Simultaneous determination of gypenoside LVI, gypenoside XLVI, 2α-OH-protopanaxadiol and their two metabolites in rat plasma by LC-MS/MS and its application to pharmacokinetic studies

Gypenoside LVI and gypenoside XLVI are the major bioactive dammarane saponins from Gynostemma pentaphyllum. Gypenoside LVI, gypenoside XLVI, and their metabolite 2α-OH-protopanaxadiol (2α-OH-PPD) possess potent non-small cell lung carcinoma A549 cell inhibitory activity. A sensitive liquid chromatography tandem mass spectrometry method was developed and validated to study the pharmacokinetics of gypenoside LVI and XLVI, 2α-OH-PPD, metabolite 1 (M1), and metabolite 2 (M2) after administration of gypenosides or 2α-OH-PPD. Plasma samples from rats were protein precipitated with methanol. Analytes were detected by triple quadrupole MS/MS with an electrospray ionization source in the positive multiple reaction monitoring mode. The transition m/z 441.4→109.2 was selected to quantify gypenoside LVI and XLVI, and 2α-OH-PPD, because of the extensive conversion of the gypenosides to aglycone in the ionization source. M1 and M2 are isomers that shared the transition m/z 493.4→143.1. To avoid interference, the baseline separation of each analyte was performed on a SunFire C18 column with a gradient of acetonitrile (0.1% formic acid, v/v) and water (0.1% formic acid, v/v). The chromatographic run time was 10min. The linearity was validated over a plasma concentration range from 2.00 to 2000ng/mL for M1 and M2, and from 10.0 to 2000 for gypenosides LVI and XLVI, and 2α-OH-protopanaxadiol. The lower limits of quantification were 10.0, 10.0, 10.0, 2.00, and 2.00ng/mL for gypenoside LVI, gypenoside XLVI, 2α-OH-PPD, M1, and M2, respectively, with acceptable intra-/inter-day precision and accuracy. The extraction recovery rates were >86.9% for each compound. No apparent matrix effect or instability was observed during each step of the bioanalysis. After full validation, this method was proved to be simple, fast, and efficient in analyzing large batches of plasma samples for the analytes.