A validated LC-MS/MS method for the simultaneous determination of 20-(S)-protopanaxatriol and its two active...

We present a validated liquid chromatography with tandem mass spectrometry method for simultaneous determination of 20-(S)-protopanaxatriol and its two oxidative stereoisomeric metabolites (20S,24S)-epoxy-dammarane-3,6,12,25-tetraol (M1) and (20S,24R)-epoxy-dammarane-3,6,12,25-tetraol (M2) in rat plasma. 20-(S)-Protopanaxatriol, M1, and M2 were extracted with methanol and separated on an ACQUITY HSS T₃ column. The mass spectrometry detection was accomplished in selected reaction monitoring mode with precursor-to-product ion transitions of m/z 493.4→143.1 for M1 and M2, m/z 475.4→391.3 for 20-(S)-protopanaxatriol, and m/z 459.4→375.3 for 20-(S)-protopanaxadiol (internal standard). The method showed good linearity over the concentration ranges of 1-1000 ng/mL for 20-(S)-protopanaxatriol and 0.5-200 ng/mL for M1 and M2, with correlation coefficients of more than 0.995. The lower limits of quantification for 20-(S)-protopanaxatriol, M1, and M2 were 1, 0.5, 0.5 ng/mL, respectively. The intra- and interday precisions (RSD, %) were less than 10.41% while the accuracy (relative error, %) ranged from -3.14 to 8.73%. Under the current conditions, M1 and M2 were completely separated within 3 min. The validated assay was successfully applied to evaluating pharmacokinetic profiles of 20-(S)-protopanaxatriol, M1, and M2 in rat.