Detection of 13 Ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, Rh2, F1, Compound K, 20(S)-Protopanaxadiol, and 20(S)-Protopanaxatriol) in Human Plasma and Application of the Analytical Method to Human Pharmacokinetic Studies Following Two Week-Repeated Administration of Red Ginseng Extract

We aimed to develop a sensitive method for detecting 13 ginsenosides using liquid chromatography–tandem mass spectrometry and to apply this method to pharmacokinetic studies in human following repeated oral administration of red ginseng extract. The chromatograms of Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, Rh2, F1, compound K (CK), protopanaxadiol (PPD), and protopanaxatriol (PPT) in human plasma were well separated. The calibration curve range for 13 ginsenosides was 0.5–200 ng/mL and the lower limit of quantitation was 0.5 ng/mL for all ginsenosides. The inter- and intra-day accuracy, precision, and stability were less than 15%. Among the 13 ginsenosides tested, nine ginsenosides (Rb1, Rb2, Rc, Rd, Rg3, CK, Rh2, PPD, and PPT) were detected in the human plasma samples. The plasma concentrations of Rb1, Rb2, Rc, Rd, and Rg3 were correlated with the content in red ginseng extract; however, CK, Rh2, PPD, and PPT were detected although they are not present in red ginseng extract, suggesting the formation of these ginsenosides through the human metabolism. In conclusion, our analytical method could be effectively used to evaluate pharmacokinetic properties of ginsenosides, which would be useful for establishing the pharmacokinetic–pharmacodymic relationship of ginsenosides as well as ginsenoside metabolism in humans.


MS/MS Analysis
The mass spectrometer was operated with electrospray ionization (ESI) in the positive ionization mode. Table 1 shows the selected precursor and product ions of analytes and respective mass spectrometric conditions in the MS/MS stage of the ginsenosides, which were optimized based on the fragmentation patterns of precursor and product ions of target ginsenoside, the specificity of target ginsenoside compared to the other ginsenosides, and the consistency with the previously published findings [11,19]. Since ginsenosides Rb2 and Rc resulted in the same m/z values of precursor and product ion, these ginsenosides should be separated each other during the elution. Retention times were 5.7 min for Rb2 and 4.8 min for Rc (Table 1 and Figure 2B).

Sample Praperations
For sample preparation, both protein precipitation and liquid-liquid extraction (LLE) methods should be applied depend on the number of glycosylation of ginsenosides. For example, we used the protein precipitation method for ginsenosides glycosylated with more than two glucose units (i.e., Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, and F2; hydrophilic ginsenosides) and the LLE method for monoglycosylated ginsenosides and their aglycones (i.e., Rh1, Rh2, CK, PPD, and PPT; lipophilic ginsenosides) based on the extraction recovery after sample preparation and the interference of endogenous peaks in human blank plasma (the plasma withdrawn from human subjects who did not

MS/MS Analysis
The mass spectrometer was operated with electrospray ionization (ESI) in the positive ionization mode. Table 1 shows the selected precursor and product ions of analytes and respective mass spectrometric conditions in the MS/MS stage of the ginsenosides, which were optimized based on the fragmentation patterns of precursor and product ions of target ginsenoside, the specificity of target ginsenoside compared to the other ginsenosides, and the consistency with the previously published findings [11,19]. Since ginsenosides Rb2 and Rc resulted in the same m/z values of precursor and product ion, these ginsenosides should be separated each other during the elution. Retention times were 5.7 min for Rb2 and 4.8 min for Rc (Table 1 and Figure 2B).

Sample Praperations
For sample preparation, both protein precipitation and liquid-liquid extraction (LLE) methods should be applied depend on the number of glycosylation of ginsenosides. For example, we used the protein precipitation method for ginsenosides glycosylated with more than two glucose units (i.e., Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, and F2; hydrophilic ginsenosides) and the LLE method for monoglycosylated ginsenosides and their aglycones (i.e., Rh1, Rh2, CK, PPD, and PPT; lipophilic ginsenosides) based on the extraction recovery after sample preparation and the interference of endogenous peaks in human blank plasma (the plasma withdrawn from human subjects who did not take ginseng or ginsenosides). The monoglycosylated ginsenoside F1 could be extracted with both the protein precipitation and LLE method; however, the detection sensitivity of analyte was better for precipitation samples than for LLE samples. Therefore, F1 were extracted with the protein precipitation method. Methyl tert-butyl ether (MTBE) was chosen as an extraction solvent based on the extraction efficiency and reproducibility of the ginsenosides Rh1, Rh2, CK, PPD, and PPT and based on previous findings [15].
The ginsenosides F2 and Rh1 were excluded in the validation process because their peaks could not be completely separated from the endogenous peaks that detected at the same m/z as F2 and Rh1 in human blank plasma, and the peak response of F2 and Rh1 at LLOQ was less than five times the response of a blank sample [20,21].

Analytical Method Validation
The method was fully validated according to the FDA Guidance for Industry: Bioanalytical Method Validation (May 2018) [21] for its specificity, accuracy, precision, matrix effect and extraction recovery, and stability.

Specificity
Representative multiple reaction-monitoring (MRM) chromatograms of the ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, Rh2, F1, F2, CK, PPD, and PPT ( Figure 2) showed that all the ginsenoside peaks obtained using the protein precipitation or LLE method were well separated with no interfering peaks at their respective retention times. The retention times of the 13 ginsenosides are shown in Table 1. The specificity of the analytes was confirmed using six different human blank plasma samples and test plasma samples obtained from human subjects at 1 h after the last oral administration of red ginseng extract ( Figure 2).

Linearity and LLOQ
To assess linearity, the standard calibration curve of eight different concentrations of 13 ginsenosides was analyzed, and the standard calibration curve and equation for each component are shown in Table 2. The LLOQ was defined as a signal-to-noise ratio of > 5.0 with a precision rate of ≤ 15% and an accuracy rate of 80-120%. The LLOQ for the ginsenosides in our analytical system was set at 0.5 ng/mL in all cases.

Linearity and LLOQ
To assess linearity, the standard calibration curve of eight different concentrations of 13 ginsenosides was analyzed, and the standard calibration curve and equation for each component are shown in Table 2. The LLOQ was defined as a signal-to-noise ratio of > 5.0 with a precision rate of ≤ 15% and an accuracy rate of 80-120%. The LLOQ for the ginsenosides in our analytical system was set at 0.5 ng/mL in all cases.

Precision and Accuracy
The inter-day and intra-day precision and accuracy were assessed using three different concentrations (1.5, 15, and 150 ng/mL) of quality control (QC) samples consisting of a specific ginsenoside mixture (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, and F1 for protein precipitation; Rh2, CK, PPD, and PPT for LLE) ( Table 3). The results showed that inter-day and intra-day precision (CV in Table 3) for the 13 ginsenosides was below 13.0%, and the inter-day and intra-day accuracy (RE in Table 3) for the 13 ginsenosides was below 15.0% (Table 3). Table 3. Intra-and inter-day precision and accuracy of 13 ginsenosides.

Extraction Recovery and Matrix Effect
The extraction recovery of the ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, and F1, which were prepared with the protein precipitation method using three different concentrations (1.5, 15, and 150 ng/mL) of QC samples, ranged from 85.5% to 99.2% with a CV of < 14.9%. In the case of the LLE method, the extraction recovery of the ginsenosides Rh2, CK, PPD, and PPT ranged from 56.3% to 81.9% with a CV of < 14.9% (Table 4). The matrix effects for the ginsenosides Rh2, CK, PPD, and PPT ranged from 77.0% to 100.1%. The matrix effects for the protein-precipitated ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, and F1) ranged from 7.0% to 92.9%. The matrix effect of ginsenosides Re, Rf, and Rg1 was in the range of 7.0%-19.5%, suggesting that Re, Rf, and Rg1 showed significant signal suppression during the ionization and protein precipitation process; however, the values of CV of Re, Rf, and Rg1 was less than 15% and the matrix effect of Re, Rf, and Rg1 was similar for the three different QC levels with an acceptable CV, and 10 other ginsenosides showed no significant interference during ionization and sample preparation. According to the EMA guideline [22], we concluded our analytical method was acceptable even though Re, Rf, and Rg1 had significant ion suppression.

Stability
The precision (CV) and accuracy (RE) of three different concentrations of QC samples consisting of a mixture of the ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, and F1, which were prepared using the protein precipitation method, were within 13.5% for short-term stability, below 14.9% for post-preparative stability, and below 12.9% for three freeze-thaw cycle stability ( Table 5). The precision (CV) and accuracy (RE) of three different concentrations of QC samples consisting of a mixture of the ginsenosides Rh2, CK, PPD, and PPT, which were prepared using the LLE method, were within 10.6% for short-term stability, below 12.4% for post-preparative stability, and below 14.7% for three freeze-thaw cycle stability (Table 5). Therefore, the 13 ginsenosides in human plasma samples had no stability issues during the storage in the freezer, sample preparation process, and analysis time after the samples were processed, as demonstrated by the three stability tests.

Contents of Ginsenosides in Red Ginseng Extract
The ginsenoside content of the red ginseng extract provided to participants daily for 14 days (three pouches of Hongsamjung All Day TM /day) is summarized in Table 6. The most abundant ginsenoside was Rb1 (18.8-23.6 mg/day), followed by Rb2, Rc, Rd, and Rg3 (12.9-5.9 mg/day). The abundance of Re, Rh1, and Rg1 was 1.6-6.6 mg/day. The daily intake of PPT-type ginsenosides was lower than that of PPD-type ginsenosides. The values of daily intake of PPD-type ginsenosides are ranged between 50.2-64.7 mg/day and those of PPT-type ginsenoside are ranged between 11.2-14.9 mg/day.  The oral administration of three pouches of red ginseng for two weeks was well tolerated and did not produce any unexpected or serious adverse events, as previously reported [17].  Figure 3. The ginsenosides Rb1, Rb2, Rc, Rd, and Rg3, which were detected in the plasma samples, are all PPD-type ginsenosides and present at a relatively high content in red ginseng extract. In contrast, the PPT-type ginsenosides Re and Rh1 were not detected in the human plasma samples despite their high content in red ginseng extract. CK, Rh2, and PPD, which are metabolites from Rb1, Rb2, and Rc, were also detected even though they are not present in red ginseng extract, suggesting that these PPD-type metabolites could be formed in the human intestine during the intestinal absorption stage (Figure 1) [11,23,24]. Among the reported PPT-type metabolites, only PPT was detected in the human plasma.    The pharmacokinetic parameters from the plasma concentration-time profiles of these ginsenosides are shown in Table 7. The plasma Rb1, Rb2, Rc, and Rd concentrations were constant over time, and they had a long terminal half-life. The AUC and C max values of Rb1, Rb2, Rc, and Rd were correlated with the content of red ginseng extract. In contrast to the plasma concentrations of Rb1, Rb2, Rc, and Rd, the plasma concentrations of Rg3, Rh2, and CK showed a bell-shaped profile ( Figure 3); this may be attributed to further metabolism to PPD. The T max of Rg3 (3.6 h) was smaller than that of Rh2 and CK (5.6-9.1 h), which may be associated with the high content of Rg3 that the absorption of Rg3 could occur following oral administration of red ginseng extract and absence of Rh2 and CK in the red ginseng extract that the absorption of Rh1 and CK could occur after they were transformed from Rb1, Rb2, Rc, and Rd. The plasma concentration profiles of PPD and PPT were similar but flatter compared with those of Rg3, Rh2, and CK. Since PPD was derived from Rg3, Rh2, and CK and could undergo further metabolism [11,23,24], the plasma profile of PPD and PPT could be attributed to the faster elimination in human body rather than intestinal formation via intestinal microbiota. Lin et al. reported that 40 metabolites of PPD were identified in human plasma and urine and the major metabolites of PPD was the hydroxylated form in human body through phase I hepatic metabolism [19].
To explain time-dependent metabolism and absorption of ginsenosides, the plasma concentrations of ginsenosides at absorption phase (from 4 to 10 h) depend on the deglycosylation states was shown in Figure 4. The plasma concentrations of Rb1, Rb2, Rc, and Rd, tri-and tetraglycosylated ginsenosides, were stable for 4-10 h of post dose ( Figure 4A), suggesting the stable absorption and slow elimination process. The plasma concentrations of Rg3 was decreased along with increasing time (4-10 h) but the monoglycosylated ginsenosides Rh2 and CK, metabolites from Rg3 and F2, increased over time ( Figure 4B,C), suggesting the gut metabolism from Rg3 to Rh2 during the absorption stage. The delayed absorption of Rh2, CK, and PPD indicated that formation and absorption of Rh2, CK, and PPD might occur in the lower part of intestine. On the other hand, the formation and absorption of PPT was faster than PPD ( Figure 4D), suggesting the rapid metabolism of PPT-type ginsenosides in human intestine and it partly attributed to the absence of Re and Rg1 in human plasma despite of the higher content in Korean red ginseng extract.

Discussion
Despite the therapeutic benefits of various ginsenosides, which include anti-cancer, anti-diabetic, anti-oxidative, and immune-stimulating effects [3][4][5][6][7][8], the plasma concentration of these ginsenosides and their pharmacokinetic-pharmacodynamic relationship need to be further investigated. As its first step, analytical methods for various ginsenosides and pharmacokinetic profile of these ginsenosides are critical. We developed an analytical method for 13 ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, and F1, Rh2, CK, PPD, and PPT) using a LC-MS/MS system, which had high sensitivity (i.e., the LLOQ of all ginsenosides was 0.5 ng/mL) and required a small plasma sample volume (100 μL). The glycosylation number of the ginsenosides was different: tetraglycosylated ginsenosides for Rb1, Rb2, and Rc; triglycosylated ginsenosides for Rd, Re, and Rg1; diglycosylated ginsenosides for F2, Rg3, and Rf; monoglycosylated ginsenosides for Rh2, CK, Rh1, and F1; aglycones for PPD and PPT ( Figure  1). Because of different extraction efficiencies, di-, tri-, and tetraglycosylated ginsenosides were extracted by protein precipitation, and aglycones were extracted by LLE. Monoglycosylated ginsenosides could be extracted using both methods; however, CK and Rh2 were extracted by LLE, and F1 was extracted by protein precipitation based on the extraction recovery and matrix effect.
We further validated our sensitive analytical method by performing a pharmacokinetic study after the oral administration of red ginseng extract (three pouches of red ginseng extract), which has demonstrated tolerability for two weeks of repeated administration [17]. We successfully measured the plasma concentration of Rb1, Rb2, Rc, Rd, Rg3, Rh2, CK, PPD, and PPT. Except for PPT, detectable ginsenosides were all PPD-type ginsenosides and their deglycosylated metabolites. Interestingly, the plasma AUC values of three glycosylated ginsenosides (Rb1, Rb2, and Rc) were correlated with the

Discussion
Despite the therapeutic benefits of various ginsenosides, which include anti-cancer, anti-diabetic, anti-oxidative, and immune-stimulating effects [3][4][5][6][7][8], the plasma concentration of these ginsenosides and their pharmacokinetic-pharmacodynamic relationship need to be further investigated. As its first step, analytical methods for various ginsenosides and pharmacokinetic profile of these ginsenosides are critical. We developed an analytical method for 13 ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, and F1, Rh2, CK, PPD, and PPT) using a LC-MS/MS system, which had high sensitivity (i.e., the LLOQ of all ginsenosides was 0.5 ng/mL) and required a small plasma sample volume (100 µL). The glycosylation number of the ginsenosides was different: tetraglycosylated ginsenosides for Rb1, Rb2, and Rc; triglycosylated ginsenosides for Rd, Re, and Rg1; diglycosylated ginsenosides for F2, Rg3, and Rf; monoglycosylated ginsenosides for Rh2, CK, Rh1, and F1; aglycones for PPD and PPT (Figure 1). Because of different extraction efficiencies, di-, tri-, and tetraglycosylated ginsenosides were extracted by protein precipitation, and aglycones were extracted by LLE. Monoglycosylated ginsenosides could be extracted using both methods; however, CK and Rh2 were extracted by LLE, and F1 was extracted by protein precipitation based on the extraction recovery and matrix effect.
We further validated our sensitive analytical method by performing a pharmacokinetic study after the oral administration of red ginseng extract (three pouches of red ginseng extract), which has demonstrated tolerability for two weeks of repeated administration [17]. We successfully measured the plasma concentration of Rb1, Rb2, Rc, Rd, Rg3, Rh2, CK, PPD, and PPT. Except for PPT, detectable ginsenosides were all PPD-type ginsenosides and their deglycosylated metabolites. Interestingly, the plasma AUC values of three glycosylated ginsenosides (Rb1, Rb2, and Rc) were correlated with the content of red ginseng extract and showed similar T max values, suggesting the similar intestinal absorption kinetics of these ginsenosides despite of the different structures and glycosidation patterns, which is consistent with the previous report [17]. The long terminal half-life suggested that the intestinal metabolism (to other PPD-type metabolites) and excretion of Rb1, Rb2, and Rc may be a slow process. The T max values of Rd, Rh2, CK, and PPD were increased according to the deglycosylated status, suggesting that deglycosylation mediated by β-glucosidase in the intestinal microbiome could occur sequentially and steadily [11,23,24], and Rh2, CK, and PPD could be detected in human plasma even though they are not present in red ginseng extract.
In the case of Rg3, its T max was smaller compared with that of Rh2 and CK because of its high content in red ginseng extract. Re and Rg1 (PPT-type ginsenosides) were not detected even though they are present in red ginseng extract; however, PPT was detected. It is possible that Re and Rg1 are metabolized to PPT by intestinal microbiota before the absorption occur [11,23,24] and biotransformation of PPT could be faster than the formation rate of PPD. However, we should note that the time-dependent gut metabolism of ginsenosides in human intestine has never been investigated, therefore we speculated time-dependent gut metabolism of ginsenoside from the plasma concentration and T max of ginsenosides and their deglycosylated metabolites. Particularly, for CK concentration, large inter-subject variation was shown in Figure 4B and previous publication [17]. This variability could be attributed to inter-subject variable metabolism related to the intestinal microbiota [25] and further studies should focus on the characterization of microorganisms that produce it and the potential beneficial effects of this metabolite.
The above stock solutions were divided and mixed according to the sample preparation method (i.e., protein precipitation and LLE). The ginsenosides for protein precipitation method (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, and F1) were mixed and diluted with methanol to a concentration of 2000 ng/mL. The ginsenosides for LLE (Rh2, CK, PPD, and PPT) were mixed and diluted with methanol to a concentration of 2000 ng/mL. Working solutions were then serially diluted with methanol to obtain calibration working solutions of 5, 10, 20, 50, 200, 500, 1000, and 2000 ng/mL. Quality control (QC) working solutions were prepared at 15, 150, and 1500 ng/mL with each ginsenoside.

Preparation of Calibration Curve and QC Samples
Calibration curve samples were prepared by spiking 10 µL of working solution into 90 µL of human blank plasma at final concentrations of 0.5, 1, 2, 5, 20, 50, 100, and 200 ng/mL. QC samples were prepared by spiking 10 µL of QC working solution into 90 µL of human blank plasma at final concentrations of 1.5, 15, and 150 ng/mL of QC samples.
For protein precipitation, 600 µL of an IS (0.05 ng/mL berberine in methanol) was added to 100 µL of calibration curve samples and QC samples. Then, the mixture was vortexed for 15 min and centrifuged at 16,100× g for 5 min. After centrifugation, 500 µL of the supernatant was transferred to a clean tube and evaporated to dryness under a nitrogen stream at 40 • C. The residue was reconstituted with 150 µL of 70% methanol consisting of 0.1% formic acid.
For LLE, 50 µL of an IS (20 ng/mL 13C-caffeine in water) and 800 µL of MTBE was added to 100 µL of calibration curve samples and QC samples. The mixture was vortexed for 10 min and centrifuged at 16,100× g for 5 min. After centrifugation, the samples were frozen at −80 • C for 4 h. The upper layer was transferred to a clean tube and evaporated to dryness under a nitrogen stream. The residue was reconfigured with 150 µL of 80% methanol consisting of 0.1% formic acid.

Specificity
The specificity of the method was assessed by comparing chromatogram responses of six lots of human blank plasma with lower limit of quantification (LLOQ) sample.

Linearity
The linearity of the method was assessed using six calibration curves analyzed on six different days. The calibration curve was obtained by plotting the peak area ratio against the concentration of each drug at eight-point levels with a weighting factor of 1/x 2 .

Precision and Accuracy
The intra-day (n = 5) and inter-day (n = 6) precision and accuracy were evaluated using three different QC samples for each analyte. The precision and accuracy at each concentration level were evaluated in terms of the coefficient of variance (CV, %) and relative error (RE, %).

Extraction Recovery and Matrix Effect
The extraction recovery and matrix effect were assessed for three different QC samples using six different blank plasma samples. The extraction recoveries were evaluated by comparing the peak areas of the extracted samples (spiked before extraction) with those of the unextracted samples (spiked after blank extraction) [26]. The matrix factor for the analyte and IS was calculated in each lot by comparing the peak responses of the post-extraction samples (spiked after blank extraction) against neat solutions, which have the same amount of analyte as the extracted sample [26].

Stability
Short-term stability was evaluated to determine whether the sample was stable during treatment. All analytes and IS of the spiked plasma samples were left for at least 6 h at 25 • C. The spiked plasma samples were also subjected to a freeze (−80 • C) and thaw cycle (25 • C and stand for 2 h) three times. After the samples were processed, it was confirmed that they were stable at 8 • C for 24 h. The stability test was conducted using three different concentrations of QC samples.

Pharmacokinetic Study
The study was approved by the Institutional Review Board of Kyungpook National University Hospital (KNUH, Daegu, Republic of Korea) and was conducted at the KNUH Clinical Trial Center in accordance with the applicable Good Clinical Practice guidelines (IRB approval no. KNUH 2018-04-028-002). All subjects provided written informed consent before study enrollment and underwent clinical evaluation including physical examination, serology tests, 12-lead electrocardiography, and clinical history assessment. A total of 11 healthy Korean male subjects aged ≥ 19 years and with a body weight of ≥ 50 kg were enrolled in this study.
The volunteers took 3 pouches of red ginseng extract per day at 9 AM for 2 weeks. On the 14th day, after taking the last dose of the red ginseng extract, blood samples (5 mL) were collected in a heparinized tube at 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h post-dose via a saline-locked angiocatheter. The plasma was collected by centrifugation for 10 min at 3000 × g and stored at −80 • C until analysis.
To analyze the ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, and F1, 600 µL of an IS (0.05 ng/mL berberine in methanol) was added to 100 µL of plasma samples. Then, the mixture was vortexed for 15 min and centrifuged at 16,100× g for 5 min. After centrifugation, 500 µL of the supernatant was transferred to a clean tube and evaporated to dryness under a nitrogen stream at 40 • C. The residue was reconstituted with 150 µL of 70% methanol consisting of 0.1% formic acid, and a 10 µL aliquot was injected into the LC-MS/MS system.
To analyze the ginsenosides Rh2, CK, PPD, and PPT, 50 µL of an IS (20 ng/mL 13C-caffeine in water) and 800 µL of MTBE were added to 100 µL of plasma samples. The mixture was vortexed for 10 min and centrifuged at 16,100× g for 5 min. After centrifugation, the samples were frozen at −80 • C for 4 h. The upper layer was transferred to a clean tube and evaporated to dryness under a nitrogen stream. The residue was reconfigured with 150 µL of 80% methanol consisting of 0.1% formic acid, and a 10 µL aliquot was injected into the LC-MS/MS system.
Similarly, the ginsenoside content in the red ginseng extract was quantified. The red ginseng extract (100 mg) was diluted 50-fold with methanol, and 100 µL of the diluted sample was prepared using the method described previously. Aliquots (10 µL) of the supernatant were directly injected into the LC-MS/MS system.

Data Analysis
Pharmacokinetic parameters were estimated using non-compartmental methods (WinNonlin version 2.0; Pharsight Co., Certara, NJ, USA). All pharmacokinetic parameters are presented as the mean ± standard deviation (SD).

Conclusions
A sensitive LC-MS/MS method for the detection of 13 ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, and F1, Rh2, CK, PPD, and PPT) in human plasma with a LLOQ of 0.5 ng/mL was developed and validated. This method can be used in the bioanalysis and pharmacokinetic studies of ginseng products administered at multiple therapeutic doses. Following repeated oral administration of red ginseng extract for two weeks, the plasma concentrations of Rb1, Rb2, Rc, Rd, Rg3, Rh2, CK, PPD, and PPT were detected. The findings can provide valuable information on ginsenoside metabolism in the human body and contribute to in vivo pharmacokinetic-pharmacodynamic correlation studies.

Conflicts of Interest:
The authors declare no conflict of interest.