Andrey Stavrianidi (Presenter)
Lomonosov Moscow State University
Authorship: Andrey Stavrianidi, Elena Stekolshchikova, Polina Turova, Igor Rodin, Irina Ananieva, Oleg Shpigun
Lomonosov Moscow State University, Moscow, Russia
| Short Abstract Ginseng saponins are used in Traditional Chinese Medicine for more than 2000 years. Developed analytical method uses mass spectrometric detection in selected ion monitoring mode for sapogenin fragmentation ions of less polar ginsenosides in urine after ginseng infusion administration. Main advantage of this technique is the ability to detect simultaneously protopanaxatriol and protopanaxatriol derivatives and its deshydroxy analogues. Limits of detection were on the level of 0.01-0.02 mg/L. Good linearity and reproducibility were observed for this method. Developed approach was applied for pharmacokinetics study of ginsenosides less polar metabolites in healthy volunteers after ginseng infusion administration. |
Long Abstract
Introduction. Ginseng based medicines help to build up general vitality and increase resistance to physical, chemical and biological stress, show antitumor activity. As a result ginseng based products are widely used as biologically active dietary supplements. Ginsenosides are considered the main active principles of the traditional medicine “ginseng”. The main metabolic pathways of these compounds in vivo include deglycosylation reactions in ginseng saponins by intestinal bacteria via stepwise cleavage of the sugar moieties. In this process less polar ginsenosides and sapogenin molecules are produced. Several methods have been established for evaluate pharmacokinetics profiles of ginseng and ginseng preparations in animals. Li et al. [1] used an HPLC/UV coupled with solid-phase extraction method for determination of four active saponins (ginsenoside Rg1, Rb1, Rd and notoginsenoside R1) from Panax notoginseng in rat urine. However, there are few reports on the determination of less polar ginsenosides in human for pharmacokinetic study. Zhao et al. [2] reported HPLC/MS/MS method for single ginsenoside Rg3 determination in human plasma and urine. Therefore multicomponent methods for less polar ginsenosides are highly demanded. The use of HPLC/ESI-MS system in our opinion has great potential for steroidal saponin analysis purposes, chemical profiling and online structural characterization of ginsenosides and other saponins. Therefore the aim of this study was to develop HPLC/ESI-MS approach for determination of structurally similar less polar ginsenoside metabolites for its future use in clinical trials and pharmacokinetics study of ginseng containing formulations.
Methods: The RP C18 column was used. Mobile phases consisted of formic acid in water and acetonitrile. Detection was accomplished simultaneously with a diode array detector and a mass spectrometer and chromatograms were recorded at 200 nm and in single ion monitoring mode for positive ions with m/z 405, 423 and m/z 407, 425 for protopanaxatriol and protopanaxadiol saponins correspondingly. Mass spectra for separated chromatographic peaks were acquired in Enhanced MS scan mode with m/z range 100–1370 amu. Peaks were identified by comparing their retention times and mass spectrum signals spectra with authentic standards. Urine samples were defrosted at a room temperature, mixed and centrifuged for 5 min at 14000 rpm. After separation of supernatant from the residue, supernatant was diluted with deionized water prior injection into LC-MS/MS system.
Results: A gradient elution system for the determination of less polar ginsenosides in urine has been developed. Good separation can be achieved within 25 min. Mass spectrometry detection in single ion monitoring (SIM) mode can be applied to successfully determine protopanaxatriol and protopanaxadiol ginsenosides with close retention time. For this purpose sapogenin fragmentation pattern ions such as [PPT-3H2O+H]+, [PPT-4H2O+H]+, [PPD-2H2O+H]+ and [PPD-3H2O+H]+ with m/z values of 423, 405, 425 and 407, correspondingly, were used. Linear range was from approximately 30 mg/L to 1000 mg/L. To test the reproducibility of the assay method, the ginsenoside standard solution and one of the spiked matrix samples were injected five times under and the observed RSD values were within 4%. To test the precision of the developed approach urine samples spiked with 3 different concentration of the ginsenosides Rg3 and C-K were injected 3 times in the HPLC-MS system. Measured amounts of the analytes were in 7% range of the expected value. This HPLC/MS/MS method was used to evaluate the pharmacokinetic profiles of less polar ginsenosides in human urine successfully. Five healthy male and five female Russian volunteers were enrolled. Urine from all subjects was sampled at specific time points after single dose administration of 500 mg of ginseng water infusion. Firstly original ginsenosides and less polar metabolites were detected using the same sapogenin fragmentation pattern ions while in urine samples taken 3 days after administration concentration of less polar ginsenosides was below limit of detection.
Conclusion: The reported analysis method for less polar ginsenoside determination in urine was developed in order to provide a useful clinical tool for proper medication. It uses a gradient elution system with MS detection in SIM mode for the determination of less polar ginsenosides. With this method good repeatability of HPL-MS analysis results was obtained, and 5 different standard ginsenosides could be determined simultaneously at low concentration levels. Developed approach is simple, sensitive and accurate and is applicable to simultaneous measurement of PPT and PPD saponins and its deshydroxy analogues which show the same fragmentation pattern ions. The method was tested in the analysis of urine samples of healthy volunteers and shown similar results among male and female subjects for the time period of 3 days after single dose oral administration.
References & Acknowledgements:
[1] L. Li, J.L. Zhang, Y.L. Sheng, G. Ye, H.Z. Guo, D.A. Guo, J. Chromatogr. B 808 (2004) 177.
[2] Q. Zhao, X. Zheng, J. Jiang, H. Zhou, P. Hu, J. Chromatogr. B 878 (2010) 2266.
This work was funded by the Russian Foundation for Basic Research (RFBR), according to the research project No. 16-33-60007 mol_à_dk
| Description | Y/N | Source |
| Grants | yes | Russian Foundation for Basic Research (RFBR) |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |