Although the current literature has recorded many reports of identifying components from herbal preparations, all of them were largely limited to target components. This paper provides a novel and generally applicable approach to identifying nontarget components from herbal preparations, based on the use of liquid chromatography ion trap time-of-flight mass spectrometry (LC/MS-IT-TOF). A simple program was originally developed for searching the common diagnostic ions from all experimentally generated ions. The components sharing the exact same ions (mass error < 5 mDa) were classified into a family. All families were then connected into a coherent network by the bridging components that are present in two or more families. With the benefit from such a network, it is feasible to sequentially characterize the structures of all diagnostic ions once a single component has been de novo identified. The structures of the diagnostic ions could then be used as "a priori" information for selecting the exact candidates containing the substructures of the corresponding diagnostic ions from the primary database hits. This strategy enables a nearly 7-fold narrowing of the database hits and thus substantially enhances the analytical efficiency and sharpness. With the use of such an approach, 43 out of 53 components incorporated into the network have been successfully identified from the test herbal preparation. For the rest, components failed to be identified using this approach; a complementary approach to screening by sequential loss of specific chemical groups, proposed from the accurate mass differences between fragments, was established to narrow the database hits. All of the 87 peaks detected have been successfully identified by combining the use of both approaches except failed to differentiate some isomers. The presently developed approach and methodology would be useful for the identifications of complicated nontarget components from various complex mixtures such as herbal preparations, biological, and environmental samples.

The paper presents a modified and universally applicable diagnostic fragment-ion-based extension strategy (DFIBES) to efficiently process the information acquired by liquid chromatography-electrospray ionization source in combination with hybrid ion trap and high-resolution time-of-flight mass spectrometry [LC-(ESI)-IT-TOF/MS], facilitating the structural determination of serial components contained in traditional Chinese medicine prescription (TCMP). The key advantage of DFIBES is that it facilitates the rapid classification of the complicated peaks into well-known chemical families, which significantly simplifies the complicated procedures of structural characterization. Moreover, considering that a certain family of compounds usually produces identical fragment ions, the DFIBES would be widely applicable to many other families of compounds identification besides the presently validated ginsenosides and lignans. Shengmai injection, composed of Panax ginseng, Radix ophiopogonis, and Schisandra chinensis, was taken as a TCMP example to conduct and validate the proposed DFIBES. Diagnostic fragment ions (DFI) for each chemical family contained in Shengmai injection was firstly determined or proposed from the separated analysis of 15 authentic standards and the extract of S. chinensis. The ESI-MSn fragmentation patterns of ginsenosides and lignans were then systematically studied for developing the 'structure extension' approach. Upon LC-IT-TOF/MS analysis and DFIBES, more than 30 ginsenosides and 20 lignans have been rapidly detected and identified from Shengmai injection, supporting that the DFIBES is a very powerful strategy and would be widely applicable for the complicated components identification from TCMP and other complicated mixtures.

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ABSTRACT:Although the biotransformation of ginsenosides in the gastrointestinal tract has been extensively studied, much less is known about hepatic cytochrome P450 (P450)-catalyzed metabolism. The major aims of this study were to clarify the metabolic pathway and P450 isoforms involved and to explore the structure-metabolism relationship of protopanaxatriol (PPT)-type ginsenosides in hepatic microsomes. Efficient depletion of ginsenoside Rh1, Rg2, Rf, and PPT was found, whereas the elimination of Re and Rg1, characterized by a glucose substitution at the C20 hydroxy group, was negligible in microsomal incubation systems. Based on high-performance liquid chromatography hybrid ion trap and time-of-flight mass spectrometry analysis, the oxygenation metabolism on the C20 aliphatic branch chain was identified as the predominant metabolic pathway of PPT ginsenosides in both human and rat hepatic microsomes. By a comparison with authentic standards, the C24-25 double bond was identified as one of the oxygenation sites to produce the metabolites of C20-24 epoxide (ocotillol-type ginsenosides). Both chemical inhibition and human recombinant P450 isoform assays indicated that CYP3A4 was the predominant isozyme responsible for the oxygenation metabolism of PPT ginsenosides. Enzyme kinetic evaluations in rat and human hepatic microsomes and human recombinant CYP3A4 isozyme incubation systems showed generally consistent results in that the intrinsic clearance ranked as Rf < Rg2 < Rh1 < PPT, closely correlating with logP values and the number of glycosyl substitutions. Results obtained from this study suggest that CYP3A4-catalyzed oxygenation metabolism plays an important role in the hepatic disposition of ginsenosides and that glycosyl substitution, especially at the C20 hydroxy group, determines their intrinsic clearances by CYP3A4.

The metabolism of tanshinone IIA was studied in rats after a single-dose intravenous administration. In the present study, 12 metabolites of tanshinone IIA were identified in rat bile, urine and feces with two LC gradients using LC-MS/MS. Seven phase I metabolites and five phase II metabolites of tanshinone IIA were characterized and their molecular structures proposed on the basis of the characteristics of their precursor ions, product ions and chromatographic retention time. The seven phase I metabolites were formed, through two main metabolic routes, which were hydroxylation and dehydrogenation metabolism. M1, M4, M5 and M6 were supposedly tanshinone IIB, hydroxytanshinone IIA, przewaquinone A and dehydrotanshinone IIA, respectively, by comparing their HPLC retention times and mass spectral patterns with those of the standard compounds. The five phase II metabolites identified in this research were all glucuronide conjugates, all of which showed a neutral loss of 176 Da. M9 and M12 were more abundant than other identified metabolites in the bile, which was the main excretion path of tanshinone IIA and the metabolites. M12 was the main metabolite of tanshinone IIA. M9 and M12 were proposed to be the glucuronide conjugates of two different semiquinones and these semiquinones were the hydrogenation products of dehydrotanshinone IIA and tanshinone IIA, respectively. This hydrogenized reaction may be catalyzed by the NAD(P)H: quinone acceptor oxidoreductase (NQO). The biotransformation pathways of tanshinone IIA were proposed on the basis of this research.

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20(S)-Ginsenoside Rh1 is one of the important protopanaxatriol ginsenosides and has been reported to be the main hydrolysis product reaching the systemic circulation after oral ingestion of ginseng. However, its pharmacokinetic characteristics and metabolic fate have never been reported. The present study was therefore designed to elucidate its pharmacokinetic profiles and metabolic pathways both in vivo and in vitro. The absolute bioavailability of 20(S)-ginsenoside Rh1 in rats was only 1.01 %. Identification of metabolites showed that, after intragastrical administration of ginsenoside Rh1, two mono-oxygenated metabolites were detected from the urine, bile, liver tissue, and intestinal tract content, while the de-glucosylated product, 20(S)-protopanaxatriol, was only found in the contents of the intestinal tract. An in vitro incubation study confirmed that the CYP450-catalyzed mono-oxygenation, the intestinal bacteria mediated de-glucosylation, and the gastric acid mediated hydration reaction were the main metabolic pathways of 20(S)-ginsenoside Rh1. The presystemic metabolism as evidenced from this study may partially explain its poor bioavailability.

Lysosomal storage disorders (LSD) are a group of heterogeneous diseases caused by compromised enzyme function leading to multiple organ failure. Therapeutic approaches involve enzyme replacement (ERT), which is effective for a substantial fraction of patients. However, there are still concerns about a number of issues including tissue penetrance, generation of host antibodies against the therapeutic enzyme, and financial aspects, which render this therapy suboptimal for many cases. Treatment with pharmacological chaperones (PC) was recognized as a possible alternative to ERT, because a great number of mutations do not completely abolish enzyme function, but rather trigger degradation in the endoplasmic reticulum. The theory behind PC is that they can stabilize enzymes with remaining function, avoid degradation and thereby ameliorate disease symptoms. We tested several compounds in order to identify novel small molecules that prevent premature degradation of the mutant lysosomal enzymes α-galactosidase A (for Fabry disease (FD)) and acid α-glucosidase (GAA) (for Pompe disease (PD)). We discovered that the expectorant Ambroxol when used in conjunction with known PC resulted in a significant enhancement of mutant α-galactosidase A and GAA activities. Rosiglitazone was effective on α-galactosidase A either as a monotherapy or when administered in combination with the PC 1-deoxygalactonojirimycin. We therefore propose both drugs as potential enhancers of pharmacological chaperones in FD and PD to improve current treatment strategies.