Short communication Received: 26 February 2014,
Revised: 1 May 2014,
Accepted: 17 May 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.3272
Simultaneous determination of seven ginsenosides in rat plasma by highperformance liquid chromatography coupled to time-of-flight mass spectrometry: application to pharmacokinetics of Shenfu injection Zhengguang Lia,b, Rui Zhangb, Xiuping Wangb, Xiaofei Huc, Yuguo Chena* and Qingfei Liud* ABSTRACT: A high-performance liquid chromatography coupled to time-of-flight mass spectrometry (HPLC-TOF MS) method was successfully developed and validated for the identification and determination of seven ginsenosides, Re, Rf, Rb1, Rc, Rb2, Ro and Rd, in a Chinese herbal preparation, Shenfu injection, and rat plasma. Based on the method, the pharmacokinetic profiles of the seven ginsenosides were investigated following intravenous administration of single dose of Shenfu injection to six rats. The established method had high linearity, selectivity, sensitivity, accuracy and precision. The pharmacokinetic results showed that Rb1, Rc and Rb2 had similar pharmacokinetic profiles and relatively long half-life values (19.29 ± 6.36, 29.54 ± 22.91 and 35.60 ± 30.66 h). The half-lives of Rf and Rd were 4.21 ± 3.68 and 8.49 ± 5.20 h, respectively, indicating that they could be metabolized more rapidly than Rb1, Rc and Rb2. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: HPLC-TOF MS; ginsenoside; pharmacokinetics; Shenfu injection
Introduction Shenfu injection is a preparation developed from an ancient traditional Chinese formulation, Shenfu decoction. It consists of the extracts of Ginseng rubra Radix and Aconiti lateralis Radix and is widely used to treat heart failure in the clinic in China (Zhu et al., 2013; Yang et al., 2013; Xiao et al., 2013; Guo and Li, 2013; Hou et al., 2013; Cui et al., 2013). It has been reported that a large number of triterpenoid saponins known as ginsenosides or panaxosides, such as Rb1, Rg, Rb1 and Re, are major components of Ginseng rubra Radix (Choi, 2002; Morisaki et al., 1995; Kitagawa et al., 1983; Zhang et al., 2002; Yang et al., 2009; Kanzaki et al., 1998; Guan et al., 2007). Since ginsenosides are also main components of Panax notoginseng, there have some articles reporting the pharmacokinetic (PK) characteristics of ginsenosides after administration of single compound, mixture, extract or preparation, like Shenmai injection (Liu et al., 2011b; Song et al., 2010; Chen et al., 2010; Xu et al., 2003). However, the types and contents of ginsenosides are often different for extracts or preparations produced by different manufacturers. It is therefore important to investigate the quality and PK characteristics of the main active components of these preparations. Since ginsenosides have poor UV radiation at a maximum absorption of 203 nm, the method of liquid chromatography coupled to mass spectrometry (LC-MS) is widely used for analyzing ginsenosides (Liu et al., 2009, 2011a; Deng et al., 2009; Ha et al., 2010; Sun et al., 2005; Feng et al., 2011; Jin et al., 2011; Ren et al., 2008; Zhao et al., 2012; Qian et al., 2009; Wan et al., 2006; Lai et al., 2006).
Biomed. Chromatogr. 2014
The objective of this study was to establish and validate a selective and sensitive high-performance liquid chromatography coupled to time-of-flight mass spectrometry (HPLC-TOF MS) for the identification and quantification of the main ginsenosides of Shenfu injection in rat plasma including Re, Rf, Rb1, Rc, Rb2, Ro and Rd (Fig. 1). Based on the established method, the PK profiles of the seven compounds could be revealed after intravenous administration of Shenfu injection to rats.
Experimental Chemicals and reagents Shenfu injection (batch no. 130201010; manufacturer, Sichuan Yaan Sanjiu Pharmaceutical Co. Ltd) was purchased from Beijing Xiyuan * Correspondence to: Department of Emergency Medicine, Qilu Hospital, Shandong University, Qingdao, Shandong 250012, China. Email: [email protected] Qingfei Liu, School of Medicine, Tsinghua University, Beijing 100084, China. Email: [email protected] a
Department of Emergency Medicine, Qilu Hospital, Shandong University, Qingdao, Shandong 250012, China
b
Qingdao Hiser Medical Center, Qingdao, Shandong 250012, China
c
The Affiliated Hospital of Qingdao University, Qingdao, Shandong 250012, China
d
School of Medicine, Tsinghua University, Beijing 100084, China Abbreviations used: PK, pharmacokinetic; PNS, Panax notoginseng saponins; TIC, total ionic current; XIC, extracted ionic currents.
Copyright © 2014 John Wiley & Sons, Ltd.
Z. Li et al.
Figure 1. Chemical structures of seven ginsenosides in Shenfu injection.
Hospital. The standard compounds of ginsenosides Re, Rf, Rb1, Rc, Rb2, Ro and Rd (purity ≥99.5%) and Digoxin (internal standard, IS) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile was of HPLC grade (Fisher, USA). HPLC-grade water was obtained using a water purification system (Milli-Q Reagent Water System, Ma, USA). All other chemicals were of analytical reagent grade. All mobile phases were filtered through 0.22 μm micropore filters (Jinteng Ltd, Tianjin, China) prior to use.
Instrumentation and chromatographic conditions The Waters HPLC-TOF MS system (Agilent Ltd, USA) was used. The column was an Agilent C18 (250 × 4.6 mm, 5 μm) maintained at 30 ± 0.15° C. The mobile phases were 0.1% HOAc solution (A) and acetonitrile (B) with gradient condition of B as follows: 0–9 min, 11–16%; 9–20 min, 16–25%; 20–35 min, 25–40%; 35–45 min, 40–60%. The flow rate was set at 1.0 mL/min.
Mass spectrometry The analytes were identified and quantified using TOF MS in negative-ion mode. Source parameters included capillary voltage of 4 kV, cone voltage of 35 V, source temperature of 120°C, desolvation temperature of 350°C,
wileyonlinelibrary.com/journal/bmc
Figure 2. The total ionic current (TIC) of Shenfu injection and the extracted ionic currents (XIC) and MS of the seven ginsenosides (A, TIC; B, Re and Rd; C, Rc and Rb2; D, Rb1; E, Ro; F, Rf). cone gas flow of 10.0 L/h, and desolvation gas flow of 10.0 L/min. Product ions were monitored in W extended mode. In the full-scan mode, the mass spectrometer was operated over a range of m/z 50–1500. Twentymicroliter samples and standards were analyzed.
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014
Simultaneous determination of seven ginsenosides of Shenfu injection
Figure 2. (Continued).
Biomed. Chromatogr. 2014
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
Conc.in Shenfu injection (mg/mL)
wileyonlinelibrary.com/journal/bmc
27.1 35.2 35.6 36.6 37.5 38.0 39.8
Formula
C48H82O18 C42H72O14 C54H92O23 C53H90O22 C53H90O22 C48H76O19 C48H82O18 Re Rf Rb1 Rc Rb2 Ro Rd
MW
[M [M [M [M [M [M [M
Fragment
Fragment used for quantification
m/z
Copyright © 2014 John Wiley & Sons, Ltd.
Compound
Six male Sprague–Dawley rats with weights of 200–220 g were purchased from Beijing Weitong Lihua Biotechnology Co. Ltd (Beijing, China). The animals were housed and cared for under a constant temperature at (22 ± 1°C) and humidity (50 ± 10%). Food was withheld prohibited for 24 h before the experiment while water was provided freely. All studies were approved by the Ethics Committee of Tsinghua University. Shenfu injection was intravenous administrated to the rats through tail vein with a dosage of 5.0 mL/kg body weight. The dosages of the seven ginsenosides were calculated based on the concentrations in the injection. For each rat, 0.3 mL blood was collected from the ophthalmic plexus vein into heparinized tubes at 0.03, 0.17, 0.33, 0.5, 1, 2, 5, 8, 12 and 24 h. A blank sample was collected before administration. The concentrations of the seven ginsenosides were calculated according to the equations of linear regression of the standards. PK parameters were obtained by DAS (version 2.0) pharmacokinetic software provided by the Chinese Pharmacology Association.
Table 1. Identification and quantification of seven ginsenosides in Shenfu injection
Application
859.49 799.45 583.30 568.29 568.29 507.24 945.51
Retention time (min)
Preparation of calibration curves. Seven calibration curves were prepared with the method above by spiking blank rat plasma with IS and the stock solution to the concentrations 0.25, 0.5, 1, 5, 10 and 20 μg/mL for Rb1, Rc and Rb2, and 0.1, 0.2, 0.5, 1, 2 and 4 μg/mL for Rf, Rd, Re and Ro. Method validation. Quality control (QC) samples were prepared at three concentrations: 0.25, 1 and 20 μg/mL for Rb1, Rc and Rb2, and 0.1, 1, 4 μg/mL for Rf, Rd, Re and Ro. Intra-day accuracy and precision (each n = 3) were tested by analysis of the QC samples at different times during the same day. Inter-day accuracy and precision were determined by repeated analysis of the same samples over three consecutive days. The concentration of each sample was determined using calibration standards prepared on the same day. The recoveries were calculated by comparing the peak areas of the plasma samples with those of the standard working samples. The limits of quantification (LOQ) and detection (LOD) of each ginsenosides were defined as the responses that yielded a signal-to-noise ratio of >10 and 3, respectively.
0.02 0.01 0.03 0.03 0.02 0.02 0.01
LOD (μg/mL)
Preparation of blood samples. The blood sample was centrifuged at ο 5000 rpm for 10 min at 4 C (TGL 16C, Medical Centrifuge Co., Beijing, China). A 120 μL aliquot of the upper layer of plasma was mixed with 12.0 μL of IS (43.4 μg/mL) and 480 μL methanol using vortex-mixing for 1 min and then centrifuged at 10,000 rpm for 10 min at 4°C. The upper layer was transferred to another tube and dried under gentle nitrogen flow. The dried substance was added to 120 μL acetonitrile, using vortex-mixing for 1 min and then centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was injected into the HPLC system for analysis. The same sample handling process was used for the recovery and precision determinations.
rha + HOAc + H2O H] H] 3glc C3H6 H] 2glc araf C3H6 + H2O H] 2glc – arap C3H6 + H2O H] 2glc glcUA + ACN H] H]
Identification and determination of ginsenosides in blood samples
0.08 0.10 0.15 0.12 0.10 0.10 0.05
LOQ (μg/mL)
Shenfu injection was diluted with methanol 10-fold before the determination. With the standard references, the main seven ginsenosides in Shenfu injection were identified and determined by the HPLC-TOF MS method established above. The standards were used to confirm and determine the compounds.
946 800 1108 1078 1078 956 946
Identification and determination of ginsenosides in Shenfu injection
0.0712 0.0206 0.1315 0.1135 0.0833 0.0319 0.1622
Z. Li et al.
Biomed. Chromatogr. 2014
Simultaneous determination of seven ginsenosides of Shenfu injection
Figure 3. The TIC of Shenfu injection in rat plasma and the XIC of seven ginsenosides and IS (A, TIC; B, Re and Rd; C, Rc and Rb2; D, Rb1; E, Ro; F, Rf; G, digoxin (IS); H, blank plasma).
Biomed. Chromatogr. 2014
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
Z. Li et al.
Figure 3. (Continued).
Results and discussion Identification and determination of the ginsenosides in Shenfu injection The total ionic current (TIC) of Shenfu injection is shown in Fig. 2 (A). The extracted ionic currents (XIC) and MS of the seven ginsenosides are shown in Fig. 2(B–F). Although there was little difference between the determined molecular weight and the theoretical molecular weight for some compounds, all seven ginsenosides were confirmed by the standards. It was interesting that even [M H] was extracted but some other fragments had much higher response and could be used for quantification. For the seven ginsenosides in Shenfu injection, there are two pairs of isomers, Re and Rd, Rc and Rb2. For Re and Rd, [M rha + HOAc + H2O H] with m/z 859.49 and [M H] with m/z 945.51 (Fig. 2B) were the main fragments with retention times of 27.1 and 39.8 min in the chromatogram, respectively. Thereafter, the two fragments were used for quantification of Re and Rd correspondingly. For Rc and Rb2, m/z 568.29 was the main fragment rather than [M H] with m/z 1077. According to the chemical structures of
wileyonlinelibrary.com/journal/bmc
Rc and Rb2, it could be inferred that the two fragments with m/z 568.29 came from [M 2glc araf C3H6 + H2O H] and [M 2glc arap C3 H6 + H2 O H] , respectively, with retention times of 36.6 and 37.5 min in the chromatogram. The fragment C 3H6 was produced from the group of isobutene (Fig. 2C). The two fragments with m/z 568.29 were therefore used for quantification of Rc and Rb2. For Rb1, the main fragment was [M 3glc C3 H6 H] with m/z 583.30 rather than [M H] with m/z 1107 (Fig. 2D) and the fragment with m/z 583.30 was used for quantification of Rb1. For Ro, the main fragment was [M 2glc glcUA + ACN H] with m/z 507.24 rather than [M H] with m/z 955 (Fig.2E) and the fragment with m/z 507.24 was used for quantification of Ro. For Rf , the main fragment [M H] with m/z 799.45 could be easily found (Fig. 2F) and was used for quantification of R f. It was interesting that there has been no any literature reporting Ro in Shenfu injection. In the present study, there was a relatively high concentration of Ro in Shenfu injection. The concentrations of the seven ginsenosides in Shenfu injection, calculated according to the regression equations of each compound (not shown in the present article) are shown in Table 1.
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014
Simultaneous determination of seven ginsenosides of Shenfu injection Method validation The calibration curves were linear over the concentration range 0.25–20 μg/mL for Rb1, Rc and Rb2, and 0.1–4 μg/mL for Rf, Rd, Re and Ro (r2 > 0.99). The results for matrix effect were in the range of 86–110% (n = 5). The intra- and inter-day accuracies were within 85–112% and the precisions were within acceptable limits at three concentrations (n = 5, RSD < 5%). The extraction recoveries were within 65–87%. The detection limit of TOF MS is generally not as good as a triple quad MS. Especially for a known compound, triple quad MS has the obvious advantage of very low limits of detection and quantitation. However, for TOF MS, the significant advantage is that it can provide exact molecular weight and molecular formula, especially for the unknown compound. Furthermore, for any compound, there must be some metabolites after administration and it is necessary to find and identify them. Therefore, TOF MS was employed in the present research to investigate the PK profiles not only of ginsenosides but also their metabolites. The results of the metabolites will be reported in a future paper.
Pharmacokinetics
Figure 4. The concentration-time profiles of seven ginsenosides, Re, Rf, Rb1, Rc, Rb2, Ro and Rd, in Shenfu injection after i.v. administration of a single dose to rats (n = 6).
Identification and determination of ginsenosides in rat plasma The TIC of the seven ginsenosides of Shenfu injection in rat plasma is shown in Fig. 3(A). The XIC and MS of the seven ginsenosides and IS are shown in Fig. 3(B–G). All seven ginsenosides were confirmed by the standards. For IS, [M + HOAc H] with m/z 839 could be easily found and there was no response of [M H] (m/z 779). [M + HOAc H] was therefore used for qualification.
The concentration–time profiles of the seven ginsenosides are shown in Figure 4(A–C) and the main PK parameters in Table 2. The dose of each ginsenoside was calculated according to the concentration in Shenfu injection. Re was metabolized rapidly and could not be determined even at 0.33 h after administration, although with a dose of 0.18 mg/kg. It was similar for Ro, which could not be detected at 0.5 h after administration. Thereafter, PK parameters of Re and Ro could not be provided in the present research. The PK parameters of Rb1, Rc and Rb2 were similar with relatively long half-lives (t1/2). The t1/2 values of Rf and Rd were 4.21 ± 3.68 and 8.49 ± 5.20 h, respectively, indicating that they were metabolized slowly but still more rapidly than Rb1, Rc and Rb2. There have been some PK investigations on ginsenosides following oral or intravenous administration of the single compound, mixture or the extract from ginseng or notoginseng, but the PK parameters were very different. For example, after oral administration of Panax notoginseng saponins (PNS) extract to rats, Ra3, Rb1, Rc and Rd had significantly longer t1/2 values (7.5–19.8 h) than the other ginsenosides (Rg1, Rg2, Rg3, F1, F2, Rh1, Rh2, Re, Rf, and 20-gluco-ginsenoside Rf; 0.2–3.2 h; Liu et al., 2009). However, another report showed that the values of t1/2 of Rb1 and Rg1 were 17.96 and 14.13 h (Xu et al., 2003). After oral administration of PNS extract to beagle dogs, values of t1/2 of R1,
Table 2. The pharmacokinetic parameters of seven ginsenosides in Shenfu injection after i.v. administration of single dose to rats (n = 6) Re Dose (mg/kg) t1/2 (h) V(L/kg) CL(L/h/kg) AUC0–t (mg/L h) MRT0–t (h)
0.18 — — — — —
Rf 0.10 4.21 ± 3.68 0.03 ± 0.03 0.03 ± 0.02 4.38 ± 3.30 2.99 ± 2.17
Rb1 0.66 19.29 ± 6.36 0.05 ± 0.04 0.01 ± 0.01 71.37 ± 31.17 9.83 ± 0.36
Rc 0.57 29.54 ± 22.91 0.04 ± 0.03 0.01 ± 0.01 65.30 ± 29.10 10.00 ± 1.30
Rb2
Ro
0.42 35.60 ± 30.66 0.04 ± 0.05 0.01 ± 0.01 44.42 ± 20.24 10.22 ± 1.51
0.08 — — — — —
Rd 0.81 8.49 ± 5.20 0.77 ± 0.04 0.39 ± 0.32 2.81 ± 2.02 6.01 ± 3.33
t1/2, Half-life; ; CL, clearance; AUC0–t, area under the concentration–time curve; MRT, mean residence time.
Biomed. Chromatogr. 2014
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
Z. Li et al. Rg1 and Rb1 were 3.35 ± 0.55, 4.55 ± 1.22 and 18.27 ± 2.55 h, respectively (Chen et al., 2010). Values of t1/2 for R1, Rg1 and Rb1 were 4.82 ± 3.13, 4.59 ± 2.95 and 60.9 ± 17.5 h after oral administration of Danshen tablets containing PNS extract to beagle dogs (Song et al., 2010). For R1, Rg1, Re and Rb1, values of t1/2 were 245.6 ± 118.5, 418.8 ± 68.0, 431.4 ± 149.5 and 1512.2 ± 1461.9 min after oral administration of Qihuotongqiao tablets to rats (Deng et al., 2009). For Rb1, Rb2 and Rb3 after intravenous and oral administration to rats with single compound, the t1/2 values were 12.5 ± 1.8, 15.4 ± 3.7, 24.9 ± 12.6 h and 9.8 ± 6.9, 23.1 ± 3.7, 21.1 ± 9.8 h, respectively (Zhao et al., 2012). As for humans, the t1/2 value of Re was 1.82 ± 0.75 h after oral administration of ginsenosides (Liu et al., 2011a) and the t1/2 of Rg1 was 2.09 ± 1.89 h following intravenous administration of Shenmai injection (Yang et al., 2009). The results of the present research show the PK profiles of seven ginsenosides in Shenfu injection for the first time following intravenous administration to rats. The PK parameters of most compounds like Rb1 were similar to those in the literature. They could provide useful reference and information for future research and clinical application.
Conclusion The PK profiles of seven ginsenosides in Shenfu injection, Re, Rf, Rb1, Rc, Rb2, Ro and Rd, were first simultaneously determined following i.v. administration of a single dose to rats. The HPLCTOF MS method was developed for identification and quantification of the seven ginsenosides in Shenfu injection and in rat plasma with high sensitivity, accuracy and precision. The PK information on the ginsenosides obtained in this research could provide significant reference for the clinical use of Shenfu injection.
Acknowledgment The project was kindly supported by Research Funding of Qingdao Hiser Medical Center.
References Chen W, Dang Y and Zhu C. Simultaneous determination of three major bioactive saponins of Panax notoginseng using liquid chromatography–tandem mass spectrometry and a pharmacokinetic study. Chinese Medicine 2010; 5: 12–17. Choi S. Epidermis proliferative effect of the Panax ginseng ginsenoside Rb-2. Archives of Pharmacal Research 2002; 25: 71–76. Cui HZ, Kim HY, Kang DG and Lee HS. Ginseng–aconite decoction + 2+ elicits a positive inotropic effect via the reverse mode Na /Ca exchanger in beating rabbit atria. Journal of Ethnopharmacology 2013; 148: 624–631. Deng GF, Wang DL, Meng MX, Hu F and Yao TW. Simultaneous determination of notoginsenoside R1, ginsenoside Rg1, Re, Rb1 and icariin in rat plasma by ultra-performance liquid chromatography–tandem mass spectrometry. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 2009; 877: 2113–2122. Feng H, Chen W and Zhu C. Pharmacokinetics study of bio-adhesive tablet of Panax notoginseng saponins. International Archives of Medicine 2011; 4: 18–25. Guan J, Lai CM and Li SP. A rapid method for the simultaneous determination of 11 saponins in Panax notoginseng using ultra performance liquid chromatography. Journal of Pharmaceutical and Biomedical Analysis 2007; 44: 996–1000. Guo ZJ and Li CS. Therapeutic effects of shenfu injection on post-cardiac arrest syndrome. Chinese Journal of Integrative Medicine 2013; 19: 716–720. Ha YW, Ahn KS, Lee JC, Kim SH, Chung BC and Choi MH. Validated quantification for selective cellular uptake of ginsenosides on
wileyonlinelibrary.com/journal/bmc
MCF-7 human breast cancer cells by liquid chromatography–mass spectrometry. Analytical and Bioanalytical Chemistry 2010; 396: 3017–3025. Hou XM, Li CS, Gu W, Guo ZJ and Yin WP. Effect of Shenfu on inflammatory cytokine release and brain edema after prolonged cardiac arrest in the swine. American Journal of Emergency Medicine 2013; 31: 1159–1164. Jin H, Seo JH, Uhm YK, Jung CY, Lee SK and Yim SV. Pharmacokinetic comparison of ginsenoside metabolite IH-901 from fermented and non-fermented ginseng in healthy Korean volunteers. Journal of Ethnopharmacology 2011; 139: 664–667. Kanzaki T, Morisaki N, Shiina R and Saito Y. Role of transforming growth factor-beta pathway in the mechanism of wound healing by saponin from Ginseng Radix rubra. British Journal of Pharmacology 1998; 125: 255–262. Kitagawa I, Yoshikawa M, Yoshihara M, Hayashi T and Taniyama T. Chemical studies on crude drug precession 1 on the constituents of ginseng radix rubra. Yakugaku Zasshi – Journal of the Pharmaceutical Society of Japan 1983; 103: 612–622. Lai CM, Li SP, Yu H, Wan JB, Kan KW and Wang YT. A rapid HPLC–ESI-MS/ MS for qualitative and quantitative analysis of saponins in ‘Xuesetong’ injection. Journal of Pharmaceutical and Biomedical Analysis 2006; 40: 669–678. Liu H, Yang J, Du F, Gao X, Ma X, Huang Y, Xu F, Niu W, Wang F, Mao Y, Sun Y, Lu T, Liu C, Zhang B and Li C. Absorption and disposition of ginsenosides after oral administration of Panax notoginseng extract to rats. Drug Metabolism and Disposition 2009; 37: 2290–2298. Liu L, Huang J, Hu X, Li K and Sun C. Simultaneous determination of ginsenosides (G-Re, G-Rg1, G-Rg2, G-F1, G-Rh1) and protopanaxatriol in human plasma and urine by LC-MS/MS and its application in a pharmacokinetics study of G-Re in volunteers. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 2011a; 879: 2011–2017. Liu M, Wang H, Zhao S, Shi X, Zhang Y, Xu H, Wang Y, Li X and Zhang L. Studies on target tissue distribution of ginsenosides and epimedium flavonoids in rats after intravenous administration of Jiweiling freeze-dried powder. Biomedical Chromatography 2011b; 25: 1260–1272. Morisaki N, Watanabe S, Tezuka M, Zenibayashi M, Shina R, Koyama N, Kanzaki T and Saito Y. Mechanism of angiogenic effects of saponin from ginseng radix-rubra in human umbilical vein endothelial-cells. British Journal of Pharmacology 1995; 115: 1188–1193. Qian ZM, Lu J, Gao QP and Li SP. Rapid method for simultaneous determination of flavonoid, saponins and polyacetylenes in Folium Ginseng and Radix Ginseng by pressurized liquid extraction and high-performance liquid chromatography coupled with diode array detection and mass spectrometry. Journal of Chromatography A 2009; 1216: 3825–3830. Ren HC, Sun JG, Wang GJ, A JY, Xie HT, Zha WB, Yan B, Sun FZ, Hao HP, Gu SH, Sheng LS, Shao F, Shi J and Zhou F. Sensitive determination of 20(S)-protopanaxadiol in rat plasma using HPLC–APCI-MS: application of pharmacokinetic study in rats. Journal of Pharmaceutical and Biomedical Analysis 2008; 48: 1476–1480. Song M, Zhang S, Xu S, Hang T and Jia L. Simultaneous determination of three Panax notoginseng saponins at sub-nanograms by LC–MS/MS in dog plasma for pharmacokinetics of compound Danshen tablets. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 2010; 878: 3331–3337. Sun JG, Wang GJ, Xie HT, Hao L, Pan GY and Tucker I. Simultaneous rapid quantification of ginsenoside Rg1 and its secondary glycoside Rh1 and aglycone protopanaxatriol in rat plasma by liquid chromatography– mass spectrometry after solid-phase extraction. Journal of Pharmaceutical and Biomedical Analysis 2005; 38: 126–132. Wan JB, Lai CM, Li SP, Lee MY, Kong LY and Wang YT. Simultaneous determination of nine saponins from Panax notoginseng using HPLC and pressurized liquid extraction. Journal of Pharmaceutical and Biomedical Analysis 2006; 41: 274–279. Xiao Y, Ma ZC, Wang YG, Tan HL, Tang XL, Liang QD, Xiao CR and Gao Y. Cardioprotection of Shenfu preparata on cardiac myocytes through cytochrome P450 2J3. Chinese Journal of Integrative Medicine 2013; 11: 327–336. Xu QF, Fang XL and Chen DF. Pharmacokinetics and bioavailability of ginsenosides Rb1 and Rg1 from Panax notoginseng in rats. Journal of Ethnopharmacology 2003; 84: 187–192.
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014
Simultaneous determination of seven ginsenosides of Shenfu injection Yang LJ, Wang J, Tian ZF and Yuan YF. Shenfu injection attenuates neonatal hypoxic–ischemic brain damage in rat. Neurological Sciences 2013; 34: 571–1574. Yang L, Xu SJ, Wu ZF, Liu YM and Zeng X. Determination of ginsenosideRg(1) in human plasma and its application to pharmacokinetic studies following intravenous administration of ‘Shenmai’ injection. Phytotherapy Research 2009; 23: 65–71. Zhang HJ, Lu ZZ, Tan GT, Qiu SX, Farnsworth NR, Pezzuto JM and Fong HHS. Polyacetyleneginsenoside-Ro, a novel triterpene saponin from Panax ginseng. Tetrahedron Letters 2002; 43: 973–977.
Biomed. Chromatogr. 2014
Zhao J, Su C, Yang CP, Liu MH, Tang L, Su WW and Liu ZQ. Determination of ginsenosides Rb1, Rb2, and Rb3 in rat plasma by a rapid and sensitive liquid chromatography tandem mass spectrometry method: application in a pharmacokinetic study. Journal of Pharmaceutical and Biomedical Analysis 2012; 64–65: 94–97. Zhu JQ, Kang LY, Ye QF, Fan GW, Liang YB, Yan C and Orgah J. Effects of Shenfu injection and its main components on the contraction of isolated rat thoracic aortic rings. Plos One 2013; 8: doi: 10.1371/journal. pone.0078026.
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
Revised: 1 May 2014,
Accepted: 17 May 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bmc.3272
Simultaneous determination of seven ginsenosides in rat plasma by highperformance liquid chromatography coupled to time-of-flight mass spectrometry: application to pharmacokinetics of Shenfu injection Zhengguang Lia,b, Rui Zhangb, Xiuping Wangb, Xiaofei Huc, Yuguo Chena* and Qingfei Liud* ABSTRACT: A high-performance liquid chromatography coupled to time-of-flight mass spectrometry (HPLC-TOF MS) method was successfully developed and validated for the identification and determination of seven ginsenosides, Re, Rf, Rb1, Rc, Rb2, Ro and Rd, in a Chinese herbal preparation, Shenfu injection, and rat plasma. Based on the method, the pharmacokinetic profiles of the seven ginsenosides were investigated following intravenous administration of single dose of Shenfu injection to six rats. The established method had high linearity, selectivity, sensitivity, accuracy and precision. The pharmacokinetic results showed that Rb1, Rc and Rb2 had similar pharmacokinetic profiles and relatively long half-life values (19.29 ± 6.36, 29.54 ± 22.91 and 35.60 ± 30.66 h). The half-lives of Rf and Rd were 4.21 ± 3.68 and 8.49 ± 5.20 h, respectively, indicating that they could be metabolized more rapidly than Rb1, Rc and Rb2. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: HPLC-TOF MS; ginsenoside; pharmacokinetics; Shenfu injection
Introduction Shenfu injection is a preparation developed from an ancient traditional Chinese formulation, Shenfu decoction. It consists of the extracts of Ginseng rubra Radix and Aconiti lateralis Radix and is widely used to treat heart failure in the clinic in China (Zhu et al., 2013; Yang et al., 2013; Xiao et al., 2013; Guo and Li, 2013; Hou et al., 2013; Cui et al., 2013). It has been reported that a large number of triterpenoid saponins known as ginsenosides or panaxosides, such as Rb1, Rg, Rb1 and Re, are major components of Ginseng rubra Radix (Choi, 2002; Morisaki et al., 1995; Kitagawa et al., 1983; Zhang et al., 2002; Yang et al., 2009; Kanzaki et al., 1998; Guan et al., 2007). Since ginsenosides are also main components of Panax notoginseng, there have some articles reporting the pharmacokinetic (PK) characteristics of ginsenosides after administration of single compound, mixture, extract or preparation, like Shenmai injection (Liu et al., 2011b; Song et al., 2010; Chen et al., 2010; Xu et al., 2003). However, the types and contents of ginsenosides are often different for extracts or preparations produced by different manufacturers. It is therefore important to investigate the quality and PK characteristics of the main active components of these preparations. Since ginsenosides have poor UV radiation at a maximum absorption of 203 nm, the method of liquid chromatography coupled to mass spectrometry (LC-MS) is widely used for analyzing ginsenosides (Liu et al., 2009, 2011a; Deng et al., 2009; Ha et al., 2010; Sun et al., 2005; Feng et al., 2011; Jin et al., 2011; Ren et al., 2008; Zhao et al., 2012; Qian et al., 2009; Wan et al., 2006; Lai et al., 2006).
Biomed. Chromatogr. 2014
The objective of this study was to establish and validate a selective and sensitive high-performance liquid chromatography coupled to time-of-flight mass spectrometry (HPLC-TOF MS) for the identification and quantification of the main ginsenosides of Shenfu injection in rat plasma including Re, Rf, Rb1, Rc, Rb2, Ro and Rd (Fig. 1). Based on the established method, the PK profiles of the seven compounds could be revealed after intravenous administration of Shenfu injection to rats.
Experimental Chemicals and reagents Shenfu injection (batch no. 130201010; manufacturer, Sichuan Yaan Sanjiu Pharmaceutical Co. Ltd) was purchased from Beijing Xiyuan * Correspondence to: Department of Emergency Medicine, Qilu Hospital, Shandong University, Qingdao, Shandong 250012, China. Email: [email protected] Qingfei Liu, School of Medicine, Tsinghua University, Beijing 100084, China. Email: [email protected] a
Department of Emergency Medicine, Qilu Hospital, Shandong University, Qingdao, Shandong 250012, China
b
Qingdao Hiser Medical Center, Qingdao, Shandong 250012, China
c
The Affiliated Hospital of Qingdao University, Qingdao, Shandong 250012, China
d
School of Medicine, Tsinghua University, Beijing 100084, China Abbreviations used: PK, pharmacokinetic; PNS, Panax notoginseng saponins; TIC, total ionic current; XIC, extracted ionic currents.
Copyright © 2014 John Wiley & Sons, Ltd.
Z. Li et al.
Figure 1. Chemical structures of seven ginsenosides in Shenfu injection.
Hospital. The standard compounds of ginsenosides Re, Rf, Rb1, Rc, Rb2, Ro and Rd (purity ≥99.5%) and Digoxin (internal standard, IS) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile was of HPLC grade (Fisher, USA). HPLC-grade water was obtained using a water purification system (Milli-Q Reagent Water System, Ma, USA). All other chemicals were of analytical reagent grade. All mobile phases were filtered through 0.22 μm micropore filters (Jinteng Ltd, Tianjin, China) prior to use.
Instrumentation and chromatographic conditions The Waters HPLC-TOF MS system (Agilent Ltd, USA) was used. The column was an Agilent C18 (250 × 4.6 mm, 5 μm) maintained at 30 ± 0.15° C. The mobile phases were 0.1% HOAc solution (A) and acetonitrile (B) with gradient condition of B as follows: 0–9 min, 11–16%; 9–20 min, 16–25%; 20–35 min, 25–40%; 35–45 min, 40–60%. The flow rate was set at 1.0 mL/min.
Mass spectrometry The analytes were identified and quantified using TOF MS in negative-ion mode. Source parameters included capillary voltage of 4 kV, cone voltage of 35 V, source temperature of 120°C, desolvation temperature of 350°C,
wileyonlinelibrary.com/journal/bmc
Figure 2. The total ionic current (TIC) of Shenfu injection and the extracted ionic currents (XIC) and MS of the seven ginsenosides (A, TIC; B, Re and Rd; C, Rc and Rb2; D, Rb1; E, Ro; F, Rf). cone gas flow of 10.0 L/h, and desolvation gas flow of 10.0 L/min. Product ions were monitored in W extended mode. In the full-scan mode, the mass spectrometer was operated over a range of m/z 50–1500. Twentymicroliter samples and standards were analyzed.
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014
Simultaneous determination of seven ginsenosides of Shenfu injection
Figure 2. (Continued).
Biomed. Chromatogr. 2014
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
Conc.in Shenfu injection (mg/mL)
wileyonlinelibrary.com/journal/bmc
27.1 35.2 35.6 36.6 37.5 38.0 39.8
Formula
C48H82O18 C42H72O14 C54H92O23 C53H90O22 C53H90O22 C48H76O19 C48H82O18 Re Rf Rb1 Rc Rb2 Ro Rd
MW
[M [M [M [M [M [M [M
Fragment
Fragment used for quantification
m/z
Copyright © 2014 John Wiley & Sons, Ltd.
Compound
Six male Sprague–Dawley rats with weights of 200–220 g were purchased from Beijing Weitong Lihua Biotechnology Co. Ltd (Beijing, China). The animals were housed and cared for under a constant temperature at (22 ± 1°C) and humidity (50 ± 10%). Food was withheld prohibited for 24 h before the experiment while water was provided freely. All studies were approved by the Ethics Committee of Tsinghua University. Shenfu injection was intravenous administrated to the rats through tail vein with a dosage of 5.0 mL/kg body weight. The dosages of the seven ginsenosides were calculated based on the concentrations in the injection. For each rat, 0.3 mL blood was collected from the ophthalmic plexus vein into heparinized tubes at 0.03, 0.17, 0.33, 0.5, 1, 2, 5, 8, 12 and 24 h. A blank sample was collected before administration. The concentrations of the seven ginsenosides were calculated according to the equations of linear regression of the standards. PK parameters were obtained by DAS (version 2.0) pharmacokinetic software provided by the Chinese Pharmacology Association.
Table 1. Identification and quantification of seven ginsenosides in Shenfu injection
Application
859.49 799.45 583.30 568.29 568.29 507.24 945.51
Retention time (min)
Preparation of calibration curves. Seven calibration curves were prepared with the method above by spiking blank rat plasma with IS and the stock solution to the concentrations 0.25, 0.5, 1, 5, 10 and 20 μg/mL for Rb1, Rc and Rb2, and 0.1, 0.2, 0.5, 1, 2 and 4 μg/mL for Rf, Rd, Re and Ro. Method validation. Quality control (QC) samples were prepared at three concentrations: 0.25, 1 and 20 μg/mL for Rb1, Rc and Rb2, and 0.1, 1, 4 μg/mL for Rf, Rd, Re and Ro. Intra-day accuracy and precision (each n = 3) were tested by analysis of the QC samples at different times during the same day. Inter-day accuracy and precision were determined by repeated analysis of the same samples over three consecutive days. The concentration of each sample was determined using calibration standards prepared on the same day. The recoveries were calculated by comparing the peak areas of the plasma samples with those of the standard working samples. The limits of quantification (LOQ) and detection (LOD) of each ginsenosides were defined as the responses that yielded a signal-to-noise ratio of >10 and 3, respectively.
0.02 0.01 0.03 0.03 0.02 0.02 0.01
LOD (μg/mL)
Preparation of blood samples. The blood sample was centrifuged at ο 5000 rpm for 10 min at 4 C (TGL 16C, Medical Centrifuge Co., Beijing, China). A 120 μL aliquot of the upper layer of plasma was mixed with 12.0 μL of IS (43.4 μg/mL) and 480 μL methanol using vortex-mixing for 1 min and then centrifuged at 10,000 rpm for 10 min at 4°C. The upper layer was transferred to another tube and dried under gentle nitrogen flow. The dried substance was added to 120 μL acetonitrile, using vortex-mixing for 1 min and then centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was injected into the HPLC system for analysis. The same sample handling process was used for the recovery and precision determinations.
rha + HOAc + H2O H] H] 3glc C3H6 H] 2glc araf C3H6 + H2O H] 2glc – arap C3H6 + H2O H] 2glc glcUA + ACN H] H]
Identification and determination of ginsenosides in blood samples
0.08 0.10 0.15 0.12 0.10 0.10 0.05
LOQ (μg/mL)
Shenfu injection was diluted with methanol 10-fold before the determination. With the standard references, the main seven ginsenosides in Shenfu injection were identified and determined by the HPLC-TOF MS method established above. The standards were used to confirm and determine the compounds.
946 800 1108 1078 1078 956 946
Identification and determination of ginsenosides in Shenfu injection
0.0712 0.0206 0.1315 0.1135 0.0833 0.0319 0.1622
Z. Li et al.
Biomed. Chromatogr. 2014
Simultaneous determination of seven ginsenosides of Shenfu injection
Figure 3. The TIC of Shenfu injection in rat plasma and the XIC of seven ginsenosides and IS (A, TIC; B, Re and Rd; C, Rc and Rb2; D, Rb1; E, Ro; F, Rf; G, digoxin (IS); H, blank plasma).
Biomed. Chromatogr. 2014
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
Z. Li et al.
Figure 3. (Continued).
Results and discussion Identification and determination of the ginsenosides in Shenfu injection The total ionic current (TIC) of Shenfu injection is shown in Fig. 2 (A). The extracted ionic currents (XIC) and MS of the seven ginsenosides are shown in Fig. 2(B–F). Although there was little difference between the determined molecular weight and the theoretical molecular weight for some compounds, all seven ginsenosides were confirmed by the standards. It was interesting that even [M H] was extracted but some other fragments had much higher response and could be used for quantification. For the seven ginsenosides in Shenfu injection, there are two pairs of isomers, Re and Rd, Rc and Rb2. For Re and Rd, [M rha + HOAc + H2O H] with m/z 859.49 and [M H] with m/z 945.51 (Fig. 2B) were the main fragments with retention times of 27.1 and 39.8 min in the chromatogram, respectively. Thereafter, the two fragments were used for quantification of Re and Rd correspondingly. For Rc and Rb2, m/z 568.29 was the main fragment rather than [M H] with m/z 1077. According to the chemical structures of
wileyonlinelibrary.com/journal/bmc
Rc and Rb2, it could be inferred that the two fragments with m/z 568.29 came from [M 2glc araf C3H6 + H2O H] and [M 2glc arap C3 H6 + H2 O H] , respectively, with retention times of 36.6 and 37.5 min in the chromatogram. The fragment C 3H6 was produced from the group of isobutene (Fig. 2C). The two fragments with m/z 568.29 were therefore used for quantification of Rc and Rb2. For Rb1, the main fragment was [M 3glc C3 H6 H] with m/z 583.30 rather than [M H] with m/z 1107 (Fig. 2D) and the fragment with m/z 583.30 was used for quantification of Rb1. For Ro, the main fragment was [M 2glc glcUA + ACN H] with m/z 507.24 rather than [M H] with m/z 955 (Fig.2E) and the fragment with m/z 507.24 was used for quantification of Ro. For Rf , the main fragment [M H] with m/z 799.45 could be easily found (Fig. 2F) and was used for quantification of R f. It was interesting that there has been no any literature reporting Ro in Shenfu injection. In the present study, there was a relatively high concentration of Ro in Shenfu injection. The concentrations of the seven ginsenosides in Shenfu injection, calculated according to the regression equations of each compound (not shown in the present article) are shown in Table 1.
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014
Simultaneous determination of seven ginsenosides of Shenfu injection Method validation The calibration curves were linear over the concentration range 0.25–20 μg/mL for Rb1, Rc and Rb2, and 0.1–4 μg/mL for Rf, Rd, Re and Ro (r2 > 0.99). The results for matrix effect were in the range of 86–110% (n = 5). The intra- and inter-day accuracies were within 85–112% and the precisions were within acceptable limits at three concentrations (n = 5, RSD < 5%). The extraction recoveries were within 65–87%. The detection limit of TOF MS is generally not as good as a triple quad MS. Especially for a known compound, triple quad MS has the obvious advantage of very low limits of detection and quantitation. However, for TOF MS, the significant advantage is that it can provide exact molecular weight and molecular formula, especially for the unknown compound. Furthermore, for any compound, there must be some metabolites after administration and it is necessary to find and identify them. Therefore, TOF MS was employed in the present research to investigate the PK profiles not only of ginsenosides but also their metabolites. The results of the metabolites will be reported in a future paper.
Pharmacokinetics
Figure 4. The concentration-time profiles of seven ginsenosides, Re, Rf, Rb1, Rc, Rb2, Ro and Rd, in Shenfu injection after i.v. administration of a single dose to rats (n = 6).
Identification and determination of ginsenosides in rat plasma The TIC of the seven ginsenosides of Shenfu injection in rat plasma is shown in Fig. 3(A). The XIC and MS of the seven ginsenosides and IS are shown in Fig. 3(B–G). All seven ginsenosides were confirmed by the standards. For IS, [M + HOAc H] with m/z 839 could be easily found and there was no response of [M H] (m/z 779). [M + HOAc H] was therefore used for qualification.
The concentration–time profiles of the seven ginsenosides are shown in Figure 4(A–C) and the main PK parameters in Table 2. The dose of each ginsenoside was calculated according to the concentration in Shenfu injection. Re was metabolized rapidly and could not be determined even at 0.33 h after administration, although with a dose of 0.18 mg/kg. It was similar for Ro, which could not be detected at 0.5 h after administration. Thereafter, PK parameters of Re and Ro could not be provided in the present research. The PK parameters of Rb1, Rc and Rb2 were similar with relatively long half-lives (t1/2). The t1/2 values of Rf and Rd were 4.21 ± 3.68 and 8.49 ± 5.20 h, respectively, indicating that they were metabolized slowly but still more rapidly than Rb1, Rc and Rb2. There have been some PK investigations on ginsenosides following oral or intravenous administration of the single compound, mixture or the extract from ginseng or notoginseng, but the PK parameters were very different. For example, after oral administration of Panax notoginseng saponins (PNS) extract to rats, Ra3, Rb1, Rc and Rd had significantly longer t1/2 values (7.5–19.8 h) than the other ginsenosides (Rg1, Rg2, Rg3, F1, F2, Rh1, Rh2, Re, Rf, and 20-gluco-ginsenoside Rf; 0.2–3.2 h; Liu et al., 2009). However, another report showed that the values of t1/2 of Rb1 and Rg1 were 17.96 and 14.13 h (Xu et al., 2003). After oral administration of PNS extract to beagle dogs, values of t1/2 of R1,
Table 2. The pharmacokinetic parameters of seven ginsenosides in Shenfu injection after i.v. administration of single dose to rats (n = 6) Re Dose (mg/kg) t1/2 (h) V(L/kg) CL(L/h/kg) AUC0–t (mg/L h) MRT0–t (h)
0.18 — — — — —
Rf 0.10 4.21 ± 3.68 0.03 ± 0.03 0.03 ± 0.02 4.38 ± 3.30 2.99 ± 2.17
Rb1 0.66 19.29 ± 6.36 0.05 ± 0.04 0.01 ± 0.01 71.37 ± 31.17 9.83 ± 0.36
Rc 0.57 29.54 ± 22.91 0.04 ± 0.03 0.01 ± 0.01 65.30 ± 29.10 10.00 ± 1.30
Rb2
Ro
0.42 35.60 ± 30.66 0.04 ± 0.05 0.01 ± 0.01 44.42 ± 20.24 10.22 ± 1.51
0.08 — — — — —
Rd 0.81 8.49 ± 5.20 0.77 ± 0.04 0.39 ± 0.32 2.81 ± 2.02 6.01 ± 3.33
t1/2, Half-life; ; CL, clearance; AUC0–t, area under the concentration–time curve; MRT, mean residence time.
Biomed. Chromatogr. 2014
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
Z. Li et al. Rg1 and Rb1 were 3.35 ± 0.55, 4.55 ± 1.22 and 18.27 ± 2.55 h, respectively (Chen et al., 2010). Values of t1/2 for R1, Rg1 and Rb1 were 4.82 ± 3.13, 4.59 ± 2.95 and 60.9 ± 17.5 h after oral administration of Danshen tablets containing PNS extract to beagle dogs (Song et al., 2010). For R1, Rg1, Re and Rb1, values of t1/2 were 245.6 ± 118.5, 418.8 ± 68.0, 431.4 ± 149.5 and 1512.2 ± 1461.9 min after oral administration of Qihuotongqiao tablets to rats (Deng et al., 2009). For Rb1, Rb2 and Rb3 after intravenous and oral administration to rats with single compound, the t1/2 values were 12.5 ± 1.8, 15.4 ± 3.7, 24.9 ± 12.6 h and 9.8 ± 6.9, 23.1 ± 3.7, 21.1 ± 9.8 h, respectively (Zhao et al., 2012). As for humans, the t1/2 value of Re was 1.82 ± 0.75 h after oral administration of ginsenosides (Liu et al., 2011a) and the t1/2 of Rg1 was 2.09 ± 1.89 h following intravenous administration of Shenmai injection (Yang et al., 2009). The results of the present research show the PK profiles of seven ginsenosides in Shenfu injection for the first time following intravenous administration to rats. The PK parameters of most compounds like Rb1 were similar to those in the literature. They could provide useful reference and information for future research and clinical application.
Conclusion The PK profiles of seven ginsenosides in Shenfu injection, Re, Rf, Rb1, Rc, Rb2, Ro and Rd, were first simultaneously determined following i.v. administration of a single dose to rats. The HPLCTOF MS method was developed for identification and quantification of the seven ginsenosides in Shenfu injection and in rat plasma with high sensitivity, accuracy and precision. The PK information on the ginsenosides obtained in this research could provide significant reference for the clinical use of Shenfu injection.
Acknowledgment The project was kindly supported by Research Funding of Qingdao Hiser Medical Center.
References Chen W, Dang Y and Zhu C. Simultaneous determination of three major bioactive saponins of Panax notoginseng using liquid chromatography–tandem mass spectrometry and a pharmacokinetic study. Chinese Medicine 2010; 5: 12–17. Choi S. Epidermis proliferative effect of the Panax ginseng ginsenoside Rb-2. Archives of Pharmacal Research 2002; 25: 71–76. Cui HZ, Kim HY, Kang DG and Lee HS. Ginseng–aconite decoction + 2+ elicits a positive inotropic effect via the reverse mode Na /Ca exchanger in beating rabbit atria. Journal of Ethnopharmacology 2013; 148: 624–631. Deng GF, Wang DL, Meng MX, Hu F and Yao TW. Simultaneous determination of notoginsenoside R1, ginsenoside Rg1, Re, Rb1 and icariin in rat plasma by ultra-performance liquid chromatography–tandem mass spectrometry. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 2009; 877: 2113–2122. Feng H, Chen W and Zhu C. Pharmacokinetics study of bio-adhesive tablet of Panax notoginseng saponins. International Archives of Medicine 2011; 4: 18–25. Guan J, Lai CM and Li SP. A rapid method for the simultaneous determination of 11 saponins in Panax notoginseng using ultra performance liquid chromatography. Journal of Pharmaceutical and Biomedical Analysis 2007; 44: 996–1000. Guo ZJ and Li CS. Therapeutic effects of shenfu injection on post-cardiac arrest syndrome. Chinese Journal of Integrative Medicine 2013; 19: 716–720. Ha YW, Ahn KS, Lee JC, Kim SH, Chung BC and Choi MH. Validated quantification for selective cellular uptake of ginsenosides on
wileyonlinelibrary.com/journal/bmc
MCF-7 human breast cancer cells by liquid chromatography–mass spectrometry. Analytical and Bioanalytical Chemistry 2010; 396: 3017–3025. Hou XM, Li CS, Gu W, Guo ZJ and Yin WP. Effect of Shenfu on inflammatory cytokine release and brain edema after prolonged cardiac arrest in the swine. American Journal of Emergency Medicine 2013; 31: 1159–1164. Jin H, Seo JH, Uhm YK, Jung CY, Lee SK and Yim SV. Pharmacokinetic comparison of ginsenoside metabolite IH-901 from fermented and non-fermented ginseng in healthy Korean volunteers. Journal of Ethnopharmacology 2011; 139: 664–667. Kanzaki T, Morisaki N, Shiina R and Saito Y. Role of transforming growth factor-beta pathway in the mechanism of wound healing by saponin from Ginseng Radix rubra. British Journal of Pharmacology 1998; 125: 255–262. Kitagawa I, Yoshikawa M, Yoshihara M, Hayashi T and Taniyama T. Chemical studies on crude drug precession 1 on the constituents of ginseng radix rubra. Yakugaku Zasshi – Journal of the Pharmaceutical Society of Japan 1983; 103: 612–622. Lai CM, Li SP, Yu H, Wan JB, Kan KW and Wang YT. A rapid HPLC–ESI-MS/ MS for qualitative and quantitative analysis of saponins in ‘Xuesetong’ injection. Journal of Pharmaceutical and Biomedical Analysis 2006; 40: 669–678. Liu H, Yang J, Du F, Gao X, Ma X, Huang Y, Xu F, Niu W, Wang F, Mao Y, Sun Y, Lu T, Liu C, Zhang B and Li C. Absorption and disposition of ginsenosides after oral administration of Panax notoginseng extract to rats. Drug Metabolism and Disposition 2009; 37: 2290–2298. Liu L, Huang J, Hu X, Li K and Sun C. Simultaneous determination of ginsenosides (G-Re, G-Rg1, G-Rg2, G-F1, G-Rh1) and protopanaxatriol in human plasma and urine by LC-MS/MS and its application in a pharmacokinetics study of G-Re in volunteers. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 2011a; 879: 2011–2017. Liu M, Wang H, Zhao S, Shi X, Zhang Y, Xu H, Wang Y, Li X and Zhang L. Studies on target tissue distribution of ginsenosides and epimedium flavonoids in rats after intravenous administration of Jiweiling freeze-dried powder. Biomedical Chromatography 2011b; 25: 1260–1272. Morisaki N, Watanabe S, Tezuka M, Zenibayashi M, Shina R, Koyama N, Kanzaki T and Saito Y. Mechanism of angiogenic effects of saponin from ginseng radix-rubra in human umbilical vein endothelial-cells. British Journal of Pharmacology 1995; 115: 1188–1193. Qian ZM, Lu J, Gao QP and Li SP. Rapid method for simultaneous determination of flavonoid, saponins and polyacetylenes in Folium Ginseng and Radix Ginseng by pressurized liquid extraction and high-performance liquid chromatography coupled with diode array detection and mass spectrometry. Journal of Chromatography A 2009; 1216: 3825–3830. Ren HC, Sun JG, Wang GJ, A JY, Xie HT, Zha WB, Yan B, Sun FZ, Hao HP, Gu SH, Sheng LS, Shao F, Shi J and Zhou F. Sensitive determination of 20(S)-protopanaxadiol in rat plasma using HPLC–APCI-MS: application of pharmacokinetic study in rats. Journal of Pharmaceutical and Biomedical Analysis 2008; 48: 1476–1480. Song M, Zhang S, Xu S, Hang T and Jia L. Simultaneous determination of three Panax notoginseng saponins at sub-nanograms by LC–MS/MS in dog plasma for pharmacokinetics of compound Danshen tablets. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 2010; 878: 3331–3337. Sun JG, Wang GJ, Xie HT, Hao L, Pan GY and Tucker I. Simultaneous rapid quantification of ginsenoside Rg1 and its secondary glycoside Rh1 and aglycone protopanaxatriol in rat plasma by liquid chromatography– mass spectrometry after solid-phase extraction. Journal of Pharmaceutical and Biomedical Analysis 2005; 38: 126–132. Wan JB, Lai CM, Li SP, Lee MY, Kong LY and Wang YT. Simultaneous determination of nine saponins from Panax notoginseng using HPLC and pressurized liquid extraction. Journal of Pharmaceutical and Biomedical Analysis 2006; 41: 274–279. Xiao Y, Ma ZC, Wang YG, Tan HL, Tang XL, Liang QD, Xiao CR and Gao Y. Cardioprotection of Shenfu preparata on cardiac myocytes through cytochrome P450 2J3. Chinese Journal of Integrative Medicine 2013; 11: 327–336. Xu QF, Fang XL and Chen DF. Pharmacokinetics and bioavailability of ginsenosides Rb1 and Rg1 from Panax notoginseng in rats. Journal of Ethnopharmacology 2003; 84: 187–192.
Copyright © 2014 John Wiley & Sons, Ltd.
Biomed. Chromatogr. 2014
Simultaneous determination of seven ginsenosides of Shenfu injection Yang LJ, Wang J, Tian ZF and Yuan YF. Shenfu injection attenuates neonatal hypoxic–ischemic brain damage in rat. Neurological Sciences 2013; 34: 571–1574. Yang L, Xu SJ, Wu ZF, Liu YM and Zeng X. Determination of ginsenosideRg(1) in human plasma and its application to pharmacokinetic studies following intravenous administration of ‘Shenmai’ injection. Phytotherapy Research 2009; 23: 65–71. Zhang HJ, Lu ZZ, Tan GT, Qiu SX, Farnsworth NR, Pezzuto JM and Fong HHS. Polyacetyleneginsenoside-Ro, a novel triterpene saponin from Panax ginseng. Tetrahedron Letters 2002; 43: 973–977.
Biomed. Chromatogr. 2014
Zhao J, Su C, Yang CP, Liu MH, Tang L, Su WW and Liu ZQ. Determination of ginsenosides Rb1, Rb2, and Rb3 in rat plasma by a rapid and sensitive liquid chromatography tandem mass spectrometry method: application in a pharmacokinetic study. Journal of Pharmaceutical and Biomedical Analysis 2012; 64–65: 94–97. Zhu JQ, Kang LY, Ye QF, Fan GW, Liang YB, Yan C and Orgah J. Effects of Shenfu injection and its main components on the contraction of isolated rat thoracic aortic rings. Plos One 2013; 8: doi: 10.1371/journal. pone.0078026.
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/bmc
