Pharmacokinetic and metabolic studies of ginsenoside Rb3 in rats using RRLC-Q-TOF-MS

Pharmacokinetic and metabolic studies of ginsenoside Rb3 in rats using RRLC-Q-TOF-MS Abstract Ginsenoside Rb3 is one of major ginsenosides in Panax ginseng with effect on cardio-vascular and central nervous system. The aim of this study is to develop a rapid resolution liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (RRLC-Q-TOF-MS) method for pharmacokinetic study of ginsenoside Rb3 and simultaneous determination of metabolites in rats. The results showed that the concentration–time profile of ginsenoside Rb3 conformed to a two-compartment pharmacokinetic model after intravenous administration at the dosage of 2.0 mg/kg for rats. The mean plasma elimination half-lives were 13.77 ± 1.23 min and 2045.70 ± 156.20 min for the distribution and exterminate phases t1/2α and t1/2β. In the metabolic study, prototype ginsenoside Rb3 and deglycosylation metabolites were characterized by comparison with the retention time of the standard compounds, accurate mass measurement and the characteristic fragment ions obtained from MS/MS. Two major metabolites Mb1 and M2’ were tentatively identified in rat urine samples after intravenous administration, and four possible metabolites Mb1, F2, M2’ and CK were detected in rat feces samples after oral administration. The deglycosylation was found to be the major metabolic pathways of ginsenoside Rb3 in rat. The in vivo metabolic pathway of ginsenoside Rb3 was summarized. Introduction The root of Panax ginseng C.A. Meyer is a well-known medicinal herb in Traditional Chinese Medicine, and has been used as herbal remedy and adaptogen that helps to maintain human’s health and restore the body to balance (1). Ginsenosides, considered as the main active components of P. ginseng, have shown various pharmacological effects on anti-tumor, diabetes mellitus, cardio-vascular system, central nervous system and immune system (2). Ginsenosides in P. ginseng present chemical diversity, and ~50 ginsenosides have been identified in P. ginseng and related processed products. (3) Pharmacokinetic and metabolic studies on selected ginsenosides have been carried on to better understand the bioactive forms, biological effects and safety of ginseng (4, 5). Ginsenoside Rb3 is one of major ginsenosides with content of ~2.1 mg/g in P. ginseng, belonging to protopanaxadiol (PPD) ginsenoside (6). It has increasingly attracted researchers’ attention for its effects on cardio-vascular and central nervous system. Studies have shown that ginsenoside Rb3 possesses the effect on myocardial injury and heart function impairment induced by isoproterenol in rats (7), and it could protect cardiomyocytes against ischemia–reperfusion (I/R) injury via the inhibition mediated NF-B pathway, suggesting that ginsenoside Rb3 has the potential to serve as a novel therapeutic agent for myocardial I/R injury (8). The results of ginsenoside Rb3 in hypertension showed that it could attenuate oxidative stress and preserve endothelial function in renal arteries from hypertensive rats (9). It was reported that ginsenoside Rb3 could protect neurons in hippocampal slices of rat while exposed to oxygen/glucose deprived (10, 11). The effect of ginsenoside Rb3 on synaptic transmission after oxygen/glucose deprived in vitro indicated that the activation of γ-aminobutyric acid receptor is correlated with its neuroprotective mechanisms (12) and has the significant protective effects on glutamate excitotoxic injury in cultured neurons of rat hippocampus (13). For the potential values of ginsenoside Rb3 as a novel therapeutic agent on cardio-vascular and central nervous system, the investigation of pharmacokinetic and metabolism of ginsenoside Rb3 will be helpful for better knowing the comprehensive pharmacokinetic parameters, the actual biotransformation and excretion pathway, and designing rational pharmacological and clinical dosage. The metabolic study of ginsenosides Rb3 has not been reported. Recently, liquid chromatography coupled with mass spectrometry (LC/MS) has been widely used for the metabolic study of ginsenosides due to the high-sensitivity and capability of structural elucidation (14, 15). Quadrupole-time-of-flight mass spectrometry (Q-TOF-MS) coupled to the fast LC systems (Ultra performance liquid chromatography or rapid resolution liquid chromatography (RRLC)) exhibits advantages of rapid analysis, high chromatographic resolution and accurate mass measurement for the metabolism studies of ginsenosides (16, 17). In this study, a sensitive and selective RRLC-Q-TOF-MS method was developed to investigate the pharmacokinetics of ginsenoside Rb3 in rat plasma after intravenous administration. And the metabolites of ginsenoside Rb3 were investigated in rat urine and feces samples after intravenous and oral administrations to explore the active component for pharmacological effect. Experimental Chemicals and reagents Ginsenoside Rb3 (purity 98.0%), Rf (internal standard, IS, purity 98.0%), F2 (purity 98.0%) and Compound K (CK; purity 98.0%) were purchased from Jilin University (Changchun, China). HPLC-grade methanol and acetonitrile were purchased from TEDIA (USA). Ultrapure water used in the experiments was prepared by a Milli-Q Ultrapure water system (Millipore, Billerica, USA). Instruments The LC analysis was carried out using an Agilent 1200 series RRLC system (Santa Clara, CA, USA) equipped with a binary pump, micro-degasser, an auto-plate sampler and a thermostatically controlled column apartment; MS detection was performed on an Agilent 6520 Q-TOF mass spectrometer (Santa Clara, CA, USA) equipped with an electrospray interface, and automatic calibration system. LC and MS conditions The LC separation was performed on an Agilent SB-C18 column (3.0 mm × 100 mm, 1.8 μm, 600 bar) at a temperature of 25°C. 0.1% formic acid (v/v) and acetonitrile were used as the mobile phases A and B, respectively. The gradient elution was programmed as follows: 0–13 min (18–46%B), 13–20 min (46–50%B), 20–23 min (50–90%B) and 23–25 min (90% B). The flow rate was 0.3 mL/min. The injected sample volume was 5 μL. This RRLC system was connected to Q-TOF mass spectrometer. The Q-TOF-MS scan range was set at m/z 100–2,200 in negative ion modes. The conditions of the electrospray ionization source were as follows: drying gas (N2) flow rate was 8.0 L/min, drying gas temperature was at 350°C, nebulizer was 255 kPa, capillary voltage was 3,500 V, fragmentor was 175 V and skimmer was 65 V. Data analysis was performed by Agilent software Mass Hunter Qualitative (version B.03.01). Preparation of standard and quality control samples The stock solutions were prepared by dissolving standard Rb3 and IS in methanol to a final concentrations of 50 and 40 μg/mL, respectively. Working solutions were obtained freshly by further diluting stock solutions in methanol. All stock solutions and working solutions were stored at 4°C. Calibration standard of Rb3 were prepared by adding appropriate amount of stock solutions and IS into 100 μL of blank rat plasma and well mixed. About 200 μL of methanol was added into each standard solution and vortex for 1 min. The sample was then centrifuged at 13,000 × g for 5 min and the supernatant was transferred into another Eppendorf tube. Calibration standards were obtained at final concentrations of 0.08, 0.10, 0.20, 0.40, 0.60, 0.80 and 1.0 μg/mL of Rb3 in plasma. The injected sample volume was 5 μL by LC/MS to obtain the calibration curve. Quality control (QC) samples in plasma were similarly prepared at concentration levels of low, middle and high (0.10, 0.36 and 0.76 μg/mL). Pharmacokinetic study The pharmacokinetic study were carried out in six Male Wistar rats (200 ± 20 g) supplied by Jilin University (Changchun, China). Rats were fasted for 12 h before dosing, with free access to water. Six rats were administrated with 2.0 mg/kg Rb3 (dissolved in 500 μL of 5% β-cyclodextrin aqueous and 500 μL of 0.9% sodium chloride) by intravenous injection. About 1 mL Rb3 dosing solution was given to each rat via tail vein. Blood samples (300 μL) were collected by punctuating the retro-orbital sinus at 0 (before dosage), 2, 5, 10, 15, 20, 30, 40, 60 and 90 min, and 2, 4, 8, 12 and 24 h after the intravenous injection. The blood samples were then centrifuged at 3,000 × g for 10 min to get plasma and then stored at −20°C until analysis. About 200 μL methanol and 100 μL IS were added into each plasma sample and vortex for 30 s, and then the sample was centrifuged at 13,000 × g for 5 min. The aliquot of 5 μL was injected into the LC/MS. Samples were then prepared for LC/MS analysis as described above. Software 3P97 (Practical Pharmacokinetic Program, 1997, China) was used to analyze pharmacokinetic study data to determine the pharmacokinetic parameters of Rb3 in rats. Metabolic studies Twelve male Wistar rats (200 ± 20 g) were divided into two groups at random for Rb3 as the intravenous injection group and the oral administration group. The intravenous dose (2 mg/kg) and oral dose (50 mg/kg) were conducted as above. Rat urine samples were collected from 0 to 48 h after the intravenous and oral administrations of Rb3. Each urine sample was concentrated under a gentle stream of nitrogen and extracted with n-butanol. The extract was dried and then the residue was dissolved in 1.0 mL 80% methanol. After passing through 0.45 μm filter, 5 μL of filtrate was injected for metabolism study by LC/MS. Rat feces samples were collected from 0 to 24 h after oral administration of Rb3. The feces sample was ground and suspended in 50 mL water, and then extracted three times with total 100 mL n-butanol. The extract was processed as above urine samples for metabolism analysis. Results Development of RRLC-Q-TOF-MS and MS/MS methods Ginsenoside Rb3 (Figure 1) was characterized by RRLC-Q-TOF-MS in full MS scan. Negative ion mode was selected in this experiment due to much lower background and less interference than that of positive ion mode. As 0.1% formic acid solution was employed as the mobile phase, the deprotonated ion [M-H]− (m/z 1077) and adduct ion [M+HCOO]− (m/z 1123) were detected in the negative ion mode, providing information about the molecular mass. MS/MS was employed for structure elucidation and the fragmentation nomenclature used in this paper is based on that described by Costello and Liu (18, 19). C20 position and C3 position substitutions are defined as α and β linkages, respectively. The mass accuracy for all molecular ions and fragment ions were <10 ppm. In the following discussion, the m/z value of ion was expressed as integer number and the accurate mass measurement is listed in Table I. Figure 2A shows the MS/MS spectra of Rb3. Y1α (m/z 945), Y0α (m/z 783), Y’1β (m/z 621), Y0β’ (m/z 459), B2α (m/z 293), B1β (m/z 161) and C1α (m/z 149) were observed. Y1α ion at m/z 945 was generated by the loss of a terminal xylose residue (132 Da) at C20 position and the corresponding C1α ion at m/z 149 indicated the terminal xylose substitution. Y0α ion at m/z 783 was produced by loss of the residue of glucose–xylose (162 + 132 Da) at C20 position and the corresponding B2α ion at m/z 293 was observed indicating the glucose–xylose linkage. Y’1β ion at m/z 621 was observed by loss of glucose–xylose residue (162 + 132 Da) at C20 position and a terminal glucose residue (162 Da) at C3 position. Y’0β ion at m/z 459 representing the PPD-type ginsenoside was produced by loss of glucose–glucose residue (162 + 162 Da) at C3 position and glucose–xylose residue (162 + 132 Da) at C20 position. The fragment information of ginsenoside Rb3 is summarized in Table I. Figure 1. View largeDownload slide Structures of ginsenosides and metabolites (Glc: glucose, xyl: xylose). Figure 1. View largeDownload slide Structures of ginsenosides and metabolites (Glc: glucose, xyl: xylose). Table I. Ginsenoside Rb3 and Identified Metabolites by RRLC-Q-TOF-MS/MS Peak (min) Identity Molecular formula MW Measured value (Mass accuracy < 10 ppm) [M-H]− [M+HCOO]− MS/MS fragment ions of [M-H]− (mass accuracy < 10 ppm) 10.19 Rb3 C53H90O22 1,078.5924 1,077.5838 1,123.5897 945.5088(Y1α), 783.4629(Y0α), 621.4125(Y1β’), 459.3637(Y0β’), 293.0716(B2α), 191.0503(0,4A2α), 161.0403 (B1β) and 149.0382 (C1α) 13.86 Mb1 C47H80O17 916.5276 915.5295 961.5247 783.4758(Y1α), 621.4123(Y0α), 459.3692(Y0β’), 293.1158(B2α), 191.0517(0,4A2α) and 161.0353 (B1β)149.0419(C1α) 15.76 M5(F2) C42H72O13 784.4973 783.4753 829.2862 621.4138(Y1α), 459.3648(Y0β’) and 161.0415(B1α) 21.94 M2’ C41H70O12 754.4863 753.4826 799.4871 621.4104(Y1α), 459.3675(Y0α), 293.1152(B2α), 191.0487(0,4A2α) and 149.0430(C1α) 23.96 C-K C36H62O8 622.4439 621.4365 667.4372 459.3642(Y0α), 161.0374(B1α) and 119.0462(2,4A1α) Peak (min) Identity Molecular formula MW Measured value (Mass accuracy < 10 ppm) [M-H]− [M+HCOO]− MS/MS fragment ions of [M-H]− (mass accuracy < 10 ppm) 10.19 Rb3 C53H90O22 1,078.5924 1,077.5838 1,123.5897 945.5088(Y1α), 783.4629(Y0α), 621.4125(Y1β’), 459.3637(Y0β’), 293.0716(B2α), 191.0503(0,4A2α), 161.0403 (B1β) and 149.0382 (C1α) 13.86 Mb1 C47H80O17 916.5276 915.5295 961.5247 783.4758(Y1α), 621.4123(Y0α), 459.3692(Y0β’), 293.1158(B2α), 191.0517(0,4A2α) and 161.0353 (B1β)149.0419(C1α) 15.76 M5(F2) C42H72O13 784.4973 783.4753 829.2862 621.4138(Y1α), 459.3648(Y0β’) and 161.0415(B1α) 21.94 M2’ C41H70O12 754.4863 753.4826 799.4871 621.4104(Y1α), 459.3675(Y0α), 293.1152(B2α), 191.0487(0,4A2α) and 149.0430(C1α) 23.96 C-K C36H62O8 622.4439 621.4365 667.4372 459.3642(Y0α), 161.0374(B1α) and 119.0462(2,4A1α) View Large Table I. Ginsenoside Rb3 and Identified Metabolites by RRLC-Q-TOF-MS/MS Peak (min) Identity Molecular formula MW Measured value (Mass accuracy < 10 ppm) [M-H]− [M+HCOO]− MS/MS fragment ions of [M-H]− (mass accuracy < 10 ppm) 10.19 Rb3 C53H90O22 1,078.5924 1,077.5838 1,123.5897 945.5088(Y1α), 783.4629(Y0α), 621.4125(Y1β’), 459.3637(Y0β’), 293.0716(B2α), 191.0503(0,4A2α), 161.0403 (B1β) and 149.0382 (C1α) 13.86 Mb1 C47H80O17 916.5276 915.5295 961.5247 783.4758(Y1α), 621.4123(Y0α), 459.3692(Y0β’), 293.1158(B2α), 191.0517(0,4A2α) and 161.0353 (B1β)149.0419(C1α) 15.76 M5(F2) C42H72O13 784.4973 783.4753 829.2862 621.4138(Y1α), 459.3648(Y0β’) and 161.0415(B1α) 21.94 M2’ C41H70O12 754.4863 753.4826 799.4871 621.4104(Y1α), 459.3675(Y0α), 293.1152(B2α), 191.0487(0,4A2α) and 149.0430(C1α) 23.96 C-K C36H62O8 622.4439 621.4365 667.4372 459.3642(Y0α), 161.0374(B1α) and 119.0462(2,4A1α) Peak (min) Identity Molecular formula MW Measured value (Mass accuracy < 10 ppm) [M-H]− [M+HCOO]− MS/MS fragment ions of [M-H]− (mass accuracy < 10 ppm) 10.19 Rb3 C53H90O22 1,078.5924 1,077.5838 1,123.5897 945.5088(Y1α), 783.4629(Y0α), 621.4125(Y1β’), 459.3637(Y0β’), 293.0716(B2α), 191.0503(0,4A2α), 161.0403 (B1β) and 149.0382 (C1α) 13.86 Mb1 C47H80O17 916.5276 915.5295 961.5247 783.4758(Y1α), 621.4123(Y0α), 459.3692(Y0β’), 293.1158(B2α), 191.0517(0,4A2α) and 161.0353 (B1β)149.0419(C1α) 15.76 M5(F2) C42H72O13 784.4973 783.4753 829.2862 621.4138(Y1α), 459.3648(Y0β’) and 161.0415(B1α) 21.94 M2’ C41H70O12 754.4863 753.4826 799.4871 621.4104(Y1α), 459.3675(Y0α), 293.1152(B2α), 191.0487(0,4A2α) and 149.0430(C1α) 23.96 C-K C36H62O8 622.4439 621.4365 667.4372 459.3642(Y0α), 161.0374(B1α) and 119.0462(2,4A1α) View Large Figure 2. View largeDownload slide MS/MS spectrum of (A) ginsenosides Rb3, (B) metabolites P1, (C) P2, (D) P3 and (E) P4 in the negative ion mode. Figure 2. View largeDownload slide MS/MS spectrum of (A) ginsenosides Rb3, (B) metabolites P1, (C) P2, (D) P3 and (E) P4 in the negative ion mode. Method validation The specificity and selectivity were investigated by comparing total ion chromatogram (TIC) of three different batches of blank plasma and samples with 5.00 μg/mL of Rb3 and IS solutions. The detection of Rb3 and IS by RRLC-Q-TOF-MS was highly selective without the interference of endogenous constituents in the biologic matrices. The retention time of Rb3 and IS were at 10.19 and 6.89 min, respectively. Calibration curves were obtained by plotting the peak area ratios of Rb3/IS versus the concentration of Rb3. The regression equation was Y = 4.5707 × −0.0065 (Y represents the peak area ratios of the analyte Rb3/IS, X represents the concentration of Rb3) with the correlation coefficient of 0.9966. Good linearity was obtained over the dynamic range of 0.08–0.90 μg/mL. The limit of detection and quantitation (LOD and LOQ) was measured as 0.05 and 0.08 μg/mL, respectively. The precision and accuracy were evaluated by analysis of variation results of intra-day (six samples at each QC level) and inter-day (three consecutive days). As shown in Table II, the intra-day and inter-day precisions were <5% for each QC level of Rb3. The accuracy determined from QC samples was range from −5% to −1%. The method showed good accuracy and precision. The stability of Rb3 was studied by analyzing QC samples at three concentrations. The samples were stable after three freeze-thaw cycles (from −20°C to room temperature) in three days and storage at room temperature in 24 h. The extraction recovery was determined by comparing the peak area of Rb3 plasma samples spiked before extraction with the peak area of Rb3 plasma samples spiked after extraction at three concentrations of 0.10, 0.36 and 0.76 μg/mL. The extraction recoveries of Rb3 were in the range of 89.32–92.78% (Table II). Table II. Precision, Accuracy and Extraction Recoveries of Ginsenoside Rb3 in Rat Plasma Nominal concentration (μg/mL) Measured concentration (μg/mL) RSD (%) Intra-day RSD (%) Inter-day RSD (%) Extraction recovery (%) 0.89 0.78 −1.53 1.74 3.18 89.32 0.38 0.35 −3.22 3.31 4.44 92.67 0.20 0.18 −4.51 2.65 2.97 92.78 Nominal concentration (μg/mL) Measured concentration (μg/mL) RSD (%) Intra-day RSD (%) Inter-day RSD (%) Extraction recovery (%) 0.89 0.78 −1.53 1.74 3.18 89.32 0.38 0.35 −3.22 3.31 4.44 92.67 0.20 0.18 −4.51 2.65 2.97 92.78 Table II. Precision, Accuracy and Extraction Recoveries of Ginsenoside Rb3 in Rat Plasma Nominal concentration (μg/mL) Measured concentration (μg/mL) RSD (%) Intra-day RSD (%) Inter-day RSD (%) Extraction recovery (%) 0.89 0.78 −1.53 1.74 3.18 89.32 0.38 0.35 −3.22 3.31 4.44 92.67 0.20 0.18 −4.51 2.65 2.97 92.78 Nominal concentration (μg/mL) Measured concentration (μg/mL) RSD (%) Intra-day RSD (%) Inter-day RSD (%) Extraction recovery (%) 0.89 0.78 −1.53 1.74 3.18 89.32 0.38 0.35 −3.22 3.31 4.44 92.67 0.20 0.18 −4.51 2.65 2.97 92.78 Pharmacokinetic study of ginsenoside Rb3 The validated RRLC-Q-TOF-MS method was used for the pharmacokinetic study of ginsenoside Rb3 in rat plasma by intravenous administration. Ginsenoside Rb3 was detected in plasma samples with a dose at 2.0 mg/kg. The mean plasma concentration–time curves of ginsenoside Rb3 (n = 6) was shown in Figure 3. Compartment model was estimated by the value of AIC (Akaike’s Information Criterion). The result of experiment demonstrated that ginsenoside Rb3 concentration–time profile follows a two-compartment pharmacokinetic model after intravenous administration for rats. The main pharmacokinetic parameters are presented in Table III. The mean plasma elimination half-lives are 13.77 ± 1.23 min and 2045.70 ± 156.20 min for distribution and exterminate phase t1/2α and t1/2β, respectively. AUC(0−t) and AUC(0−∞) (the area under the curve) were calculated as 366.58 ± 18.25 and 1,047.42 ± 152.04 μg/(L·min). Plasma clearance (CL) was 1.91 ± 0.02 (L/min/kg). Figure 3. View largeDownload slide (A) Plasma concentration–time profiles of ginsenoside Rb3 after intravenous administration (2 mg/kg) in Wistar rats (n = 6) and (B) the related ln concentration–time profiles. Figure 3. View largeDownload slide (A) Plasma concentration–time profiles of ginsenoside Rb3 after intravenous administration (2 mg/kg) in Wistar rats (n = 6) and (B) the related ln concentration–time profiles. Table III. Main Pharmacokinetic Parameters of Rb3 in Rat Plasma after Intravenous Administration (2 mg/kg) to Six Rats (mean ± SD) Parameter Ginsenoside Rb3 C0 (μg/mL) 0.93 ± 0.05 t1/2α (min) 13.77 ± 1.23 t1/2β (min) 2,045.70 ± 156.20 K10 (1/min) 0.001 ± 0.0002 K12 (1/min) 0.033 ± 0.004 K21 (1/min) 0.017 ± 0.001 AUC(0−t) (μg/L ∗ min) 366.58 ± 18.25 AUC (0−∞) (μg/L ∗ min) 1,047.43 ± 152.04 Vc (L/kg) 2,144.02 ± 174.12 CL (L/min/kg) 1.91 ± 0.02 Parameter Ginsenoside Rb3 C0 (μg/mL) 0.93 ± 0.05 t1/2α (min) 13.77 ± 1.23 t1/2β (min) 2,045.70 ± 156.20 K10 (1/min) 0.001 ± 0.0002 K12 (1/min) 0.033 ± 0.004 K21 (1/min) 0.017 ± 0.001 AUC(0−t) (μg/L ∗ min) 366.58 ± 18.25 AUC (0−∞) (μg/L ∗ min) 1,047.43 ± 152.04 Vc (L/kg) 2,144.02 ± 174.12 CL (L/min/kg) 1.91 ± 0.02 Table III. Main Pharmacokinetic Parameters of Rb3 in Rat Plasma after Intravenous Administration (2 mg/kg) to Six Rats (mean ± SD) Parameter Ginsenoside Rb3 C0 (μg/mL) 0.93 ± 0.05 t1/2α (min) 13.77 ± 1.23 t1/2β (min) 2,045.70 ± 156.20 K10 (1/min) 0.001 ± 0.0002 K12 (1/min) 0.033 ± 0.004 K21 (1/min) 0.017 ± 0.001 AUC(0−t) (μg/L ∗ min) 366.58 ± 18.25 AUC (0−∞) (μg/L ∗ min) 1,047.43 ± 152.04 Vc (L/kg) 2,144.02 ± 174.12 CL (L/min/kg) 1.91 ± 0.02 Parameter Ginsenoside Rb3 C0 (μg/mL) 0.93 ± 0.05 t1/2α (min) 13.77 ± 1.23 t1/2β (min) 2,045.70 ± 156.20 K10 (1/min) 0.001 ± 0.0002 K12 (1/min) 0.033 ± 0.004 K21 (1/min) 0.017 ± 0.001 AUC(0−t) (μg/L ∗ min) 366.58 ± 18.25 AUC (0−∞) (μg/L ∗ min) 1,047.43 ± 152.04 Vc (L/kg) 2,144.02 ± 174.12 CL (L/min/kg) 1.91 ± 0.02 Metabolic study of Rb3 in rat urine after intravenous administration Related metabolites were tentatively identified as deglycosylation products based on fragmentation information and accurate mass measurement. Figure 4A shows a typical RRLC-Q-TOF-MS-TIC of a rat urine sample after intravenous administration in the negative ion mode. Prototype ginsenoside Rb3 and two possible metabolites were detected. Ginsenoside Rb3 was identified by comparing the retention time and fragmentation patterns with that of the standard compound. The extracted ion chromatograms of the two deglycosylated metabolites of Rb3 (P1 and P2) were shown in Figure 4B and C. As 0.1% formic acid solution was employed as the mobile phase, detected metabolite showed adduct ion [M+HCOO]− in negative ion mode. P1 and P2 exhibited [M+HCOO]− ions at m/z 961 and m/z 799 in RRLC-Q-TOF-MS, determined as metabolites by loss of glucose and glucose–glucose substitution at C3 position from ginsenoside Rb3, respectively. In Figure 2B, the MS/MS spectrum of metabolites P1, characteristic fragment ions Y1α (m/z 783), Y0α (m/z 621), Y0β’ (m/z 459), B2α (m/z 293), 0,4A2α (m/z 191.0517), B1β (m/z 161) and C1α (m/z 149) were all observed, which are coincident with the MS/MS fragment ions of ginsenoside Rb3. Product ions at m/z 783 was produced by the loss of xylose residue and HCOOH (132 + 46 Da) and m/z 621 was generated by loss of glucose–xylose residue and HCOOH (162 + 132 + 46 Da) from the [M+HCOO]− ion at m/z 961. That indicated the terminal sugar was xylose and the chain substitution was glucose–xylose, corresponding to C20 position of ginsenoside Rb3. Ion at m/z 459 was characteristic ion of PPD-type ginsenoside helpful for aglycone determination, which was produced by lose of glucose–xylose, glucose residue and HCOOH (162 + 132 + 162 + 46 Da) from the [M+HCOO]− ion at m/z 961. The corresponding glucose–xylose, glucose and xylose residue ions at m/z 293 (B2α), m/z 161 (B1β) and m/z 149 (C1α) were also observed in the MS/MS spectrum. Thus, P1 was tentatively identified as metabolite by loss of terminal glucose (162 Da) at C3 position from Rb3. Ginsenosides Rb2 and Rb3 are isomers with C-20 terminal sugar substitution as arabinose and xylose, respectively. The deglycosylation product by loss of C-3 terminal substitution glucose form ginsenoside Rb2 was identified as Mb in stomach and large intestine (20, 21). Thus, the metabolite P1 of Rb3 by loss of terminal glucose (162 Da) at C3 position was defined as Mb1 in this experiment (Figures 1 and 2B). Figure 2C shows the MS/MS spectrum of P2. The ion at m/z 753 was produced by loss of the HCOOH (46 Da) from [M+HCOO]− ions at m/z 799. The fragments ion at m/z 621 (Y1α) was produced by loss HCOOH and a xylose residue (132 Da) at C20 position, and the corresponding ion at m/z 149 (C1α) was observed in the MS/MS spectra, which indicated that the terminal substitution was xylose. PPD-type characteristic ion at m/z 459 (Y0α) was generated by loss of HCOOH, glucose–xylose residues (46 + 162 + 132 Da) from [M+HCOO]− ions. That indicated that the substitution sugar linkage was glucose–xylose at C20 position, and ions at m/z 293 (B2α) and m/z 191 (0,4A2α) also proved that. Thus, P2 was deduced as metabolite deglucosylated from ginsenoside Rb3 by loss the substitution saccharide at C3 position (Figures 1 and 2C). According to reference’s reports about the metabolite M2 produced by loss of the saccharide substitution at C3 position form ginsenoside Rb2 (20, 22). This metabolite produced from ginsenoside Rb3 is an isomer of M2 with difference of xylose as terminal substitution at C20 position, so it is named as M2’ in this experiment. The result of experiment showed that part of ginsenoside Rb3 was excreted through urine as prototype and metabolites Mb1 and M2’ after intravenous administration. The identified compounds are summarized in Table I. Figure 4. View largeDownload slide (A) RRLC-Q-TOF-MS TIC of rat urine sample after intravenous administration of Rb3 in the negative ion mode, (B) EIC of the metabolite P1 and (C) EIC of the metabolite P2. Figure 4. View largeDownload slide (A) RRLC-Q-TOF-MS TIC of rat urine sample after intravenous administration of Rb3 in the negative ion mode, (B) EIC of the metabolite P1 and (C) EIC of the metabolite P2. Metabolic study of Rb3 in rat feces after oral administration Ginsenoside Rb3 and four possible metabolites were detected in rat feces samples collected from 0 to 24 h after oral administration by using RRLC-Q-TOF-MS and MS/MS. The TIC of rat feces samples after oral administration is shown in Figure 5A. Ginsenoside Rb3 was identified by comparing retention time and fragmentations with that of standard compound (Figure 5A, Table I). Figure 5B–D and E showed the extracted ion chromatograms of the four metabolites (P1, P2, P3 and P4). In full MS negative ion mode, P1, P2, P3 and P4 exhibited [M+HCOO]− ions at m/z 961, m/z 829, m/z 799 and m/z 667. These four metabolites were further analyzed by MS/MS to give the structural information (Figure 2, Table I). MS/MS fragment ions of P1 and P2 were the same with the identified metabolites Mb1 and M2’ in rat urine after intravenous administration (Table I). The retention time of P1 and P2 were 13.86 and 15.76 min, respectively. Therefore, metabolites P1 and P2 were tentatively identified as Mb1 and M2’ by analysis of retention time and MS/MS fragmentations. Figure 2D shows the MS/MS spectrum of metabolite P3. [M+HCOO]− ion at m/z 829. The fragments ion at m/z 621 (Y0α) was produced by loss of HCOOH (46 Da) and a glucose residue (162 Da) at C20 position and the corresponding ion at m/z 161 (B1α) was detected in the MS/MS spectra. Characteristic ion at m/z 459 (Y0α) of PPD-type ginsenoside was generated by loss of two glucose residues (162 + 162 Da) substitution. Metabolite P3 has the same retention time and MS/MS fragmentation patterns with the reference standard of F2. Thus, the metabolite P3 was identified as ginsenoside F2. The MS/MS spectra of metabolite P4 is shown in Figure 2E. The [M+HCOO]− ion at m/z 667 generate the product ion at m/z 621 by loss of HCOOH (46 Da). The ion at m/z 459 was observed by loss of one glucose residue (162 Da), indicating the aglycone is PPD-type. And the corresponding ion at 161 m/z (B1α) was observed in the MS/MS spectra. Compared with the reference standard of CK, Metabolite P4 has the same retention time and MS/MS fragmentation patterns. P4 was identified as CK. The metabolic pathway of ginsenoside Rb3 was proposed in Figure 6 according to the major metabolites detected in rat urine and feces samples. Figure 5. View largeDownload slide RRLC-Q-TOF-MS chromatograms of rat feces samples after oral administrations of Rb3: (A) TIC of feces sample, (B) EIC of metabolites P1, (C) EIC of metabolites P2, (D) EIC of metabolites P3 and (E) EIC of metabolites P4. Figure 5. View largeDownload slide RRLC-Q-TOF-MS chromatograms of rat feces samples after oral administrations of Rb3: (A) TIC of feces sample, (B) EIC of metabolites P1, (C) EIC of metabolites P2, (D) EIC of metabolites P3 and (E) EIC of metabolites P4. Figure 6. View largeDownload slide Proposed metabolic pathway of ginsenoside Rb3 in rats. Figure 6. View largeDownload slide Proposed metabolic pathway of ginsenoside Rb3 in rats. Discussion Ginsenoside Rb3 was firstly analyzed to develop RRLC-Q-TOF-MS and MS/MS methods for pharmacokinetics and metabolism studies. Both chromatographic and mass spectrometric conditions were optimized to gain rapid detection and efficient determination. The chromatographic conditions including mobile phase, gradient elution program, column temperature and flow rate were optimized to ensure good separation. A gradient elution with acetonitrile and water containing 0.1% formic acid was finally chosen. The fragment information of ginsenoside Rb3 was summarized. These MS/MS fragmentation provide structural information helpful for elucidation of metabolite of ginsenosides Rb3 in complex mixtures. Then, the established method was validated for evaluating specificity and selectivity, linearity, limit of detection and quantitation, precision and accuracy, and extraction recovery for pharmacokinetic study. The pharmacokinetic result was consistent with the reported pharmacokinetic features of PPD ginsenosides with the relatively higher plasma protein binding (14, 15). Metabolic studies of Rb3 in rat urine after intravenous administration and in rat feces after oral administration were carried out by RRLC-Q-TOF-MS and MS/MS to reveal possible active compounds and to better understand the biotransformation and excretion. In intravenous administration study, metabolites in rat urine collected from 0 to 72 h after administration were analyzed. In the oral administration study, Rb3 was not detected in plasma and urine samples from 0 to 24 h with a dose of 50 mg/kg. This result may be caused by the poor oral absorption of natural ginsenosides or be metabolized as prototype or metabolites (such as CK, PPD) in feces by the gastric acid and intestinal flora after oral administration (14, 21). Then, the metabolites of ginsenoside Rb3 were detected in the feces samples. Conclusions In this study, a RRLC-Q-TOF-MS method was developed for pharmacokinetic studies of ginsenoside Rb3 and simultaneous determination of its metabolites in rat urine and feces samples. The validated method was applied for pharmacokinetic study of ginsenoside Rb3 after intravenous administration. The half-life of ginsenoside Rb3 (t1/2α=13.77 ± 1.23 min and t1/2β = 2,045.70 ± 156.20 min) suggested that the distribution of ginsenoside Rb3 in vivo was quick and the extermination was slow after administration. The further metabolic studies of Rb3 in rat urine after intravenous administration and in rat feces after oral administration were carried out by RRLC-Q-TOF-MS and MS/MS. The deglycosylation was found to be the major metabolic pathways of ginsenoside Rb3 in rat. The experiments demonstrated that part of ginsenoside Rb3 was excreted through urine as prototype and some changed into metabolites Mb1 and M2’ after intravenous administration, and ginsenoside Rb3 could be transformed into Mb1, F2, M2’ and CK in the gastrointestinal tract after oral administration. It is expected that this experiment results would provide some basis for the further developing and understanding the bioactive form of the ginsenoside Rb3. Funding This research work about analytical method was supported by the Science and Technology Development Plan Project of Jilin Province (No. 20170204010YY) and the pharmacokinetic and metabolic studies were supported by project of Key Lab of Ginseng Chemistry and Pharmacology of Jilin Province (No. 20160101334JC). 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Pharmacokinetic and metabolic studies of ginsenoside Rb3 in rats using RRLC-Q-TOF-MS

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Abstract

Abstract Ginsenoside Rb3 is one of major ginsenosides in Panax ginseng with effect on cardio-vascular and central nervous system. The aim of this study is to develop a rapid resolution liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (RRLC-Q-TOF-MS) method for pharmacokinetic study of ginsenoside Rb3 and simultaneous determination of metabolites in rats. The results showed that the concentration–time profile of ginsenoside Rb3 conformed to a two-compartment pharmacokinetic model after intravenous administration at the dosage of 2.0 mg/kg for rats. The mean plasma elimination half-lives were 13.77 ± 1.23 min and 2045.70 ± 156.20 min for the distribution and exterminate phases t1/2α and t1/2β. In the metabolic study, prototype ginsenoside Rb3 and deglycosylation metabolites were characterized by comparison with the retention time of the standard compounds, accurate mass measurement and the characteristic fragment ions obtained from MS/MS. Two major metabolites Mb1 and M2’ were tentatively identified in rat urine samples after intravenous administration, and four possible metabolites Mb1, F2, M2’ and CK were detected in rat feces samples after oral administration. The deglycosylation was found to be the major metabolic pathways of ginsenoside Rb3 in rat. The in vivo metabolic pathway of ginsenoside Rb3 was summarized. Introduction The root of Panax ginseng C.A. Meyer is a well-known medicinal herb in Traditional Chinese Medicine, and has been used as herbal remedy and adaptogen that helps to maintain human’s health and restore the body to balance (1). Ginsenosides, considered as the main active components of P. ginseng, have shown various pharmacological effects on anti-tumor, diabetes mellitus, cardio-vascular system, central nervous system and immune system (2). Ginsenosides in P. ginseng present chemical diversity, and ~50 ginsenosides have been identified in P. ginseng and related processed products. (3) Pharmacokinetic and metabolic studies on selected ginsenosides have been carried on to better understand the bioactive forms, biological effects and safety of ginseng (4, 5). Ginsenoside Rb3 is one of major ginsenosides with content of ~2.1 mg/g in P. ginseng, belonging to protopanaxadiol (PPD) ginsenoside (6). It has increasingly attracted researchers’ attention for its effects on cardio-vascular and central nervous system. Studies have shown that ginsenoside Rb3 possesses the effect on myocardial injury and heart function impairment induced by isoproterenol in rats (7), and it could protect cardiomyocytes against ischemia–reperfusion (I/R) injury via the inhibition mediated NF-B pathway, suggesting that ginsenoside Rb3 has the potential to serve as a novel therapeutic agent for myocardial I/R injury (8). The results of ginsenoside Rb3 in hypertension showed that it could attenuate oxidative stress and preserve endothelial function in renal arteries from hypertensive rats (9). It was reported that ginsenoside Rb3 could protect neurons in hippocampal slices of rat while exposed to oxygen/glucose deprived (10, 11). The effect of ginsenoside Rb3 on synaptic transmission after oxygen/glucose deprived in vitro indicated that the activation of γ-aminobutyric acid receptor is correlated with its neuroprotective mechanisms (12) and has the significant protective effects on glutamate excitotoxic injury in cultured neurons of rat hippocampus (13). For the potential values of ginsenoside Rb3 as a novel therapeutic agent on cardio-vascular and central nervous system, the investigation of pharmacokinetic and metabolism of ginsenoside Rb3 will be helpful for better knowing the comprehensive pharmacokinetic parameters, the actual biotransformation and excretion pathway, and designing rational pharmacological and clinical dosage. The metabolic study of ginsenosides Rb3 has not been reported. Recently, liquid chromatography coupled with mass spectrometry (LC/MS) has been widely used for the metabolic study of ginsenosides due to the high-sensitivity and capability of structural elucidation (14, 15). Quadrupole-time-of-flight mass spectrometry (Q-TOF-MS) coupled to the fast LC systems (Ultra performance liquid chromatography or rapid resolution liquid chromatography (RRLC)) exhibits advantages of rapid analysis, high chromatographic resolution and accurate mass measurement for the metabolism studies of ginsenosides (16, 17). In this study, a sensitive and selective RRLC-Q-TOF-MS method was developed to investigate the pharmacokinetics of ginsenoside Rb3 in rat plasma after intravenous administration. And the metabolites of ginsenoside Rb3 were investigated in rat urine and feces samples after intravenous and oral administrations to explore the active component for pharmacological effect. Experimental Chemicals and reagents Ginsenoside Rb3 (purity 98.0%), Rf (internal standard, IS, purity 98.0%), F2 (purity 98.0%) and Compound K (CK; purity 98.0%) were purchased from Jilin University (Changchun, China). HPLC-grade methanol and acetonitrile were purchased from TEDIA (USA). Ultrapure water used in the experiments was prepared by a Milli-Q Ultrapure water system (Millipore, Billerica, USA). Instruments The LC analysis was carried out using an Agilent 1200 series RRLC system (Santa Clara, CA, USA) equipped with a binary pump, micro-degasser, an auto-plate sampler and a thermostatically controlled column apartment; MS detection was performed on an Agilent 6520 Q-TOF mass spectrometer (Santa Clara, CA, USA) equipped with an electrospray interface, and automatic calibration system. LC and MS conditions The LC separation was performed on an Agilent SB-C18 column (3.0 mm × 100 mm, 1.8 μm, 600 bar) at a temperature of 25°C. 0.1% formic acid (v/v) and acetonitrile were used as the mobile phases A and B, respectively. The gradient elution was programmed as follows: 0–13 min (18–46%B), 13–20 min (46–50%B), 20–23 min (50–90%B) and 23–25 min (90% B). The flow rate was 0.3 mL/min. The injected sample volume was 5 μL. This RRLC system was connected to Q-TOF mass spectrometer. The Q-TOF-MS scan range was set at m/z 100–2,200 in negative ion modes. The conditions of the electrospray ionization source were as follows: drying gas (N2) flow rate was 8.0 L/min, drying gas temperature was at 350°C, nebulizer was 255 kPa, capillary voltage was 3,500 V, fragmentor was 175 V and skimmer was 65 V. Data analysis was performed by Agilent software Mass Hunter Qualitative (version B.03.01). Preparation of standard and quality control samples The stock solutions were prepared by dissolving standard Rb3 and IS in methanol to a final concentrations of 50 and 40 μg/mL, respectively. Working solutions were obtained freshly by further diluting stock solutions in methanol. All stock solutions and working solutions were stored at 4°C. Calibration standard of Rb3 were prepared by adding appropriate amount of stock solutions and IS into 100 μL of blank rat plasma and well mixed. About 200 μL of methanol was added into each standard solution and vortex for 1 min. The sample was then centrifuged at 13,000 × g for 5 min and the supernatant was transferred into another Eppendorf tube. Calibration standards were obtained at final concentrations of 0.08, 0.10, 0.20, 0.40, 0.60, 0.80 and 1.0 μg/mL of Rb3 in plasma. The injected sample volume was 5 μL by LC/MS to obtain the calibration curve. Quality control (QC) samples in plasma were similarly prepared at concentration levels of low, middle and high (0.10, 0.36 and 0.76 μg/mL). Pharmacokinetic study The pharmacokinetic study were carried out in six Male Wistar rats (200 ± 20 g) supplied by Jilin University (Changchun, China). Rats were fasted for 12 h before dosing, with free access to water. Six rats were administrated with 2.0 mg/kg Rb3 (dissolved in 500 μL of 5% β-cyclodextrin aqueous and 500 μL of 0.9% sodium chloride) by intravenous injection. About 1 mL Rb3 dosing solution was given to each rat via tail vein. Blood samples (300 μL) were collected by punctuating the retro-orbital sinus at 0 (before dosage), 2, 5, 10, 15, 20, 30, 40, 60 and 90 min, and 2, 4, 8, 12 and 24 h after the intravenous injection. The blood samples were then centrifuged at 3,000 × g for 10 min to get plasma and then stored at −20°C until analysis. About 200 μL methanol and 100 μL IS were added into each plasma sample and vortex for 30 s, and then the sample was centrifuged at 13,000 × g for 5 min. The aliquot of 5 μL was injected into the LC/MS. Samples were then prepared for LC/MS analysis as described above. Software 3P97 (Practical Pharmacokinetic Program, 1997, China) was used to analyze pharmacokinetic study data to determine the pharmacokinetic parameters of Rb3 in rats. Metabolic studies Twelve male Wistar rats (200 ± 20 g) were divided into two groups at random for Rb3 as the intravenous injection group and the oral administration group. The intravenous dose (2 mg/kg) and oral dose (50 mg/kg) were conducted as above. Rat urine samples were collected from 0 to 48 h after the intravenous and oral administrations of Rb3. Each urine sample was concentrated under a gentle stream of nitrogen and extracted with n-butanol. The extract was dried and then the residue was dissolved in 1.0 mL 80% methanol. After passing through 0.45 μm filter, 5 μL of filtrate was injected for metabolism study by LC/MS. Rat feces samples were collected from 0 to 24 h after oral administration of Rb3. The feces sample was ground and suspended in 50 mL water, and then extracted three times with total 100 mL n-butanol. The extract was processed as above urine samples for metabolism analysis. Results Development of RRLC-Q-TOF-MS and MS/MS methods Ginsenoside Rb3 (Figure 1) was characterized by RRLC-Q-TOF-MS in full MS scan. Negative ion mode was selected in this experiment due to much lower background and less interference than that of positive ion mode. As 0.1% formic acid solution was employed as the mobile phase, the deprotonated ion [M-H]− (m/z 1077) and adduct ion [M+HCOO]− (m/z 1123) were detected in the negative ion mode, providing information about the molecular mass. MS/MS was employed for structure elucidation and the fragmentation nomenclature used in this paper is based on that described by Costello and Liu (18, 19). C20 position and C3 position substitutions are defined as α and β linkages, respectively. The mass accuracy for all molecular ions and fragment ions were <10 ppm. In the following discussion, the m/z value of ion was expressed as integer number and the accurate mass measurement is listed in Table I. Figure 2A shows the MS/MS spectra of Rb3. Y1α (m/z 945), Y0α (m/z 783), Y’1β (m/z 621), Y0β’ (m/z 459), B2α (m/z 293), B1β (m/z 161) and C1α (m/z 149) were observed. Y1α ion at m/z 945 was generated by the loss of a terminal xylose residue (132 Da) at C20 position and the corresponding C1α ion at m/z 149 indicated the terminal xylose substitution. Y0α ion at m/z 783 was produced by loss of the residue of glucose–xylose (162 + 132 Da) at C20 position and the corresponding B2α ion at m/z 293 was observed indicating the glucose–xylose linkage. Y’1β ion at m/z 621 was observed by loss of glucose–xylose residue (162 + 132 Da) at C20 position and a terminal glucose residue (162 Da) at C3 position. Y’0β ion at m/z 459 representing the PPD-type ginsenoside was produced by loss of glucose–glucose residue (162 + 162 Da) at C3 position and glucose–xylose residue (162 + 132 Da) at C20 position. The fragment information of ginsenoside Rb3 is summarized in Table I. Figure 1. View largeDownload slide Structures of ginsenosides and metabolites (Glc: glucose, xyl: xylose). Figure 1. View largeDownload slide Structures of ginsenosides and metabolites (Glc: glucose, xyl: xylose). Table I. Ginsenoside Rb3 and Identified Metabolites by RRLC-Q-TOF-MS/MS Peak (min) Identity Molecular formula MW Measured value (Mass accuracy < 10 ppm) [M-H]− [M+HCOO]− MS/MS fragment ions of [M-H]− (mass accuracy < 10 ppm) 10.19 Rb3 C53H90O22 1,078.5924 1,077.5838 1,123.5897 945.5088(Y1α), 783.4629(Y0α), 621.4125(Y1β’), 459.3637(Y0β’), 293.0716(B2α), 191.0503(0,4A2α), 161.0403 (B1β) and 149.0382 (C1α) 13.86 Mb1 C47H80O17 916.5276 915.5295 961.5247 783.4758(Y1α), 621.4123(Y0α), 459.3692(Y0β’), 293.1158(B2α), 191.0517(0,4A2α) and 161.0353 (B1β)149.0419(C1α) 15.76 M5(F2) C42H72O13 784.4973 783.4753 829.2862 621.4138(Y1α), 459.3648(Y0β’) and 161.0415(B1α) 21.94 M2’ C41H70O12 754.4863 753.4826 799.4871 621.4104(Y1α), 459.3675(Y0α), 293.1152(B2α), 191.0487(0,4A2α) and 149.0430(C1α) 23.96 C-K C36H62O8 622.4439 621.4365 667.4372 459.3642(Y0α), 161.0374(B1α) and 119.0462(2,4A1α) Peak (min) Identity Molecular formula MW Measured value (Mass accuracy < 10 ppm) [M-H]− [M+HCOO]− MS/MS fragment ions of [M-H]− (mass accuracy < 10 ppm) 10.19 Rb3 C53H90O22 1,078.5924 1,077.5838 1,123.5897 945.5088(Y1α), 783.4629(Y0α), 621.4125(Y1β’), 459.3637(Y0β’), 293.0716(B2α), 191.0503(0,4A2α), 161.0403 (B1β) and 149.0382 (C1α) 13.86 Mb1 C47H80O17 916.5276 915.5295 961.5247 783.4758(Y1α), 621.4123(Y0α), 459.3692(Y0β’), 293.1158(B2α), 191.0517(0,4A2α) and 161.0353 (B1β)149.0419(C1α) 15.76 M5(F2) C42H72O13 784.4973 783.4753 829.2862 621.4138(Y1α), 459.3648(Y0β’) and 161.0415(B1α) 21.94 M2’ C41H70O12 754.4863 753.4826 799.4871 621.4104(Y1α), 459.3675(Y0α), 293.1152(B2α), 191.0487(0,4A2α) and 149.0430(C1α) 23.96 C-K C36H62O8 622.4439 621.4365 667.4372 459.3642(Y0α), 161.0374(B1α) and 119.0462(2,4A1α) View Large Table I. Ginsenoside Rb3 and Identified Metabolites by RRLC-Q-TOF-MS/MS Peak (min) Identity Molecular formula MW Measured value (Mass accuracy < 10 ppm) [M-H]− [M+HCOO]− MS/MS fragment ions of [M-H]− (mass accuracy < 10 ppm) 10.19 Rb3 C53H90O22 1,078.5924 1,077.5838 1,123.5897 945.5088(Y1α), 783.4629(Y0α), 621.4125(Y1β’), 459.3637(Y0β’), 293.0716(B2α), 191.0503(0,4A2α), 161.0403 (B1β) and 149.0382 (C1α) 13.86 Mb1 C47H80O17 916.5276 915.5295 961.5247 783.4758(Y1α), 621.4123(Y0α), 459.3692(Y0β’), 293.1158(B2α), 191.0517(0,4A2α) and 161.0353 (B1β)149.0419(C1α) 15.76 M5(F2) C42H72O13 784.4973 783.4753 829.2862 621.4138(Y1α), 459.3648(Y0β’) and 161.0415(B1α) 21.94 M2’ C41H70O12 754.4863 753.4826 799.4871 621.4104(Y1α), 459.3675(Y0α), 293.1152(B2α), 191.0487(0,4A2α) and 149.0430(C1α) 23.96 C-K C36H62O8 622.4439 621.4365 667.4372 459.3642(Y0α), 161.0374(B1α) and 119.0462(2,4A1α) Peak (min) Identity Molecular formula MW Measured value (Mass accuracy < 10 ppm) [M-H]− [M+HCOO]− MS/MS fragment ions of [M-H]− (mass accuracy < 10 ppm) 10.19 Rb3 C53H90O22 1,078.5924 1,077.5838 1,123.5897 945.5088(Y1α), 783.4629(Y0α), 621.4125(Y1β’), 459.3637(Y0β’), 293.0716(B2α), 191.0503(0,4A2α), 161.0403 (B1β) and 149.0382 (C1α) 13.86 Mb1 C47H80O17 916.5276 915.5295 961.5247 783.4758(Y1α), 621.4123(Y0α), 459.3692(Y0β’), 293.1158(B2α), 191.0517(0,4A2α) and 161.0353 (B1β)149.0419(C1α) 15.76 M5(F2) C42H72O13 784.4973 783.4753 829.2862 621.4138(Y1α), 459.3648(Y0β’) and 161.0415(B1α) 21.94 M2’ C41H70O12 754.4863 753.4826 799.4871 621.4104(Y1α), 459.3675(Y0α), 293.1152(B2α), 191.0487(0,4A2α) and 149.0430(C1α) 23.96 C-K C36H62O8 622.4439 621.4365 667.4372 459.3642(Y0α), 161.0374(B1α) and 119.0462(2,4A1α) View Large Figure 2. View largeDownload slide MS/MS spectrum of (A) ginsenosides Rb3, (B) metabolites P1, (C) P2, (D) P3 and (E) P4 in the negative ion mode. Figure 2. View largeDownload slide MS/MS spectrum of (A) ginsenosides Rb3, (B) metabolites P1, (C) P2, (D) P3 and (E) P4 in the negative ion mode. Method validation The specificity and selectivity were investigated by comparing total ion chromatogram (TIC) of three different batches of blank plasma and samples with 5.00 μg/mL of Rb3 and IS solutions. The detection of Rb3 and IS by RRLC-Q-TOF-MS was highly selective without the interference of endogenous constituents in the biologic matrices. The retention time of Rb3 and IS were at 10.19 and 6.89 min, respectively. Calibration curves were obtained by plotting the peak area ratios of Rb3/IS versus the concentration of Rb3. The regression equation was Y = 4.5707 × −0.0065 (Y represents the peak area ratios of the analyte Rb3/IS, X represents the concentration of Rb3) with the correlation coefficient of 0.9966. Good linearity was obtained over the dynamic range of 0.08–0.90 μg/mL. The limit of detection and quantitation (LOD and LOQ) was measured as 0.05 and 0.08 μg/mL, respectively. The precision and accuracy were evaluated by analysis of variation results of intra-day (six samples at each QC level) and inter-day (three consecutive days). As shown in Table II, the intra-day and inter-day precisions were <5% for each QC level of Rb3. The accuracy determined from QC samples was range from −5% to −1%. The method showed good accuracy and precision. The stability of Rb3 was studied by analyzing QC samples at three concentrations. The samples were stable after three freeze-thaw cycles (from −20°C to room temperature) in three days and storage at room temperature in 24 h. The extraction recovery was determined by comparing the peak area of Rb3 plasma samples spiked before extraction with the peak area of Rb3 plasma samples spiked after extraction at three concentrations of 0.10, 0.36 and 0.76 μg/mL. The extraction recoveries of Rb3 were in the range of 89.32–92.78% (Table II). Table II. Precision, Accuracy and Extraction Recoveries of Ginsenoside Rb3 in Rat Plasma Nominal concentration (μg/mL) Measured concentration (μg/mL) RSD (%) Intra-day RSD (%) Inter-day RSD (%) Extraction recovery (%) 0.89 0.78 −1.53 1.74 3.18 89.32 0.38 0.35 −3.22 3.31 4.44 92.67 0.20 0.18 −4.51 2.65 2.97 92.78 Nominal concentration (μg/mL) Measured concentration (μg/mL) RSD (%) Intra-day RSD (%) Inter-day RSD (%) Extraction recovery (%) 0.89 0.78 −1.53 1.74 3.18 89.32 0.38 0.35 −3.22 3.31 4.44 92.67 0.20 0.18 −4.51 2.65 2.97 92.78 Table II. Precision, Accuracy and Extraction Recoveries of Ginsenoside Rb3 in Rat Plasma Nominal concentration (μg/mL) Measured concentration (μg/mL) RSD (%) Intra-day RSD (%) Inter-day RSD (%) Extraction recovery (%) 0.89 0.78 −1.53 1.74 3.18 89.32 0.38 0.35 −3.22 3.31 4.44 92.67 0.20 0.18 −4.51 2.65 2.97 92.78 Nominal concentration (μg/mL) Measured concentration (μg/mL) RSD (%) Intra-day RSD (%) Inter-day RSD (%) Extraction recovery (%) 0.89 0.78 −1.53 1.74 3.18 89.32 0.38 0.35 −3.22 3.31 4.44 92.67 0.20 0.18 −4.51 2.65 2.97 92.78 Pharmacokinetic study of ginsenoside Rb3 The validated RRLC-Q-TOF-MS method was used for the pharmacokinetic study of ginsenoside Rb3 in rat plasma by intravenous administration. Ginsenoside Rb3 was detected in plasma samples with a dose at 2.0 mg/kg. The mean plasma concentration–time curves of ginsenoside Rb3 (n = 6) was shown in Figure 3. Compartment model was estimated by the value of AIC (Akaike’s Information Criterion). The result of experiment demonstrated that ginsenoside Rb3 concentration–time profile follows a two-compartment pharmacokinetic model after intravenous administration for rats. The main pharmacokinetic parameters are presented in Table III. The mean plasma elimination half-lives are 13.77 ± 1.23 min and 2045.70 ± 156.20 min for distribution and exterminate phase t1/2α and t1/2β, respectively. AUC(0−t) and AUC(0−∞) (the area under the curve) were calculated as 366.58 ± 18.25 and 1,047.42 ± 152.04 μg/(L·min). Plasma clearance (CL) was 1.91 ± 0.02 (L/min/kg). Figure 3. View largeDownload slide (A) Plasma concentration–time profiles of ginsenoside Rb3 after intravenous administration (2 mg/kg) in Wistar rats (n = 6) and (B) the related ln concentration–time profiles. Figure 3. View largeDownload slide (A) Plasma concentration–time profiles of ginsenoside Rb3 after intravenous administration (2 mg/kg) in Wistar rats (n = 6) and (B) the related ln concentration–time profiles. Table III. Main Pharmacokinetic Parameters of Rb3 in Rat Plasma after Intravenous Administration (2 mg/kg) to Six Rats (mean ± SD) Parameter Ginsenoside Rb3 C0 (μg/mL) 0.93 ± 0.05 t1/2α (min) 13.77 ± 1.23 t1/2β (min) 2,045.70 ± 156.20 K10 (1/min) 0.001 ± 0.0002 K12 (1/min) 0.033 ± 0.004 K21 (1/min) 0.017 ± 0.001 AUC(0−t) (μg/L ∗ min) 366.58 ± 18.25 AUC (0−∞) (μg/L ∗ min) 1,047.43 ± 152.04 Vc (L/kg) 2,144.02 ± 174.12 CL (L/min/kg) 1.91 ± 0.02 Parameter Ginsenoside Rb3 C0 (μg/mL) 0.93 ± 0.05 t1/2α (min) 13.77 ± 1.23 t1/2β (min) 2,045.70 ± 156.20 K10 (1/min) 0.001 ± 0.0002 K12 (1/min) 0.033 ± 0.004 K21 (1/min) 0.017 ± 0.001 AUC(0−t) (μg/L ∗ min) 366.58 ± 18.25 AUC (0−∞) (μg/L ∗ min) 1,047.43 ± 152.04 Vc (L/kg) 2,144.02 ± 174.12 CL (L/min/kg) 1.91 ± 0.02 Table III. Main Pharmacokinetic Parameters of Rb3 in Rat Plasma after Intravenous Administration (2 mg/kg) to Six Rats (mean ± SD) Parameter Ginsenoside Rb3 C0 (μg/mL) 0.93 ± 0.05 t1/2α (min) 13.77 ± 1.23 t1/2β (min) 2,045.70 ± 156.20 K10 (1/min) 0.001 ± 0.0002 K12 (1/min) 0.033 ± 0.004 K21 (1/min) 0.017 ± 0.001 AUC(0−t) (μg/L ∗ min) 366.58 ± 18.25 AUC (0−∞) (μg/L ∗ min) 1,047.43 ± 152.04 Vc (L/kg) 2,144.02 ± 174.12 CL (L/min/kg) 1.91 ± 0.02 Parameter Ginsenoside Rb3 C0 (μg/mL) 0.93 ± 0.05 t1/2α (min) 13.77 ± 1.23 t1/2β (min) 2,045.70 ± 156.20 K10 (1/min) 0.001 ± 0.0002 K12 (1/min) 0.033 ± 0.004 K21 (1/min) 0.017 ± 0.001 AUC(0−t) (μg/L ∗ min) 366.58 ± 18.25 AUC (0−∞) (μg/L ∗ min) 1,047.43 ± 152.04 Vc (L/kg) 2,144.02 ± 174.12 CL (L/min/kg) 1.91 ± 0.02 Metabolic study of Rb3 in rat urine after intravenous administration Related metabolites were tentatively identified as deglycosylation products based on fragmentation information and accurate mass measurement. Figure 4A shows a typical RRLC-Q-TOF-MS-TIC of a rat urine sample after intravenous administration in the negative ion mode. Prototype ginsenoside Rb3 and two possible metabolites were detected. Ginsenoside Rb3 was identified by comparing the retention time and fragmentation patterns with that of the standard compound. The extracted ion chromatograms of the two deglycosylated metabolites of Rb3 (P1 and P2) were shown in Figure 4B and C. As 0.1% formic acid solution was employed as the mobile phase, detected metabolite showed adduct ion [M+HCOO]− in negative ion mode. P1 and P2 exhibited [M+HCOO]− ions at m/z 961 and m/z 799 in RRLC-Q-TOF-MS, determined as metabolites by loss of glucose and glucose–glucose substitution at C3 position from ginsenoside Rb3, respectively. In Figure 2B, the MS/MS spectrum of metabolites P1, characteristic fragment ions Y1α (m/z 783), Y0α (m/z 621), Y0β’ (m/z 459), B2α (m/z 293), 0,4A2α (m/z 191.0517), B1β (m/z 161) and C1α (m/z 149) were all observed, which are coincident with the MS/MS fragment ions of ginsenoside Rb3. Product ions at m/z 783 was produced by the loss of xylose residue and HCOOH (132 + 46 Da) and m/z 621 was generated by loss of glucose–xylose residue and HCOOH (162 + 132 + 46 Da) from the [M+HCOO]− ion at m/z 961. That indicated the terminal sugar was xylose and the chain substitution was glucose–xylose, corresponding to C20 position of ginsenoside Rb3. Ion at m/z 459 was characteristic ion of PPD-type ginsenoside helpful for aglycone determination, which was produced by lose of glucose–xylose, glucose residue and HCOOH (162 + 132 + 162 + 46 Da) from the [M+HCOO]− ion at m/z 961. The corresponding glucose–xylose, glucose and xylose residue ions at m/z 293 (B2α), m/z 161 (B1β) and m/z 149 (C1α) were also observed in the MS/MS spectrum. Thus, P1 was tentatively identified as metabolite by loss of terminal glucose (162 Da) at C3 position from Rb3. Ginsenosides Rb2 and Rb3 are isomers with C-20 terminal sugar substitution as arabinose and xylose, respectively. The deglycosylation product by loss of C-3 terminal substitution glucose form ginsenoside Rb2 was identified as Mb in stomach and large intestine (20, 21). Thus, the metabolite P1 of Rb3 by loss of terminal glucose (162 Da) at C3 position was defined as Mb1 in this experiment (Figures 1 and 2B). Figure 2C shows the MS/MS spectrum of P2. The ion at m/z 753 was produced by loss of the HCOOH (46 Da) from [M+HCOO]− ions at m/z 799. The fragments ion at m/z 621 (Y1α) was produced by loss HCOOH and a xylose residue (132 Da) at C20 position, and the corresponding ion at m/z 149 (C1α) was observed in the MS/MS spectra, which indicated that the terminal substitution was xylose. PPD-type characteristic ion at m/z 459 (Y0α) was generated by loss of HCOOH, glucose–xylose residues (46 + 162 + 132 Da) from [M+HCOO]− ions. That indicated that the substitution sugar linkage was glucose–xylose at C20 position, and ions at m/z 293 (B2α) and m/z 191 (0,4A2α) also proved that. Thus, P2 was deduced as metabolite deglucosylated from ginsenoside Rb3 by loss the substitution saccharide at C3 position (Figures 1 and 2C). According to reference’s reports about the metabolite M2 produced by loss of the saccharide substitution at C3 position form ginsenoside Rb2 (20, 22). This metabolite produced from ginsenoside Rb3 is an isomer of M2 with difference of xylose as terminal substitution at C20 position, so it is named as M2’ in this experiment. The result of experiment showed that part of ginsenoside Rb3 was excreted through urine as prototype and metabolites Mb1 and M2’ after intravenous administration. The identified compounds are summarized in Table I. Figure 4. View largeDownload slide (A) RRLC-Q-TOF-MS TIC of rat urine sample after intravenous administration of Rb3 in the negative ion mode, (B) EIC of the metabolite P1 and (C) EIC of the metabolite P2. Figure 4. View largeDownload slide (A) RRLC-Q-TOF-MS TIC of rat urine sample after intravenous administration of Rb3 in the negative ion mode, (B) EIC of the metabolite P1 and (C) EIC of the metabolite P2. Metabolic study of Rb3 in rat feces after oral administration Ginsenoside Rb3 and four possible metabolites were detected in rat feces samples collected from 0 to 24 h after oral administration by using RRLC-Q-TOF-MS and MS/MS. The TIC of rat feces samples after oral administration is shown in Figure 5A. Ginsenoside Rb3 was identified by comparing retention time and fragmentations with that of standard compound (Figure 5A, Table I). Figure 5B–D and E showed the extracted ion chromatograms of the four metabolites (P1, P2, P3 and P4). In full MS negative ion mode, P1, P2, P3 and P4 exhibited [M+HCOO]− ions at m/z 961, m/z 829, m/z 799 and m/z 667. These four metabolites were further analyzed by MS/MS to give the structural information (Figure 2, Table I). MS/MS fragment ions of P1 and P2 were the same with the identified metabolites Mb1 and M2’ in rat urine after intravenous administration (Table I). The retention time of P1 and P2 were 13.86 and 15.76 min, respectively. Therefore, metabolites P1 and P2 were tentatively identified as Mb1 and M2’ by analysis of retention time and MS/MS fragmentations. Figure 2D shows the MS/MS spectrum of metabolite P3. [M+HCOO]− ion at m/z 829. The fragments ion at m/z 621 (Y0α) was produced by loss of HCOOH (46 Da) and a glucose residue (162 Da) at C20 position and the corresponding ion at m/z 161 (B1α) was detected in the MS/MS spectra. Characteristic ion at m/z 459 (Y0α) of PPD-type ginsenoside was generated by loss of two glucose residues (162 + 162 Da) substitution. Metabolite P3 has the same retention time and MS/MS fragmentation patterns with the reference standard of F2. Thus, the metabolite P3 was identified as ginsenoside F2. The MS/MS spectra of metabolite P4 is shown in Figure 2E. The [M+HCOO]− ion at m/z 667 generate the product ion at m/z 621 by loss of HCOOH (46 Da). The ion at m/z 459 was observed by loss of one glucose residue (162 Da), indicating the aglycone is PPD-type. And the corresponding ion at 161 m/z (B1α) was observed in the MS/MS spectra. Compared with the reference standard of CK, Metabolite P4 has the same retention time and MS/MS fragmentation patterns. P4 was identified as CK. The metabolic pathway of ginsenoside Rb3 was proposed in Figure 6 according to the major metabolites detected in rat urine and feces samples. Figure 5. View largeDownload slide RRLC-Q-TOF-MS chromatograms of rat feces samples after oral administrations of Rb3: (A) TIC of feces sample, (B) EIC of metabolites P1, (C) EIC of metabolites P2, (D) EIC of metabolites P3 and (E) EIC of metabolites P4. Figure 5. View largeDownload slide RRLC-Q-TOF-MS chromatograms of rat feces samples after oral administrations of Rb3: (A) TIC of feces sample, (B) EIC of metabolites P1, (C) EIC of metabolites P2, (D) EIC of metabolites P3 and (E) EIC of metabolites P4. Figure 6. View largeDownload slide Proposed metabolic pathway of ginsenoside Rb3 in rats. Figure 6. View largeDownload slide Proposed metabolic pathway of ginsenoside Rb3 in rats. Discussion Ginsenoside Rb3 was firstly analyzed to develop RRLC-Q-TOF-MS and MS/MS methods for pharmacokinetics and metabolism studies. Both chromatographic and mass spectrometric conditions were optimized to gain rapid detection and efficient determination. The chromatographic conditions including mobile phase, gradient elution program, column temperature and flow rate were optimized to ensure good separation. A gradient elution with acetonitrile and water containing 0.1% formic acid was finally chosen. The fragment information of ginsenoside Rb3 was summarized. These MS/MS fragmentation provide structural information helpful for elucidation of metabolite of ginsenosides Rb3 in complex mixtures. Then, the established method was validated for evaluating specificity and selectivity, linearity, limit of detection and quantitation, precision and accuracy, and extraction recovery for pharmacokinetic study. The pharmacokinetic result was consistent with the reported pharmacokinetic features of PPD ginsenosides with the relatively higher plasma protein binding (14, 15). Metabolic studies of Rb3 in rat urine after intravenous administration and in rat feces after oral administration were carried out by RRLC-Q-TOF-MS and MS/MS to reveal possible active compounds and to better understand the biotransformation and excretion. In intravenous administration study, metabolites in rat urine collected from 0 to 72 h after administration were analyzed. In the oral administration study, Rb3 was not detected in plasma and urine samples from 0 to 24 h with a dose of 50 mg/kg. This result may be caused by the poor oral absorption of natural ginsenosides or be metabolized as prototype or metabolites (such as CK, PPD) in feces by the gastric acid and intestinal flora after oral administration (14, 21). Then, the metabolites of ginsenoside Rb3 were detected in the feces samples. Conclusions In this study, a RRLC-Q-TOF-MS method was developed for pharmacokinetic studies of ginsenoside Rb3 and simultaneous determination of its metabolites in rat urine and feces samples. The validated method was applied for pharmacokinetic study of ginsenoside Rb3 after intravenous administration. The half-life of ginsenoside Rb3 (t1/2α=13.77 ± 1.23 min and t1/2β = 2,045.70 ± 156.20 min) suggested that the distribution of ginsenoside Rb3 in vivo was quick and the extermination was slow after administration. The further metabolic studies of Rb3 in rat urine after intravenous administration and in rat feces after oral administration were carried out by RRLC-Q-TOF-MS and MS/MS. The deglycosylation was found to be the major metabolic pathways of ginsenoside Rb3 in rat. The experiments demonstrated that part of ginsenoside Rb3 was excreted through urine as prototype and some changed into metabolites Mb1 and M2’ after intravenous administration, and ginsenoside Rb3 could be transformed into Mb1, F2, M2’ and CK in the gastrointestinal tract after oral administration. It is expected that this experiment results would provide some basis for the further developing and understanding the bioactive form of the ginsenoside Rb3. Funding This research work about analytical method was supported by the Science and Technology Development Plan Project of Jilin Province (No. 20170204010YY) and the pharmacokinetic and metabolic studies were supported by project of Key Lab of Ginseng Chemistry and Pharmacology of Jilin Province (No. 20160101334JC). 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Journal of Chromatographic ScienceOxford University Press

Published: Apr 18, 2018

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