Development of a novel sectional multiple filtering scheme for rapid screening and classifying metabolites of ziyuglycoside II in rat liver and excreta specimen based on high-resolution mass spectrometry.

Ziyuglycoside II, one of the major effective ingredients of Sanguisorba officinalis L., had various pharmacological activities including anticancer, anti-inflammation and anti-oxidation, etc. Better understanding of the pharmacology and toxicology of ziyuglycoside II requires the detailed elucidation of its biologic fates in vivo. Herein, the metabolic fate of ziyuglycoside II in rats was investigated based on liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS). To accelerate and simplify the process of metabolite identification from complicated biological matrix, the sectional multiple filtering (SMF) scheme was designed according to the relationship among the molecular weight (MW), mass defect (MD) and retention time (tR) of the metabolites. SMF-I (MW: 700-850Da, MD: 0.40-0.45Da, tR: 4.0-10.0min), SMF-II (MW: 550-700Da, MD: 0.30-0.40Da, tR: 6.0-14.0min) and SMF-III (MW: 400-550Da, MD at 0.25-0.35Da, tR at 9.5-16.0min) were built and utilized to screen phase II conjugations and phase I redox metabolites and deglycosylated derivatives, respectively. As a result, dozens of metabolites, including glucuronic conjugates, hydroxylation, oxidization, dehydration and deglycosylation products, were rapidly discovered, classified and structural identified in rat urine and feces based on SMF scheme and accurate MS(1)/MS(2) information. Obviously, the SMF technique showed superior efficiency and selectivity in ziyuglycoside II metabolite identification. More importantly, SMF would find its extensive application in, but not limited to, the metabolic study for single drug or homologous compounds in traditional Chinese medicine.

Comprehensive characterization of the in vitro and in vivo metabolites of ziyuglycoside I in rat microsome, intestinal flora, excretion specimen and fresh tissues based on LC-Q-TOF/MS.

Ziyuglycoside I is one of the major active ingredients in Sanguisorba officinalis, a popular medicinal plant in China. In the present study, the metabolites of ziyuglycoside I in rat liver microsome and intestinal flora were identified and structurally characterized, and the metabolic rules were summed based on the LC-Q-TOF/MS system. Then, the metabolites in rat excreta samples were rapidly screened and identified according to the in vitro metabolic rules. Finally, ziyuglycoside I was incubated with fresh liver/lung/kidney/stomach homogenates to further explore the source of the metabolites and reveal the possible metabolic organs involved. Four metabolites in liver microsome were identified as M0-Glu, M0-CH2OH, M0-Glu+CH3, M0-Glu-Ara+CH3. In intestinal flora incubation system, 6 degradation products including M0-Glu-Ara+O, M0-Ara, M0-Glu-COOH, M0-Glu, M0-Glu-Ara+O and M0-Ara+H2O were tentatively identified by interpretation of their accurate MS(1) and MS(2) data. Fifteen metabolites in rat urine and feces were identified, and most of the metabolites were attributed to the transformation in liver microsome and intestinal flora. Specifically, more than a dozen of new metabolites were identified in rat fresh tissues, and ziyuglycoside II was confirmed as the major metabolite in rats.

Appropriate choice of collision-induced dissociation energy for qualitative analysis of notoginsenosides based on liquid chromatography hybrid ion trap time-of-flight mass spectrometry.

Liquid chromatography hybrid ion trap/time-of-flight mass spectrometry possessesd both the MS(n) ability of ion trap and the excellent resolution of a time-of-flight, and has been widely used to identify drug metabolites and determine trace multi-components for in natural products. Collision energy, one of the most important factors in acquiring MS(n) information, could be set freely in the range of 10%-400%. Herein, notoginsenosides were chosen as model compounds to build a novel methodology for the collision energy optimization. Firstly, the fragmental patterns of the representatives for the authentic standards of protopanaxadiol-type and protopanaxatriol-type notoginsenosides authentic standards were obtained based on accurate MS(2) and MS(3) measurements via liquid chromatography hybrid ion trap/time-of-flight mass spectrometry. Then the extracted ion chromatograms of characteristic product ions of notoginsenosides in Panax Notoginseng Extract, which were produced under a series of collision energies and, were compared to screen out the optimum collision energies values for MS(2) and MS(3). The results demonstrated that the qualitative capability of liquid chromatography hybrid ion trap/time-of-flight mass spectrometry was greatly influenced by collision energies, and 50% of MS(2) collision energy was found to produce the highest collision-induced dissociation efficiency for notoginsenosides. BesidesAddtionally, the highest collision-induced dissociation efficiency appeared when the collision energy was set at 75% in the MS(3) stage.

Development and validation of an UFLC-MS/MS assay for the absolute quantitation of nine notoginsenosides in rat plasma: Application to the pharmacokinetic study of Panax Notoginseng Extract.

Notoginsenosides, the main active gradients of Chinese traditional medicine Panax notoginseng, possesses a variety of biological activities including antioxidant property, anti-hyperglycemic, anti-obese, etc. However, pharmacokinetic evaluation for notoginsenosides is still a formidable task due to their low concentrations and complex components in vivo. The summation of this work generated a rapid and sensitive method for quantitative analysis of multi-notoginsenoside in rat plasma based on ultra fast liquid chromatographic-tandem mass spectrometric. After liquid-liquid extraction by n-butanol, notoginsenoside R1, Rg3, Rd, Rg2, Rb2, Rf, Rg1, Rb1 and Re were simultaneously monitored in negative ionization mode after separating on a Thermo ODS C18 column (5mm 50mm×2.1mm) by a binary gradient elution, and all compounds were analyzed within 9min. Multiple reaction monitoring (MRM) was performed as follows: R1 (m/z 967.7→637.4), Rg3 (m/z 819.6→621.4), Rd (m/z 981.6→783.5), Rg2 (m/z 819.6→475.4), Rb2 (m/z 1113.4→783.4), Rf (m/z 835.6→475.4), Rg1 (m/z 835.6→637.6), Rb1 (m/z 1143.7→945.6), Re (m/z 981.6→637.4), internal standard (digoxin, m/z 815.5→779.4). Validation parameters (linearity, sensitivity, intra-and inter-assay precision and accuracy, recovery and matrix effect) were within acceptable ranges and biological extracts were stable during the entire storing and preparing process. This UFLC-MS/MS approach was further validated by being applied to the pharmacokinetic study for P. Notoginseng extract in rats, and the pharmacokinetic parameters were calculated by Winolin software. Thus, the presently developed methodology was simple, robust, accurate, precise, and would be useful for the pharmacokinetic studies for all kinds of notoginsenosides and other herbal saponins.

Development of a systematic approach to rapid classification and identification of notoginsenosides and metabolites in rat feces based on liquid chromatography coupled triple time-of-flight mass spectrometry.

The present work contributes to the development of a powerful technical platform to rapidly identify and classify complicated components and metabolites for traditional Chinese medicines. In this process, notoginsenosides, the main active ingredients in Panaxnotoginseng, were chosen as model compounds. Firstly, the fragmental patterns, diagnostic product ions and neutral loss of each subfamily of notoginsenosides were summarized by collision-induced dissociation analysis of representative authentic standards. Next, in order to maximally cover low-concentration components which could otherwise be omitted from previous diagnostic fragment-ion method using only single product ion of notoginsenosides, a multiple product ions filtering strategy was proposed and utilized to identify and classify both non-target and target notoginsenosides of P.notoginseng extract (in vitro). With this strategy, 13 protopanaxadiol-type notoginsenosides and 30 protopanaxatriol-type notoginsenosides were efficiently extracted. Then, a neutral loss filtering technique was employed to trace prototype components and metabolites in rats (in vivo) since diagnostic product ions might shift therefore become unpredictable when metabolic reactions occurred on the mother skeleton of notoginsenosides. After comparing the constitute profiles in vitro with in vivo, 62 drug-related components were identified from rat feces, and these components were classified into 27 prototype compounds and 35 metabolites. Lastly, all the metabolites were successfully correlated to their parent compounds based on chemicalome-metabolome matching approach which was previously built by our group. This study provided a generally applicable approach to global metabolite identification for the complicated components in complex matrices.

Pharmacokinetic compatibility of ginsenosides and Schisandra Lignans in Shengmai-san: from the perspective of p-glycoprotein.

BACKGROUND: Phytochemical-mediated alterations in P-glycoprotein (P-gp) activity may result in herb-drug interactions by altering drug pharmacokinetics. Shengmai-san, a traditional Chinese herbal medicine composed by Panax Ginseng, Ophiopogon Japonicus, and Schisandra Chinensis, is routinely being used for treating various coronary heart diseases. In our previous studies, Schisandra Lignans Extract (SLE) was proved as a strong P-gp inhibitor, and herein, the compatibility of Shengmai-san was studied by investigating the influence of SLE on the pharmacokinetics of the ginsenosides from the perspective of P-gp. METHODOLOGY: Pharmacokinetic experiments were firstly performed based on in vitro uptake, efflux and transport experiments in Caco-2, LLC-PK1 wild-type and MDR1-overexpressing L-MDR1 cells. During the whole experiment, digoxin, a classical P-gp substrate, was used as a positive control drug to verify the cells used are the valid models. Meanwhile, the effects of SLE on the pharmacokinetics of ginsenosides were further investigated in rats after single-dose and multi-dose of SLE. RESULTS AND CONCLUSIONS: The efflux ratios of ginsenoside Rb2, Rc, Rg2, Rg3, Rd and Rb1 were found more than 3.5 in L-MDR1 cells and can be decreased significantly by verapamil (a classical P-gp inhibitor). Contrarily, the efflux ratios of other ginsenosides (Rh1, F1, Re, and Rg1) were lower than 2.0 and not affected by verapamil. Then, the effects of SLE on the uptake and transport of ginsenosides were investigated, and SLE was found can significantly enhance the uptake and inhibit the efflux ratio of ginsenoside Rb2, Rc, Rg2, Rg3, Rd and Rb1 in Caco-2 and L-MDR1 cells. Besides, In vivo experiments showed that single-dose and multi-dose of SLE at 500 mg/kg could increase the area under the plasma concentration time curve of Rb2, Rc and Rd significantly without affecting terminal elimination half-time. In conclusion, SLE could enhance the exposure of ginsenosides Rb2, Rc, Rg2, Rg3, Rd and Rb1 significantly.

Evaluation of liquid chromatography-ion trap-time of flight hybrid mass spectrometry on the quantitative analysis for ginsenosides.

It is ideal and desirable for a single instrument to meet the requirement of both qualitative and quantitative analysis of complicated components in pharmacokinetic research for herbal medicine. Liquid chromatography combined with hybrid ion trap and time-of-flight mass spectrometry (LCMS-IT-TOF) was recently confirmed as a very powerful tool for the qualitative analysis of both target and nontarget components in herbal medicines. The present study was designed to investigate the feasibility of LCMS-IT-TOF on quantitative analysis of ginsenosides in biological matrices. A simple liquid-liquid extraction procedure was followed by injection of the extracts onto a C18 column with gradient elution and detection based on LCMS-IT-TOF system in negative scan mode. The developed method was validated with respect to the limit of quantification, linear dynamic range, precision, accuracy, matrix effects and stabilities. All the results suggested that the presently developed method was sufficiently sensitive and robust enough to simultaneously monitor 15 ginsenosides with diverse properties and a large range of concentration differences. Therefore, this method would be expected to be highly useful for comprehensive studies of ginsenosides in complicated matrix.