*2.5. LC-MS*/*MS Analysis of Compound K*

Compound K and PPD concentrations were analyzed using a modified LC-MS/MS method of Jin et al. [8] with an Agilent 6430 triple quadrupole LC-MS/MS system (Agilent, Wilmington, DE, USA). Compound K and PPD were separated using an Eclipse Plus C18 RRHD (1.8 µm particle size, 3.0 × 5.0 mm, Agilent, Wilmington, DE, USA). The mobile phase consisted of 0.1% formic acid in water (8%) and 0.1% formic acid in methanol (92%) at a flow rate of 0.15 mL/min.

Quantification of a separated analyte peak was performed at *m*/*z* 645.5 → 203.1 for compound K (*T*<sup>R</sup> (retention time) 6.9 min), *m*/*z* 425.3 → 109.1 for PPD (*T*<sup>R</sup> 13.9 min), and *m*/*z* 198.2 → 140.1 for 13C-caffeine (IS) (*T*<sup>R</sup> 2.9 min) in the positive ionization mode with collision energy (CE) of 35, 25 and 20 eV, respectively. The analytical data were quantified using Mass Hunter (version B.06.00, Agilent, Wilmington, DE, USA).

The calibration standards and quality control (QC) samples were prepared by spiking a 5 µL aliquot of the working solution with 45 µL aliquot of blank matrix (plasma, liver, kidney, heart, lung, pancreas, brain, testis, and urine). The final concentrations of the compound K and PPD calibration standards for plasma and urine samples were 5, 10, 20, 50, 200, 500, 2000 ng/mL, and the concentrations of QC samples of compound K and PPD were 15, 100, and 1500 ng/mL. The concentrations of calibration standards and QC samples of compound K and PPD for bile and fecal homogenates were 25, 50, 100, 250, 1000, 2500, and 10,000 ng/mL and 75, 500, and 7500 ng/mL, respectively. The concentrations of calibration standards and QC samples of compound K for liver, kidney, heart, lung, pancreas, brain, and testis homogenates were 5, 10, 20, 50, 200, 500, and 2000 ng/mL and 15, 100, and 1500 ng/mL, respectively. The standard calibration curves for compound K and PPD was linear in the concentration range of 5–2000 ng/mL in the plasma, urine, and tissue homogenates samples and in the concentration range of 25–10,000 ng/mL in the bile and feces homogenates samples, respectively. The inter-day and intra-day precision and accuracy (%CV) for compound K and PPD in all biological samples was less than 15%.

#### *2.6. Data Analysis*

Pharmacokinetic parameters were estimated using non-compartmental methods (WinNonlin version 2.0, Pharsight Co., Certara, NJ, USA).

All pharmacokinetic parameters are given as the mean ± standard deviation. All statistical analyses were performed using SAS (ver. 9.4; SAS Institute Inc., Cary, NC, USA). A *p*-value < 0.05 was considered statistically significant.

#### **3. Results**

#### *3.1. Comparative Pharmacokinetics of Compound K in Rats and Mice*

The plasma concentration-time profile of compound K was compared between the mice and the rats. Since the intestinal absorption of compound K is low and variable [3,4], intravenous injection of compound K was used in this study intead of oral administration. The plasma concentrations of compound K in mice were greater than those in rats (Figure 1). The pharmacokinetic parameters of compound K such as AUC and plasma concentration were about 5–6-fold greater in mice than in rats, while there was no significant difference in the half-life (*T*1/2) and mean residence time (MRT) between rats and mice (Table 3). However, the clearance (CL) and volume of distribution (Vd) values were about five-fold larger in rats than in mice (Table 3). Taken together, the results suggest that the distribution and elimination of compound K differ between rats and mice.

**Figure 1.** (**A**) Plasma concentration vs. time profile of compound K following intravenous injection of compound K at a single dose of 2 mg/kg in rats (**A**) and mice (**B**). Plasma concentration of compound K (*Y*-axis) was represented using a logarithmic scale. Data expressed as mean± standard deviation from four rats or four mice at different time points.


**Table 3.** Pharmacokinetic parameters of compound K in rat and mouse.

∞ AUC48 h or AUC∞: area under the plasma concentration-time curve from 0 to 48 h or to infinity; *C*0: initial plasma concentration; *T*1/2: half-life; MRT: mean residence time; CL: clearance; Vd: Volume of distribution. Data expressed as mean ± standard deviation (*n* = 4). \*: *p* < 0.05 compared with the rat group.

To compare the elimination pathway of compound K, we measured the renal and fecal recovery of compound K following intravenous administration of compound K in both rats and mice. The recovery of compound K from the urine was about 0.02% of the intravenous dose in both mice and rats. In contrast, the fecal recovery of compound K was about 28.4 ± 5.9% in mice and 13.8 ± 7.1% in rats (Table 1). The results suggest that fecal excretion is a major excretion route for compound K in both rats and mice but also that compound K may undergo in vivo metabolism in both rats and mice.

#### *3.2. Tissue Distribution of Compound K*

To analyze the tissue distribution of compound K, we measured the temporal profile of compound K in various tissues in rats and mice following intravenous administration of compound K. As shown in Figures 2 and 3, the tissue distribution pattern of compound K was comparable in both mice and rats. Compound K was predominantly distributed to the liver in both rats and mice, and the liver to plasma AUC ratio was 11.1 in rats and 16.7 in mice. The AUC ratios of other tissues (i.e., kidney, heart, lung, pancreas, testis, and brain) to plasma, however, were much lower than the liver/plasma AUC ratio for both species (Figures 2B and 3B). These results suggest the involvement of the uptake

transport system, which is dominantly expressed in the liver. Previously, Jiang et al. [12] reported the involvement of the Oatp transporters (OATP1B3 in humans and Oatp1b2 in rats) in the hepatic uptake of ginsenosides Rg1, Re, and R1. Therefore, we investigated the effect of rifampin, a representative inhibitor of the Oatp transporter [11], on the liver distribution of compound K to further assess the involvement of the Oatp uptake transporter.

**Figure 2.** (**A**) Tissue concentration vs. time profile of compound K following intravenous injection of compound K at a single dose of 2 mg/kg in rats. (**B**) AUC ratios (AUCtissue/AUCplasma) of compound K in the liver, kidney, heart, lung, pancreas, testis, and brain following intravenous injection (2 mg/kg) in rats. AUC was calculated from the data shown in (**A**) and the dotted line in (**B**) represents unity. Data are expressed as mean± standard deviation from four rats at different time points.

**Figure 3.** (**A**) Tissue concentration vs. time profile of compound K following intravenous injection of compound K at a single dose of 2 mg/kg in mice. (**B**) AUC ratios of compound K in the liver, kidney, heart, lung, pancreas, testis, and brain to plasma following intravenous injection (2 mg/kg) in mice. AUC was calculated from the data shown in (**A**) and the dotted line in (**B**) represents unity. Data are expressed as mean ± standard deviation from four rats at different time points.

When rifampin was given orally at 1 h prior to compound K injection, the plasma concentration of compound K increased and the liver to plasma concentration ratios decreased when compared to the control group (no rifampin pre-treatment) in both mice and rats (Figure 4). This can be explained by the blocking of Oatp-mediated hepatic uptake of compound K by the rifampin pre-treatment.

**Figure 4.** Effect of rifampin pre-treatment on the liver to plasma concentration ratios of compound K in (**A**) rats and (**B**) mice. Liver and plasma compound K concentration were measured following intravenous injection of compound K at a single dose of 2 mg/kg in the presence or absence of rifampin pre-treatment (20 mg/kg, per oral). Data expressed as mean ± standard deviation from four rats or four mice at different time points. \*: *p* < 0.05 compared with control group.
