Short communicationSimultaneous determination of seven ginsenosides in Du Shen Tang decoction by rapid resolution liquid chromatography (RRLC) coupled with tandem mass spectrometry
Introduction
It is estimated that about 65–80% of the world’s population is using traditional medicine as the primary form of healthcare (Akerele, 1992). Decoction is the traditional prescription of traditional Chinese medicines (TCMs). One single herb or combining several kinds of herbs prescribed is based on TCM theory and boiled in water makes the decoction. Du-Shen-Tang, the decoction of the root of single Panax ginseng, is a commonly used TCM prescription about 1600 years. According to TCM theory, ginseng has actions of reinforcing qi, promoting blood circulation, benefiting the spleen and lungs and anchoring the mind, and has been traditionally used as a tonic and a panacea that promotes longevity (Leung & Wong, 2010). As summarised in many review articles, ginsenosides, which were generally classified into four groups, namely protopanaxadiol, protopanaxatriol, ocotillol and oleanolic acid type (Chu and Zhang, 2009, Jia and Zhao, 2009, Jia et al., 2009), are known to be bioactive for the prevention and treatment of cardiovascular and cerebrovascular diseases, immunoregulation, hepatoprotection and anti-carcinogenesis (Ackloo et al., 2000, Jang et al., 2008, Lu et al., 2009, Luo and Luo, 2009, Park et al., 2010, Wan et al., 2006). Up to now more than 150 ginsenosides have been isolated and identified according to the reports, among these ginsenosides, ginsenosides-Rb1, Rb2, Rc, Rd, Rg1, Re and Rf (Fig. 1) are the most important compounds in chemical analysis of ginsengs. The chemical profiles and clinical studies of Du Shen Tang decoction have been reported (Li, Lai, et al., 2010, Liu, 2005, Zhen and Yang, 2002). However, there were no studies on simultaneously quantitative determination of ginsenosides in the decoction. Moreover, it is well known that the quantity of active compounds vary as the harvest time of Panax ginseng. Obviously, this has a significant impact on the consistency and efficacy of decoctions produced. Thus, it is very important to develop a direct and rapid approach for assessment of the quantity of Du Shen Tang decoction.
The study on the qualitative and quantitative analysis of ginsenosides using HPLC–DAD (ELSD) and LC–MS (Fuzzati, 2004, Hu et al., 2009, Lai et al., 2006, Li, Luo, et al., 2010, Liu et al., 2006, Popovich et al., 2005, Qi et al., 2011, Qi et al., 2012, Shangguan et al., 2001, Song et al., 2005, Toh et al., 2010, Xie et al., 2008) were reported previously. In comparison, triple quadrupole tandem mass spectrometer (QQQ-MS/MS) can achieve high sensitivity and selectivity by using the multiple reaction monitoring (MRM) scan mode (Wang et al., 2008) without establish the baseline chromatographic separation of target analytes. This method greatly facilitates the quantification of chemical markers in the complex matrixes with only a small amount of sample. RRLC is a relatively new technique giving new possibilities in liquid chromatography, especially concerning on decrease separation time and solvent consumption. Meanwhile, the development of particle columns with small diameters (e.g. 1.8 mm) has turned rapid separation into a reality (Guillarme et al., 2009).
The aim of the present study was to develop a direct and rapid RRLC–MS/MS method for simultaneously quantifying the seven constituents including ginsenosides-Rb1, Rb2, Rc, Rd, Rg1, Re and Rf in Du-Shen-Tang. In addition, the concentration of seven compounds in the decoctions made by the root of Panax ginseng with different years were also investigated and evaluated.
Section snippets
Chemicals, standards and samples
HPLC grade acetonitrile was purchased from Merck (Germany) and MS grade formic acid from Sigma–Aldrich. All other chemicals and solvents were of an analytical grade. Ultra-pure water (18.2 MΩ) was prepared with a Milli-Q water purification system (Millipore, Bedford, MA, USA). The reference standards of ginsenosides-Rb1, Rb2, Rc, Rd, Rg1, Re and Rf were purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China). The structures of these compounds
Chromatographic conditions and MS/MS method development
Different kinds of mobile phases, such as acetonitrile and methanol 0.05%, 0.1% and 0.2% aqueous formic acid and acetic acid were tested. The best peak shape and resolution was obtained from a mixture of acetonitrile and aqueous 0.05% formic acid solution. By an optimised gradient elution in the column, the main components were separately eluted within 11 min. The typical RRLC-QQQ MS/MS chromatograms of marker chemicals in Du Shen Tang decoction were shown in Fig. 2. In order to increase the
Conclusion
This was the first report of simultaneous determination of the major compounds in Du Shen Tang decoction. By using RRLC coupled with an ESI triple quadrupole tandem spectrometer, a rapid, simple and reliable method to simultaneously determine seven marker chemicals (ginsenosides-Re, Rg1, Rb1, Rc, Rb2, Rd and Rf) in Du Shen Tang decoction was first developed and validated. This method provides an excellent quantitative tool for quality assessments of TCM formulae due to its high capacity, high
Acknowledgements
This work was financially supported by the Autonomic Project (project number zz2012011) of China Academy of Chinese Medicine Sciences, National Science and Technology Major Project (No. 20112X09201-201-04) , 2013 Program for Liaoning Innovative Research Team in University (LT2013020) and the National Natural Science Foundation of China (Grant 2009 211A02 and Grant 81001597).
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