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A specific and reliable liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed for the simultaneous determination of timosaponin H1 (TH1), timosaponin E1 (TE1), timosaponin E (TE), timosaponin B-II (TB-II), timosaponin B-III (TB-III) and anemarrhenasaponin I (AS-I) in rat plasma. After addition of internal standard (IS) ginsenoside Rh1, plasma samples were pretreated by protein precipitation with acetonitrile. Chromatographic separation was performed on a reverse phase ACQUITY™ BEH C18 column (100mm×2.1mm i.d., 1.7μm) using a gradient mobile phase system of acetonitrile-water containing 0.05% formic acid and 5mM ammonium formate. The triple quadruple mass spectrometer was set in negative electrospray ionization mode and multiple reaction monitoring (MRM) was used for six steroidal saponins quantification. The precursors to produce ion transitions monitored for TH1, TE1, TE, TB-II, TB-III, AS-I and IS were m/z 1211.5>1079.6, 935.5>773.4, 935.4>773.5, 919.6>757.4, 901.5>739.3, 757.4>595.3 and 637.3>475.3, respectively. The method validation was conducted over the curve range of 0.5-400ng/mL for the six saponins. The intra- and inter-day precisions (RSD%) were less than 9.4% and the average extraction recoveries ranged from 82.5% to 97.8% for each analyte. Six steroidal saponins were proved to be stable during sample storage, preparation and analytical procedures. The validated method was successfully applied for the first time to determine the concentrations of six main steroidal saponins in incurred rat plasma samples, after intragastric administration of the extract of Anemarrhena asphodeloides Bge. for a rat pharmacokinetic study., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Although various techniques have been employed to analyze drug metabolites, the metabolism of multicomponent herbal medicine has seldom been fully addressed. In contrast to chemical drugs, a number of compounds in herbal medicine could get into circulation and then be metabolized. Metabolism study on active constituents in herbal medicine is a good way for us to explain and predict a variety of events related to the efficacy and toxicity of herbal medicine. The present work aims to elucidate the multicomponent metabolic characteristics of a herbal medicine by the combination of plasma pharmacochemistry and microdialysis sampling. Anemarrhena asphodeloides, a well-known traditional Chinese medicine, was chosen as a model. After oral administration of A. asphodeloides saponin extract to rats, microdialysis samples were collected continuously in the jugular vein and analyzed by ultrahigh-performance LC/quadrupole-TOF MS. The identification of compounds in biosamples was achieved by accurate mass measurement and detailed fragmentation pathway analysis. The results showed that unbound constituents in blood circulation of the rat included seven parent saponins and six metabolites, which might be the potential active components in vivo. Among which, three metabolites have not been previously reported and were identified in this study. It is the first report on systemic metabolism of total saponins of A. asphodeloides in mammalian plasma., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The aim of this study was to compare the pharmacokinetics of timosaponin B-II and timosaponin A-III in rat plasma after oral administration of Zhimu-Baihe herb-pair, Zhimu extract, free timosaponin B-II and free timosaponin A-III. After addition of internal standard (IS) ginsenoside Rh1, plasma samples were pretreated by protein precipitation. Chromatographic separation was carried out on a SHISEIDO CAPCELL PAK C18 column (100 mm × 3 mm i.d., 3.0 μm) with the mobile phase consisting of acetonitrile and 0.05% (v/v) formic acid by linear gradient elution. The detection was performed on an Agilent G1946D single quadrupole mass spectrometer with negative electrospray ionization (ESI) interface in select-ion-monitoring (SIM) mode. The following ions: m/z 919 for timosaponin B-II, m/z 739 for timosaponin A-III and m/z 683 for the IS were used for quantitative determination. Good linearity was achieved over the concentration ranged from 4.0-793.3 ng/mL to 3.9-781.3 ng/mL for the two saponins. The precision of the in vivo study was evaluated by intra- and inter-day assays and the percentages of relative standard deviation were all within 15%. The plasma concentrations of timosaponin B-II and timosaponin A-III in rats at designated time periods were successfully determined using this fully validated method, and statistically significant differences (p<0.5) in pharmacokinetic parameters including AUC0-t, AUC0-∞ and MRT (mean residence time) were obtained. Compared to these pharmacokinetic parameters after oral administration of Zhimu extract and monomer solution, higher peak concentration (Cmax is higher), slower elimination (MRT is longer) and larger AUC values could be observed after giving Zhimu-Baihe herb-pair in our study. Therefore, this result not only elucidated the steady and long-lasting pharmacological properties but also revealed the practical value of the compatibility of herb-pair remedy., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Rationale: Timosaponin B-II (TB-II) is one of the major bioactive steroid glycosides isolated from Anemarrhena asphodeloides Bge. (Fam. Liliaceae). It has been regarded as a potential lead compound, which may be further developed into a promising new drug for preventing dementia. To fully understand the action mechanism of TB-II, it is important to study the metabolism profile of this compound in vivo., Methods: Herein, a rapid and sensitive method based on ultrahigh-performance liquid chromatography (UHPLC)/quadrupole-time-of-flight mass spectrometry (QTOFMS) was established to comprehensively investigate the metabolism of TB-II in Sprague-Dawley rat urine following oral administration of a single dose of TB-II at 500.4 mg·kg(-1)., Results: A total of twelve metabolites were detected and identified by means of comparing molecular mass, retention time and spectral pattern of the analytes with those of the parent drug. A possible metabolic pathway on the biotransformation of TB-II was also investigated and proposed., Conclusions: Oxidation, deglycosylation and E-ring cleavage were found to be the major metabolic processes of the compound in rat. It is the first report on a mammalian metabolism study of timosaponin, a common member of steroid glycosides, in rat urine., (Copyright © 2012 John Wiley & Sons, Ltd.)