Liquid Chromatography-hybrid Ion Trap Research Articles

Systematically identifying the hepatoprotective ingredients of schisandra lignan extract from pharmacokinetic and pharmacodynamic perspectives

BackgroundHerbal medicines (HMs) have been proven to be productive sources of leads for the development of drugs. To date approximately 150 lignans have been identified from Schisandra sphenanthera. Hepatoprotective activity is a well-known characteristic of schisandra lignans, yet the authentic types of active lignans are still not well known. PurposeThe present study aimed to develop a reliable and efficient strategy for identifying the hepatoprotective ingredients of schisandra lignan extract (SLE). MethodsSLEs were prepared by extracting Schisandra sphenanthera powder using 10%, 50% and 90% ethanol (w/w 1:10) combining 5-fold volume of ethyl acetate. The schisandra lignans in SLEs were qualitatively analyzed based on liquid chromatography hybrid ion trap time-of-flight mass spectrometry (LCMS-IT-TOF). Preparative liquid chromatography (PLC) was used to collect ingredient fractions. The hepatoprotective activity of schisandra lignans was systematically investigated on in vivo and in vitro models. ResultsThe SLE extracted by 50% ethanol and 5-fold volume of ethyl acetate (50%SLE) had the highest lignan content and exhibited significantly stronger hepatoprotective activity than other SLEs (P < 0.01). The hepatoprotective effect of 50%SLE mainly attributed to the SLE segment which collected from 12 to 22 min by PLC. Schisantherin A (Sth A) was confirmed as the most promising hepatoprotective drug in Schisandra sphenanthera due to high content in crude materials, high exposure level in vivo and high efficiency on APAP-induced hepatotoxicity. ConclusionThe hepatoprotective ingredients of SLEs were systematically investigated based on the presently developed approach, and Sth A was identified as the optimum hepatoprotective candidate in Schisandra sphenanthera.

Fast screening of flavonoids from switchgrass and Mikania micrantha by liquid chromatography hybrid-ion trap time-of-flight mass spectrometry

A strategy for fast screening of flavonoids in switchgrass and M. micrantha by LC/MS-IT-TOF has been established and applied.

Identification, Synthesis, and Safety Assessment of Thidiazuron [1-Phenyl-3-(1,2,3-thidiazol-5-yl)urea] and Its Metabolites in Kiwifruits.

The quality of kiwifruit became worse as a result of the abuse of plant growth regulators (PGRs). The safety of the fruits treated with PGRs also worried consumers. Therefore, the present study analyzed the structure of thidiazuron [TDZ, (1-phenyl-3-(1,2,3-thidiazol-5-yl)-urea)] (1) and its metabolites of biotransformation in kiwifruits using liquid chromatography hybrid ion trap time-of-flight mass spectrometry (LC-IT-TOF-MS). Standard compounds were also synthesized and used for structural identification of those metabolites. In addition, cytotoxicity of TDZ and its metabolites was tested through sulforhodamine B assays against normal Chinese hamster ovary (CHO) cells. Four metabolites were identified. They were 4-hydroxy-thidiazuron (2), 3-hydroxy-thidiazuron (3), thidiazuron-4-O-β-d-glucoside (4), and thidiazuron-3-O-β-d-glucoside (5). Values of IC50 of compounds 1, 2, and 3 to CHO cells were 18.3 ± 1.8, 37.56 ± 1.5, and 23.36 ± 1.59 μM, respectively. Compounds 4 and 5 had no effect on CHO cells.

Global identification strategy based on mass spectrometry for studying protostane triterpenoids in rhizomes of Alisma orientalis

The liquid chromatography hybrid ion trap time-of-flight mass spectrometry (LCMS-IT-TOF) combined with a global identification strategy was applied to studying the protostane triterpenoids (PTs) in the rhizomes of Alisma orientalis in this paper. Initially, the MSn analyses of four reference compounds were performed to summarize the fragmentation rules of PTs. Subsequently, a series of ions of PTs were presented by LC–MSn analyses of the crude extract of alismatis rhizome, from which six ions at m/z 339, 353, 365, 381, 383 and 397 with high frequency of occurrence were selected as diagnostic ions to classify the peaks. Twenty peaks were classified into six families based on common ions. A network was established for the six families via diagnostic ions. 20 PTs including 1 new compound and 19 known ones were characterized through such strategy, fragmentation rules and references. Ultimately, the detected new compound and two known compounds were isolated from the total extract of A. orientalis under the guide of above method, of which the new compound was identified as 11-deoxylalisol F by MS and NMR spectrum. This systematical investigation on the PTs demonstrated that such a strategy could be widely used in the analysis and separation of natural products.

In vitro metabolism study of saikosaponin d and its derivatives in rat liver microsomes

1. Saikosaponins, one of the representative bioactive ingredients in Radix Bupleuri, possess hepatoprotective, anti-inflammatory, antiviral, antitumor, and other pharmacological activities. Up to now, few studies focused on the further metabolism of saikosaponins and their secondary metabolites absorbed into the circulatory system.2. To understand the in vivo efficacy of saikosaponin d, the in vitro metabolism of saikosaponin d, and its two derivatives formed in the gastrointestinal tract, prosaikogenin G and saikogenin G was investigated in rat liver microsomes, respectively.3. Fifteen metabolites were detected using high-performance liquid chromatography hybrid ion trap and time-of-flight mass spectrometry and triple-quadrupole mass spectrometry, and the predominant metabolic reactions were hydroxylation, carboxylation and combinations of these steps on the aglycone moiety.4. The metabolic pathways of saikosaponin d, prosaikogenin G, and saikogenin G were proposed in vitro and the results contribute to the understanding of saikosaponins in vivo metabolism.

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 MSn 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 MSn 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 MS2 and MS3 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 MS2 and MS3. 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 MS2 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 MS3 stage.

Identification, synthesis, and safety assessment of forchlorfenuron (1-(2-chloro-4-pyridyl)-3-phenylurea) and its metabolites in kiwifruits.

Identification and evaluation of safety of forchlorfenuron ((1-(2-chloro-4-pyridyl)-3-phenylurea)), 1, metabolites after biotransformation in kiwifruit is the objective of this study. To elucidate properties of these metabolites, liquid chromatography hybrid ion trap time-of-flight mass spectrometry (LC-IT-TOF-MS) was applied, with MetID Solution and Formula Predictor Software in positive mode. Cytotoxicity of forchlorfenuron and its metabolites were tested through sulforhodamine B assays against normal Chinese hamster ovary cells (CHO). As deduced from characteristic fragment ions of forchlorfenuron, then confirmed by comparison with synthetic standards, as well as characterized by NMR and mass spectrometry techniques, results indicate the presence of 4-hydroxyphenyl-forchlorfenuron, 2, 3-hydroxyphenyl-forchlorfenuron, 3, and forchlorfenuron-4-O-β-D-glucoside, 5. Forchlorfenuron (IC50 = 12.12 ± 2.14 μM) and 4-hydroxyphenyl-forchlorfenuron (IC50 = 36.15 ± 1.59 μM), exhibits significant cytotoxicity against CHO, while 3-hydroxyphenyl-forchlorfenuron and forchlorfenuron-4-O-β-D-glucoside show no cytotoxicity.

Global detection and identification of components from Yunnan Baiyao based on liquid chromatography hybrid ion trap time‐of‐flight mass spectrometry

Yunnan Baiyao is a widely used herbal prescription in traditional medicine for the treatment of bleeding and hematological diseases, while its chemical profile remains elusive. In this work, a novel methodology combining polarity-directed extraction technique with a diagnostic ion filtering strategy based on LC hybrid ion trap TOF-MS analysis was developed for global, efficient, and rapid characterization of components in Yunnan Baiyao. Di-ethyl ether, n-butanol, and ethanol/water (70:30, v/v) covering low-to-high polarity ranges were chosen as the extraction solvent, respectively. The results clearly showed that, compared with conventional single extraction solvent, collaboratively using extraction solvents with different polarities can effectively increase the number of detected peaks and enrich the product ions information in multistage mass spectra analysis. By further matching diagnostic ions and fragmental pathways, a total of 34 components were successfully identified. Our work clearly demonstrates that integrating polarity-directed extraction and diagnostic ion filtering techniques is a powerful and reliable strategy for global detection and identification of complex chemicalome from herbal prescriptions, and may open new avenues for chemical analysis in other complex mixtures.

Identification and dynamic analysis of the purine alkaloids in rat plasma after oral administration of green tea by liquid chromatography hybrid ion trap time-of-flight mass spectrometry

A liquid chromatography hybrid ion trap time-of-flight mass spectrometric (LC–IT-TOF-MS) method was developed and validated for identification and simultaneous determination of the potential bioactive components from green tea in rat plasma. The plasma samples were extracted by liquid–liquid extraction with ethyl acetate and separated on Shim-pack XR-ODS II column by a gradient elution within a runtime of 8.0min. The mobile phase consisted of A (0.1% formic acid in acetonitrile) and B (0.1% formic acid in water) at a flow rate of 0.4ml/min. Two prototype components and one metabolite were successfully identified as caffeine, theobromine and theophylline according to their retention times, accurate molecule weight, and major fragment ions. Then they were determined with the addition of two internal standards, hypoxanthine and paracetamol. The linear range was 10–10,000ng/ml for caffeine, 2.0–2000ng/ml for theobromine and 1.0–1000ng/ml for theophylline, respectively. Intra-day and inter-day precision were within 6.0% and 10.9%, and accuracy was less than 4.8% and 6.5%, respectively. The validated method was successfully applied to investigate the dynamic change rules of caffeine, theobromine and theophylline in rat plasma after oral administration of caffeine, theobromine and green tea extract. The comparative analysis of the pharmacokinetic parameters indicated that there were obvious differences between green tea extract administration and single substances administration.

Quantification of Lansoprazole in Oral Suspension by Ultra-High-Performance Liquid Chromatography Hybrid Ion-Trap Time-of-Flight Mass Spectrometry.

An LC-MS/MS method was developed and validated to be used as a stability indicating assay for the study of a 3 mg/mL lansoprazole oral suspension. The method utilizes a UPLC (ultra-performance liquid chromatography) column and unique mass spectrometric detection (ion-trap time-of-flight (IT-TOF)) to achieve a sensitive (LOD 2 ng/mL), accurate, and reproducible quantification of lansoprazole. This method reports an intraday and interday coefficient of variation of 2.98 ± 2.17% (n = 5 for each concentration for each day) and 3.07 ± 0.89% (n = 20 for each concentration), respectively. Calibration curves (5–25 μg/mL) were found to be linear with an R2 value ranging from 0.9972 to 0.9991 on 4 different days. Accuracy of the assay, expressed as % error, ranged from 0.30 to 5.22%. This method is useful for monitoring the stability of lansoprazole in oral suspension.

Microsomal Cytochrome P450-Mediated Metabolism of Protopanaxatriol Ginsenosides: Metabolite Profile, Reaction Phenotyping, and Structure-Metabolism Relationship

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.

Development of a Systematic Approach to Identify Metabolites for Herbal Homologs Based on Liquid Chromatography Hybrid Ion Trap Time-of-Flight Mass Spectrometry: Gender-Related Difference in Metabolism of SchisandraLignans in Rats

Metabolic research for herbal medicine (HM) is a formidable task, which is still in its infancy due to complicated components in HM, complex metabolic pathways, and lack of authentic standards. The present work contributes to the development of a powerful technical platform to rapidly identify and classify metabolites of herbal components based on a liquid chromatography hybrid ion trap time-of-flight mass spectrometry. Taking Schisandra lignans extract as an example, the metabolic studies were completed both in vitro and in vivo. In the in vitro study, metabolites for five representative Schisandra lignans were identified and structurally characterized. The major metabolic pathways were summed as demethylation, hydroxylation, and demethylation and hydroxylation. In the in vivo study, 44 metabolites were detected in rat urine. These metabolites were identified and classified rapidly according to the metabolic rules obtained in the in vitro studies, and hydroxylation was confirmed as the primacy metabolic pathway for lignans in rat urine. In addition, "relative cumulative excretion" (RCE) for the metabolites in female and male rats were calculated according to their relative intensities in the urine samples collected at 0 to 12, 12 to 24, and 24 to 36 h. As a result, great gender-related difference on RCE was observed. For most metabolites, RCE in female rats was significantly lower than that in male rats. In conclusion, the presently developed methodology and approach on metabolic research for Schisandra lignans will find its wide use in metabolic studies for herbal medicines.

Qualitative and quantitative determination of complicated herbal components by liquid chromatography hybrid ion trap time-of-flight mass spectrometry and a relative exposure approach to herbal pharmacokinetics independent of standards

To date, the pharmacokinetic research of herbal medicines (HMs) is still in its infancy and is facing critical technical challenges on the qualitative and quantitative analysis of complicated components from biological matrices. Additionally, the lack of authentic standards constitutes another bottleneck on assessing herbal pharmacokinetics. This present work contributes to the development of a powerful technical platform for both qualitative and quantitative pharmacokinetic analysis of herbal components, and a strategy of relative exposure that provides a practicable pharmacokinetic assessment independent of authentic standards, based on the use of liquid chromatography hybrid ion trap time-of-flight mass spectrometry (LC–IT-TOF/MS). Taking schisandra lignans extract (SLE) as an example, the LC–IT-TOF/MS assay was initially applied to the global qualitative analysis of components contained in SLE per se and in the rat plasma post SLE dosing. Afterwards, this study focused on validating the quantitative performance of LC–IT-TOF/MS assay by comparison with a well-established LC–Q/MS assay. For the absolute quantification of five lignans components with authentic standards, both assays showed very similar analytical figures of merit such as linearity, precision, accuracy, and pharmacokinetic parameters. Compared with LC–Q/MS, the prominent advantage of LC–IT-TOF/MS assay is its much higher sensitivity. Moreover, a ‘relative exposure approach’ (REA) that entails the use of sequentially diluted original herbal preparations to prepare the ‘mixed calibration curves’ was developed to assessing herbal pharmacokinetics independent of specific authentic compounds for each component. Such an approach was found capable of providing virtually identical pharmacokinetic parameters as that from the typical pharmacokinetic assay calibrated by authentic standards, except for the absolute plasma concentrations. The presently developed methodology and approach will find its wide use in, but not limited to, the qualitative and quantitative pharmacokinetic analysis of herbal medicines.

An approach to identifying sequential metabolites of a typical phenylethanoid glycoside, echinacoside, based on liquid chromatography–ion trap-time of flight mass spectrometry analysis

Metabolite identification for the compounds that undergo multiple and sequential metabolism is still a great challenge. Echinacoside (ECH), a typical phenylethanoid glycoside, contains multiple unstable chemical bonds and high reactive functional groups which are susceptible to multiple pathways of degradation and metabolism, leading great difficulties for its metabolite identification. This study proposed a novel approach for rapidly identifying the complicated and unpredictable metabolites of ECH, based on the powerful liquid chromatography hybrid ion trap and time of flight mass spectrometry (LC/MS-IT-TOF) analysis. Four degradation products were rapidly identified via the “fragmentation–degradation” comparisons. Five phase I and phase II metabolites of the degradation products were rapidly characterized via the crossover mass differences comparisons of their quasi-molecular ions with the potential precursors. Four direct phase I and phase II metabolites of the parent compound were identified by the mass differences analysis of the molecular ions between metabolites and the parent compound. Multiple stages of fragmentation patterns were used to confirm the metabolites characterizations. This study provides a novel approach to characterizing the complicated metabolites, and would be widely applicable for the metabolite identification of natural products.

Simultaneous characterization of quaternary alkaloids, 8-oxoprotoberberine alkaloids, and a steroid compound in Coscinium fenestratum by liquid chromatography hybrid ion trap time-of-flight mass spectrometry

Simultaneous characterization of quaternary alkaloids, 8-oxoprotoberberine alkaloids, and a steroid compound in Coscinium fenestratum was successfully performed by liquid chromatography hybrid ion trap time-of-flight mass spectrometry (LC/IT-TOF MS). A total of 32 compounds, including 2 benzylisoquinoline alkaloids, 3 aporphine alkaloids, 12 quaternary protoberberine alkaloids, 10 8-oxoprotoberberine alkaloids, 3 tetrahydroprotoberberine alkaloids, and a steroid compound were simultaneously separated and characterized by matching the empirical molecular formulae with those published in literature and the multi-stage mass spectrometry (MS n ) data obtained using structural information from IT, accurate mass measurement obtained from TOF MS, and HPLC separation. A total of 20 compounds, including 4 novel natural products were identified or tentatively identified for the first time from Coscinium fenestratum. In the positive-ion mode, 8-oxoprotoberberines produced [M + H] + and [M + Na] +; the fragmentation of 8-oxodihydroprotoberberines produced [M + H– CH 3] + , [M + H– CH 3– CH 3] +, and [M + H– CH 3– CH 3–CO] +, while 8-oxotetrahydroprotoberberines generated [M + H– CH 3] + , [M + H– CH 3– CH 3] +, [M + H– CH 3– H] +, and iminium ions from the cleavage of the protoberberine skeleton. The method can be applied for the analysis of 8-oxoberberine and other alkaloids in Coptis japonica, Phellodendron amurense, and other herbal medicines.

Global Detection and Identification of Nontarget Components from Herbal Preparations by Liquid Chromatography Hybrid Ion Trap Time-of-Flight Mass Spectrometry and a Strategy

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.