3,12,25-triol (M2) and (20S,24R)-epoxydammarane-3,12,25-triol (M3), in rat plasma. Precipitation with acetonitrile was employed for sample preparation and chromatographic separations were achieved on a C-18 column. The sample was detected using triple quadrupole tandem mass spectrometer with selected reaction monitoring mode. The monitored precursor-to-product ion transitions were m/z 459.4375.3 for PPD, m/z 635.4113.0 for M1, m/z 477.4441.4 for M2 and M3 and m/z 475.4391.3 for IS. The developed assay was validated according to the guidelines of the US Food and Drug Administration. The calibration curves showed good linearity over the tested concentration ranges (r>0.9993), with the LLOQ being 1ng/mL for all analytes. The intra- and inter-day precisions (RSD) were<9.51% while the accuracy (RE) ranged from -8.91 to 12.84%. The extraction recovery was >80% and no obvious matrix effect was detected. The analytes were stable in rat plasma with the RE ranging from -12.34 to 9.77%. The validated assay has been successfully applied to the pharmacokinetic study of PPD as well as its metabolites in rat plasma. According to the pharmacokinetic parameters, the in vivo exposures of M1, M2 and M3 were 11.91, 47.95 and 22.62% of that of PPD, respectively.