Determination of Gypenoside A and Gypenoside XLIX in Rat Plasma by UPLC-MS/MS and Applied to the Pharmacokinetics and Bioavailability

In this work, a UPLC-MS/MS method was developed for the determination of gypenoside A and gypenoside XLIX in rat plasma. For chromatographic separation, a UPLC BEH C18 column was employed, the mobile phase comprised acetonitrile: water (w/0.1% formic acid), and the elution time was 4 min. Detection of each compound was enabled by electrospray ionization in negative-ion mode, and quantitative analysis was enabled by operating in multiple reaction monitoring (MRM) mode by monitoring the transitions of m/z 897.5⟶403.3 for gypenoside A, m/z 1045.5⟶118.9 for gypenoside XLIX, and m/z 825.4⟶617.5 for the internal standard. The calibration curves for gypenoside A and gypenoside XLIX demonstrated excellent linearity (r > 0.995) over the range of 2–3000 ng/mL. The intraday and interday precisions of gypenoside A and gypenoside XLIX were within 14.9%, the intraday and interday accuracies ranged from 90.1% to 113.9%, the recoveries were all greater than 88.3%, and the matrix effect ranged from 87.1% to 94.1%. The developed method was successfully applied in the determination of the pharmacokinetics of gypenoside A and gypenoside XLIX. Gypenoside A and gypenoside XLIX had very short half-lives in rats, with oral t1/2z of 1.4 ± 0.2 h and 1.8 ± 0.6 h, respectively, and low bioavailabilities (0.90% and 0.14%, respectively).

Gypenoside A and gypenoside XLIX are the two main saponins found in Gynostemma pentaphyllum [15,16] and have been isolated from the plant and purified by LC-MS/MS. Guo et al. performed a solid-phase extraction method and developed an LC-MS/MS method to detect gypenoside XLIX in rat plasma [15]. ey then studied the rat pharmacokinetics of gypenoside XLIX after intravenous administration. Hu et al. developed a quantitative UPLC-MS method to detect gypenoside A in rat plasma [17]. Each sample required 5 min, the proteins were precipitated with methanol to avoid interference from matrix effects, and the gypenoside A concentration in rats was determined after oral administration of Gelanxinning soft capsules. However, both methods only analyzed the in vivo concentration of gypenoside A or gypenoside XLIX separately, and neither detected them simultaneously nor did they conduct bioavailability studies.
In this paper, we developed a UPLC-MS/MS method to enable the simultaneous determination of gypenoside A and gypenoside XLIX in rat plasma and then utilized the method to study the pharmacokinetics of both gypenosides under different administration routes (oral and intravenous administration) to determine their bioavailability.
For the mass spectrometer, nitrogen was used as the cone gas (50 L/h flow rate) and desolvation gas (1000 L/h flow rate). e capillary voltage was set to 3.2 kV, the ion source temperature was 145°C, and the desolvation temperature was 500°C. e mass spectrometer was operated in electrospray (ESI) negative-ion mode, and the quantitation of the two gypenosides was enabled by operating in multiple reaction monitoring modes (MRM) by monitoring the transitions of m/z 897.5⟶403.3 (cone voltage 76 V,   International Journal of Analytical Chemistry 3

Method Development.
e mass spectrometry conditions were obtained after optimizing the spray needle voltage, drying gas temperature, capillary voltage, and collision energy [18][19][20][21]. Comparing the positive and negative modes, gypenoside A and gypenoside XLIX were best suited for detection in ESI negative-ion mode because the sensitivity was significantly higher than detection in other modes. Gypenoside A and gypenoside XLIX were prepared in rat blank plasma at a concentration of 100 ng/mL. During the method development, a variety of different solvents and solvent mixtures were assessed for their ability to efficiently precipitate the proteins in the rat plasma. Acetonitrile, methanol-acetonitrile (1 : 9, v/v), 10% trichloroacetic acid, methanol-acetonitrile (1 : 1, v/v), and methanol were employed, and it was determined that methanol-acetonitrile (1 : 9, v/v) had the highest extraction efficiency, so it was chosen as the solvent for the protein precipitation step. Figure 3, the retention times of gypenoside A, gypenoside XLIX, and the internal standard were 1.86, 1.72, and 1.91 min, respectively, and there was no interference from the endogenous components within the plasma, indicating that the developed method was highly selective for the two natural compounds.

Standard Curve.
e calibration curves of gypenoside A and gypenoside XLIX in rat plasma that were generated over the concentration range of 2-3000 ng/mL demonstrated excellent linearity, indicating that they could reliably be used for calculating the concentration of the natural compounds in the rat plasma. e regression equation for gypenoside A was y1 � 0.0096x1 + 0.0023 (R 2 � 0.9984), wherein x1 represented the concentration of gypenoside A in the plasma, and y1 represented the ratio of the peak area of gypenoside A to the internal standard. e regression equation for gypenoside XLIX was y2 � 0.0024x2 + 0.0014 (R 2 � 0.9971), wherein x2 represented the concentration of gypenoside XLIX in plasma, and y2 represented the ratio of the peak area of gypenoside XLIX to the internal standard. Based on these two equations, the lower limit of quantification of gypenoside A and gypenoside XLIX in rat plasma was 2 ng/mL, and the detection limit was 1 ng/mL.

Precision, Accuracy, Recovery, and Matrix Effects.
e intraday and interday precisions of gypenoside A were within 14.9%, the intraday and interday accuracies were 90.1-107.5%, the recovery was greater than 88.3%, and the matrix effects were 87.1-93.9%. e intra-and interday precisions of gypenoside XLIX were within 12.9%, the intraand interday accuracies were 91.8-113.9%, the recovery was greater than 93.2%, and the matrix effects were in the range of 89.3-94.1% (Table 1).

Stability.
e accuracy of gypenoside A was between 92.2% and 110.3%, and the RSD was within 14.8%; the accuracy of gypenoside XLIX was between 87.9% and 112.2%, and the RSD was within 14.5% (Table 2). ese      International Journal of Analytical Chemistry 5 results indicated that gypenoside A and gypenoside XLIX had excellent stability.

Pharmacokinetic
Studies. e noncompartmental model was used to fit the main pharmacokinetic parameters (Table 3), and the concentration-time curves for gypenoside A and gypenoside XLIX in rat plasma are shown in Figure 4. After intravenous administration, the half-lives (t 1/2z ) of gypenoside A and gypenoside XLIX in the rats were 0.8 ± 0.2 h and 1.6 ± 1.7 h, respectively, while the oral t 1/2z were 1.4 ± 0.2 h and 1.8 ± 0.6 h, respectively, indicating that the compounds were metabolized very quickly. e t 1/2 of gypenoside A in rats reported in the literature was 6.247 ± 2.039 h [17], which is significantly longer than the values we reported, but this difference was likely due to the different dosage forms. However, the t 1/2 of gypenoside XLIX in rats reported in the literature (3.17 ± 1.01) h was closer to the values we calculated [15]. Based on the pharmacokinetics data, gypenoside A and gypenoside XLIX had very low oral bioavailabilities of 0.90% and 0.14%, respectively, indicating that the concentration of the drug in systemic circulation was low.

Conclusion
In this study, a UPLC-MS/MS method was established for the determination of gypenoside A and gypenoside XLIX in rat plasma. e UPLC-MS/MS method required only 4 min for each sample, and a simple and inexpensive protein precipitation method was used. e accuracy, precision, selectivity, and linearity of this method were highly robust, corroborating the application of this method in studying the pharmacokinetics and bioavailability of these compounds and others alike in rats.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.