Characterization of alkaloids and phenolics in Nitraria roborowskii Kom. fruit by UHPLC-triple-TOF-MS/MS and its sucrase and maltase inhibitory effects.

Nitraria roborowskii Kom (NRK), with high economic and ecological value, is mainly distributed in the Qaidam Basin, China. However, research on its chemical components and bioactivities is still rare. In this study, its chemical constituents (52) including 10 ß-carboline alkaloids, nine cyclic peptides, three indole alkaloids, five pyrrole alkaloids, eight phenolic acids and 17 flavonoids were identified tentatively using UPLC-triple-TOF-MS/MS. Notablely, one new ß-carboline alkaloid and five new cyclic peptides were confirmed using MS/MS fragmentation pathways. In addition, experiments in vitro indicated that NRK-C had strong maltase and sucrase inhibitory activities (IC50 of 0.202 and 0.103 mg/mL, respectively). Polysaccharide tolerance experiments confirmed NRK-C (400 mg/kg) was associated with decreased postprandial blood glucose (PBG) in diabetic mice. These results suggested that NRK fruit might be used as a functional ingredient in food products.

Effect of Chemical Refining on the Reduction of ß-Carboline Content in Sesame Seed Oil.

ß-carbolines (harman and norharman) are potentially mutagenic and have been reported in some vegetable oils. Sesame seed oil is obtained from roasted sesame seeds. During sesame oil processing, roasting is the key procedure to aroma enhancement, in which ß-carbolines are produced. Pressed sesame seed oils cover most market share, while leaching solvents are used to extract oils from the pressed sesame cake to improve the utilization of the raw materials. ß-carbolines are nonpolar heterocyclic aromatic amines with good solubility in leaching solvents (n-hexane); therefore, the ß-carbolines in sesame cake migrated to the leaching sesame seed oil. The refining procedures are indispensable for leaching sesame seed oil, in which some small molecules can be reduced. Thus, the critical aim is to evaluate the changes in ß-carboline content during the refining of leaching sesame seed oil and the key process steps for the removal of ß-carbolines. In this work, the levels of ß-carbolines (harman and norharman) in sesame seed oil during chemical refining processes (degumming, deacidification, bleaching and deodorization) have been determined using solid phase extraction and high performance liquid chromatography-mass spectrometry (LC-MS). The results indicated that in the entire refining process, the levels of total ß-carbolines greatly decreased, and the adsorption decolorization was the most effective process in reducing ß-carbolines, which might be related to the adsorbent used in the decolorization process. In addition, the effects of adsorbent type, adsorbent dosage and blended adsorbent on ß-carbolines in sesame seed oil during the decolorization process were investigated. It was concluded that oil refining can not only improve the quality of sesame seed oil, but also reduce most of the harmful ß-carbolines.

ß-Carboline Alkaloids in Soy Sauce and Inhibition of Monoamine Oxidase (MAO).

Monoamine oxidase (MAO) oxidizes neurotransmitters and xenobiotic amines, including vasopressor and neurotoxic amines such as the MPTP neurotoxin. Its inhibitors are useful as antidepressants and neuroprotectants. This work shows that diluted soy sauce (1/3) and soy sauce extracts inhibited human MAO-A and -B isozymes in vitro, which were measured with a chromatographic assay to avoid interferences, and it suggests the presence of MAO inhibitors. Chromatographic and spectrometric studies showed the occurrence of the ß-carboline alkaloids harman and norharman in soy sauce extracts inhibiting MAO-A. Harman was isolated from soy sauce, and it was a potent and competitive inhibitor of MAO-A (0.4 µM, 44 % inhibition). The concentrations of harman and norharman were determined in commercial soy sauces, reaching 243 and 52 µg/L, respectively. Subsequently, the alkaloids 1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid (THCA) and 1-methyl-1,2,3,4-tetrahydro-ß-carboline-3-carboxylic acid (MTCA) were identified and analyzed in soy sauces reaching concentrations of 69 and 448 mg/L, respectively. The results show that MTCA was a precursor of harman under oxidative and heating conditions, and soy sauces increased the amount of harman under those conditions. This work shows that soy sauce contains bioactive ß-carbolines and constitutes a dietary source of MAO-A and -B inhibitors.

Evaluation of the In Vitro Wound-Healing Potential of Ayahuasca.

Ayahuasca is an Amazonian drink, which contains ß-carboline alkaloids and N,N-dimethyltryptamine. The aim of this study was to evaluate the healing potential of decoctions of a commercial mixture, four individual plants and four mixtures of two plants used in the ayahuasca preparation. Thus, the cytotoxic potential of the samples was evaluated and a wound-healing assay was performed with a NHDF cell line. Subsequently, a parallel artificial membrane permeability assay was also performed, to verify if any psychoactive compound could be absorbed by skin fibroblasts. The integrity and permeability of the cell layer were also evaluated, using the transepithelial electrical resistance assay and Lucifer yellow permeability assay, respectively. The compounds absorbed by the cell layer were quantified by high-performance liquid chromatography coupled to a diode array detector. The results showed that only one sample showed cytotoxicity and all the others promoted the migration of skin fibroblasts. Additionally, it was also verified that ß-carbolynic alkaloids and N,N-dimethyltriptamine were not absorbed by the cell layer, and in general, did not interfere with its permeability and integrity. To the best of our knowledge, this is the first study where ayahuasca's wound-healing potential was evaluated.

Ayahuasca Lyophilization (Freeze-drying) Protocol with Pre- and Post-procedure Alkaloids Quantification.

Ayahuasca is a psychoactive brew from the decoction of different Amazonian plants, traditionally used in several cultures, religions, and rituals. Scientific studies with ayahuasca are rapidly increasing due to its subjective effects and therapeutic potential. Although ayahuasca is traditionally used in its liquid presentation, lyophilized (freeze-dried) ayahuasca is often used in scientific experimentation settings. However, there is no standard process or guideline to freeze-dry ayahuasca nor comparison of the chemical profile between the liquid and freeze-dried presentations. Therefore, we describe a reproducible five-day protocol for ayahuasca lyophilization with alkaloids quantification by liquid chromatography coupled to tandem mass spectrometry of both the liquid and the final freeze-dried ayahuasca. By the end of the protocol, approximately 295 g of freeze-dried extract with similar alkaloids concentration were obtained from two liters of ayahuasca (dry matter: 14.75 %). The final extract was stored for three years inside a vacuum desiccator (approximately 6°C) with its texture quality preserved. Further studies should address the impact of different storage conditions and the lyophilization on the alkaloids' quantity of the freeze-dried ayahuasca, especially the use of heat in regards to the ß-carbolines.

Identification of Compounds with Potential Therapeutic Uses from Sweet Pepper (Capsicum annuum L.) Fruits and Their Modulation by Nitric Oxide (NO).

Plant species are precursors of a wide variety of secondary metabolites that, besides being useful for themselves, can also be used by humans for their consumption and economic benefit. Pepper (Capsicum annuum L.) fruit is not only a common food and spice source, it also stands out for containing high amounts of antioxidants (such as vitamins C and A), polyphenols and capsaicinoids. Particular attention has been paid to capsaicin, whose anti-inflammatory, antiproliferative and analgesic activities have been reported in the literature. Due to the potential interest in pepper metabolites for human use, in this project, we carried out an investigation to identify new bioactive compounds of this crop. To achieve this, we applied a metabolomic approach, using an HPLC (high-performance liquid chromatography) separative technique coupled to metabolite identification by high resolution mass spectrometry (HRMS). After chromatographic analysis and data processing against metabolic databases, 12 differential bioactive compounds were identified in sweet pepper fruits, including quercetin and its derivatives, L-tryptophan, phytosphingosin, FAD, gingerglycolipid A, tetrahydropentoxylin, blumenol C glucoside, colnelenic acid and capsoside A. The abundance of these metabolites varied depending on the ripening stage of the fruits, either immature green or ripe red. We also studied the variation of these 12 metabolites upon treatment with exogenous nitric oxide (NO), a free radical gas involved in a good number of physiological processes in higher plants such as germination, growth, flowering, senescence, and fruit ripening, among others. Overall, it was found that the content of the analyzed metabolites depended on the ripening stage and on the presence of NO. The metabolic pattern followed by quercetin and its derivatives, as a consequence of the ripening stage and NO treatment, was also corroborated by transcriptomic analysis of genes involved in the synthesis of these compounds. This opens new research perspectives on the pepper fruit's bioactive compounds with nutraceutical potentiality, where biotechnological strategies can be applied for optimizing the level of these beneficial compounds.

Kinetic profile of N,N-dimethyltryptamine and ß-carbolines in saliva and serum after oral administration of ayahuasca in a religious context.

Ayahuasca is a beverage obtained from Banisteriopsis caapi plus Psychotria viridis. B. caapi contains the ß-carbolines harmine, harmaline, and tetrahydroharmine that are monoamine oxidase inhibitors and P. viridis contains N,N-dimethyltryptamine (DMT) that is responsible for the visionary effects of the beverage. Ayahuasca use is becoming a global phenomenon, and the recreational use of DMT and similar alkaloids has also increased in recent years; such uncontrolled use can lead to severe intoxications. In this investigation, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to study the kinetics of alkaloids over a 24 h period in saliva and serum of 14 volunteers who consumed ayahuasca twice a month in a religious context. We compared the area under the curve (AUC), maximum concentration (Cmax ), time to reach Cmax (Tmax ), mean residence time (MRT), and half-life (t1/2 ), as well as the serum/saliva ratios of these parameters. DMT and ß-carboline concentrations (Cmax ) and AUC were higher in saliva than in serum and the MRT was 1.5-3.0 times higher in serum. A generalized estimation equations (GEEs) model suggested that serum concentrations could be predicted by saliva concentrations, despite large individual variability in the saliva and serum alkaloid concentrations. The possibility of using saliva as a biological matrix to detect DMT, ß-carbolines, and their derivatives is very interesting because it allows fast noninvasive sample collection and could be useful for detecting similar alkaloids used recreationally that have considerable potential for intoxication.

ß-carbolines norharman and harman in vegetable oils in China.

The beta-carbolines norharman and harman, two heterocyclic aromatic amines with potential mutagenicity, have been determined in vegetable oils. Identification and analysis were carried out by ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS/MS). In 88 samples analysed, the concentrations of norharman and harman were < LOD to 336.22 ng/g and < LOD to 505.14 ng/g, respectively. A high variability of norharman and harman levels among different oil types was observed. Sesame-, flaxseed-, sunflower seed-, peanut- and rapeseed oils were most contaminated. Both ß-carbolines were most likely formed during roasting of the oilseeds. Oil consumption, especially of oils obtained after roasting of the seeds, was a major dietary source of the ß-carbolines norharman and harman. Under existing oil risk factors, this investigation contributes to the unprecedented and essential information for dietary assessments associated with oil consumption.

Bioactive ß-Carbolines in Food: A Review.

Harman and norharman, two neuroactive ß-carbolines, are present in several plants and in thermally processed foods. They exhibited a wide spectrum of biological and pharmacological effects, including antioxidant, neuroprotective, and anti-inflammatory effects. In this article, we review the progress of recent research on the presence of these compounds in food, as well as their various biological and neuroactive properties. Our findings strongly suggest that some foods, especially coffee, can act as a rich source of ß-carbolines, which may possibly be associated with a reduced risk for serious neurodegenerative diseases, such as Parkinson's and Alzheimer's.

Use of liquid chromatography quadrupole time-of-flight mass spectrometry and metabolomic approach to discriminate coffee brewed by different methods.

An untargeted metabolomic method based on UPLC-QTOF were used to investigate the differences in coffee brewed by boiled, pour-over and cold-brew methods here. Distinctive separation among the three groups could be seen from principal component analysis and hierarchical clustering analysis. Analysis of variance, fold change and orthogonal projection to latent structures discriminant mode were conducted to find the characteristic potential markers, subsequently, nine potential markers were putatively identified using general chemical databases, and five of them were further confirmed by acquisition of reference standards. This work provides an efficient way for discrimination of coffee brewed by different methods. Interestingly, the result of this work also suggested that the contents of two selected markers, norharman and harman, were higher in the pour-over and boiled methods, compared to the cold-brew method. This content difference were further verified by the quantitative analysis data of commercial coffee samples.

Large-Scale Screening of Foods for Glucose-Derived ß-Carboline Alkaloids by Stable Isotope Dilution LC-MS/MS.

The occurrence of glucose-derived ß-carboline alkaloids tangutorid E (Tan E) and tangutorid F (Tan F) as well as their dehydroxy-derivatives (DH-Tan E/F) was investigated in a broad variety of foodstuffs by LC-MS/MS-based stable isotope dilution analysis (SIDA). For that purpose, the target compounds and their 13C6-stable isotope-labeled analogues were synthesized from l-tryptophan and (13C6-)d-glucose and used to develop a rapid LC-MS/MS-SIDA method. After validation for several food matrices, the method was applied to the analysis of these ß-carbolines in 80 food items. Quantitative amounts were detected in 46.3, 50.0, and 42.5% of the samples regarding Tan E, Tan F, and DH-Tan E/F, respectively, with corresponding ranges of 0.01-6.75, 0.01-5.07, and 0.01-0.75 mg/kg; the highest amounts were found in processed tomato products. A combination of the obtained occurrence data in foods with average food consumption data led to the calculation of rough estimates for the chronic daily intake of those alkaloids, yielding values of 0.44, 0.36, and 0.13 µg/kg body weight/day for Tan E, Tan F, and DH-Tan E/F, respectively. Evidently, the consumption of processed tomato-based products accounts for the majority of the total daily intake of the investigated ß-carbolines; the potential bioactivities of Tan E, Tan F, and DH-Tan E/F have yet to be investigated.

High-Performance Thin-Layer Chromatography Method for Simultaneous Determination of Antipsychotic and Medicinally Important Five ß-Carboline Alkaloids.

This is the first report on the development and validation of high-performance thin-layer chromatography (HPTLC) method for simultaneous analysis of five antipsychotic and medicinally important ß-carboline alkaloids (ßCAs), namely, harmalol, harmaline, harmine, harmane and norharmane. These ßCAs occurs in both plant and animal system including human being. In the present investigation, their best separation was achieved using an optimized mobile phase, chloroform: methanol: glacial acetic acid (7.8:2.2:0.2, v/v/v) on aluminum TLC plates precoated with silica gel 60 F254. The quantification was performed by densitometric scanning in fluorescence mode at 366 nm. The calibration curves were drawn using linear regression, plotted over the range 25-250 ng band-1 of standard ßCAs with correlation coefficient (R2) between 0.97 and 0.992. Accuracy in terms of recovery (83.95-112.40%), repeatability of application (0.61-2.42%), repeatability of measurement (1.94-3.05%) and intermediate precision (0.62-11.16%) of developed method were simultaneously determined. The limit of detection and limit of quantification were between 4.95-6.59 and 16.50-21.93 ng band-1, respectively. The method was validated according to ICH guidelines and was simple, cost-effective, precise, sensitive and specific for the determination of ßCAs in herbs, Fagonia schweinfurthii, Peganum harmala and Tribulus terrestris. The developed HPTLC method would have importance in forensic and industrial chromatographic analysis and fingerprinting of various herbs and drug formulations containing ßCAs.

Spatio-Temporal Variation of Alkaloids in Annona purpurea and the Associated Influence on Their Antifungal Activity.

Annona purpurea grows in the areas of low elevation in deciduous forests of Mexico, those areas have marked rainy and dry seasons. This species produces more than 30 bioactive alkaloids that could have potential in the control of phytopathogens. This research provides data on the variation of the content and number of alkaloids during an annual cycle and the associated inhibitory potential of the compounds against three phytopathogenic fungi. For one year, alkaloidal extracts of stems and leaves were obtained every two months. The extract profiles were determined by gas chromatography with tandem mass spectrometry and their antifungal activity was examined in vitro. The alkaloids, annomontine and oxopurpureine, obtained from the roots and leaves, respectively, were also evaluated individually. The yields, profiles and activities of the extracts, as well as the abundance of annomontine and oxopurpureine in the extracts, were contrasted with the seasonality and phenological phases of the plant. The data indicate that the alkaloid content was higher at the height of the dry season. High yields also occurred during flowering. The strongest inhibitory effect was obtained from the root extracts during the last month of dry season. This finding seems to be explained by the higher chemodiversity of alkaloids in extracts from this season. Annomontine and oxopurpureine inhibited all three phytopathogens; however, they were not solely responsible for the activity of A. purpurea.

Quality Assessment of Kumu Injection, a Traditional Chinese Medicine Preparation, Using HPLC Combined with Chemometric Methods and Qualitative and Quantitative Analysis of Multiple Alkaloids by Single Marker.

Kumu injection (KMI) is a common-used traditional Chinese medicine (TCM) preparation made from Picrasma quassioides (D. Don) Benn. rich in alkaloids. An innovative technique for quality assessment of KMI was developed using high performance liquid chromatography (HPLC) combined with chemometric methods and qualitative and quantitative analysis of multi-components by single marker (QAMS). Nigakinone (PQ-6, 5-hydroxy-4-methoxycanthin-6-one), one of the most abundant alkaloids responsible for the major pharmacological activities of Kumu, was used as a reference substance. Six alkaloids in KMI were quantified, including 6-hydroxy-ß-carboline-1-carboxylic acid (PQ-1), 4,5-dimethoxycanthin-6-one (PQ-2), ß-carboline-1-carboxylic acid (PQ-3), ß-carboline-1-propanoic acid (PQ-4), 3-methylcanthin-5,6-dione (PQ-5), and PQ-6. Based on the outcomes of twenty batches of KMI samples, the contents of six alkaloids were used for further chemometric analysis. By hierarchical cluster analysis (HCA), radar plots, and principal component analysis (PCA), all the KMI samples could be categorized into three groups, which were closely related to production date and indicated the crucial influence of herbal raw material on end products of KMI. QAMS combined with chemometric analysis could accurately measure and clearly distinguish the different quality samples of KMI. Hence, QAMS is a feasible and promising method for the quality control of KMI.

Quantitation of ß-carboline and quercetin in alligator weed (Alternanthera philoxeroides (Mart.) Griseb.) by LC-MS/MS and evaluation of cardioprotective effects of the methanol extracts.

Plant invasion is one of the major threats to natural ecosystems. The alligator weed grows rapidly within a small span of time and is easily available all over the world. ß-Carboline and quercetin are considered as excellent bioactive components of the alligator weed. In our study LC-MS/MS methods were performed for the detection and determination of the bioactive constituents, ꞵ-carboline and quercetin in leaves, in multiple reaction monitoring (MRM) mode. The effects of methanol extract on cardiomyocyte apoptosis induced by doxorubicin using H9c2 cells were evaluated by MTT assay and Annexin V-FITC/PI staining assay. A sensitive and selective liquid chromatography tandem mass spectrometry was developed and validated for the determination of ꞵ-carboline and quercetin in this plant. According to in vitro cell evaluation experiments, methanol extracts significantly prevented cardiomyocyte apoptosis induced by doxorubicin.

Trace anti-inflammatory ß-carboline alkaloid identified in Arenaria kansuensis by two-dimensional chromatography coupled with UniElut C18AEX based solid-phase extraction re-enrichment technology.

Traditional Chinese medicine is important for discovery of drug precursors. However, information about trace chemical composition of them is very limited due to the lack of appropriate enrichment and chromatographic purification methods In our work, A. kansuensis was taken as an example, a novel two-dimensional reversed-phase/hydrophilic interaction liquid chromatography coupled with UniElut C18AEX solid-phase extraction re-enrichment method based on anti-inflammatory bioactivity-guided assay was developed for gathering and purifying trace ß-carboline alkaloids with high purity from the ethyl acetate extract of A. kansuensis. Extraction with ethyl acetate as the first enrichment method, then, a UniElut C18AEX column was employed to re-enrich trace fraction which was hardly detected by diode array detector during high performance liquid chromatography analysis, eighteen grams of UniElut C18AEX was used as sorbent material to pack a 60mL pipette tip for the extraction of ß-carboline alkaloids from 100mL of ethyl acetate sample. The whole extraction process was finished in 10min, and the volume of eluent used was only 120mL. The enriching fraction (100mg) was used for the following two-dimensional purification. First-dimensional preparation was carried on a RP-Megress-C18 prep column, and four anti-inflammatory fractions were obtained from the 100mg re-enriching sample with a recovery of 66.9%. A HILIC-XAmide prep column was selected for the second dimensional preparation. Finally, two pair of analogue ß-carboline alkaloids and one other ß-carboline alkaloid were purified from A. kansuensis. The purity of the isolated compounds was ≫>98%, which indicated that the method was efficient to re-enrich and manufacture single trace ß-carboline alkaloids with high purity from A. kansuensis. Additionally, this method showed great potential to serve as a good example for the purification and enrichment of analogue structure anti-inflammation carboline alkaloids from other plant materials.

A self-feedback network based on liquid chromatography-quadrupole-time of flight mass spectrometry for system identification of ß-carboline alkaloids in Picrasma quassioides.

Profiling chemical components in herbs by mass spectrometry is a challenging work because of the lack of standard compounds, especially for position isomers. This paper provides a strategy based on a self-feedback network of mass spectra (MS) data to identify chemical constituents in herbs by liquid chromatography-quadrupole-time of flight mass spectrometry without compound standards. Components sharing same skeleton were screened and all ions were classified into a database. All candidates were connected by the selected bridging ions to establish a primary MS network. Benefited from such a network, it is feasible to characterize sequentially the structures of all diagnostic ions and candidates once single component has been de novo identified. Taking Picrasma quassioides as an example, the primary network of ß-carbolines was established with 65 ions (selected from 76 ß-carbolines), each of which appeared at least in four compounds. Once an alkaloid has been identified, its logical ions could feedback into primary network to build pathways with other unknown compounds. Moreover, the position of the substituent groups could be deduced through the secondary metabolic pathways of alkaloids (plant secondary metabolism). The network therefore can be utilized for identification of unknown compounds and even their position isomers.

A rapid and simple method for the determination of psychoactive alkaloids by CE-UV: application to Peganum Harmala seed infusions.

The ß-carboline alkaloids of the harmala (HAlks) group are compounds widely spread in many natural sources, but found at relatively high levels in some specific plants like Peganum harmala (Syrian rue) or Banisteriopsis caapi. HAlks are a reversible Mono Amino Oxidase type A Inhibitor (MAOI) and, as a consequence, these plants or their extracts can be used to produce psychotropic effects when are combined with psychotropic drugs based on amino groups. Since the occurrence and the levels of the HAlks in natural sources are subject to significant variability, more widespread use is not clinical but recreational or ritual, for example B. caapi is a known part of the Ayahuasca ritual mixture. The lack of simple methods to control the variable levels of these compounds in natural sources restricts the possibilities to dose in strict quantities and, as a consequence, limits its use with pharmacological or clinical purposes. In this work, we present a fast, simple, and robust method of quantifying simultaneously the six HAlks more frequently found in plants, i.e., harmine, harmaline, harmol, harmalol, harmane, and norharmane, by capillary electrophoresis instruments equipped with the more common detector UV. The method is applied to analyze these HAlks in P. Harmala seeds infusion which is a frequent intake form for these HAlks. The method is validated in three different instruments in order to evaluate the transferability and to compare the performances between them. In this case, harmaline, harmine, and harmol were found in the infusion samples. Copyright © 2016 John Wiley & Sons, Ltd.

Phytoelectrochemical analysis of Zanthoxylum chiloperone.

INTRODUCTION: An innovative application of the voltammetry of microparticles methodology to characterize the phytochemical composition of extracts of different parts of Zanthoxylum chiloperone var. angustifolium Engl. is described. OBJECTIVE: Characterize the phytochemical composition of extracts of different parts of plants by electrochemical methodologies. METHODS: The voltammetry of microparticles methodology was applied to alcoholic extracts from leaves, seeds, fruits, roots and stem bark of Zanthoxylum chiloperone. RESULTS: In contact with aqueous phosphate buffer, characteristic cathodic signals of its main natural products (canthin-6-one, 5-methoxycanthin-6-one and trans-avicennol) were recorded. The study of the voltammograns allows the estimation of the relative amounts of canthin-6-one, 5-methoxycanthin-6-one and trans-avicennol from the different parts of Zanthoxylum chiloperone. CONCLUSION: The voltammetric responses of alcoholic extracts from different parts of Zanthoxylum chiloperone var. angustifolium allows their phytochemical characterization without need of sample pretreatment thus illustrating the capabilities of the voltammetry of microparticles methodology to increase the tools applied to phytochemical analysis. Copyright © 2016 John Wiley & Sons, Ltd.

Metabolic profile of 5-hydroxy-4-methoxycanthin-6-one, a typical canthinone alkaloid, in rats determined by liquid chromatography-quadrupole time-of-flight tandem mass spectrometry together with multiple data processing techniques.

Picrasma quassioides (D. Don) Benn. is a traditional Chinese medicine used clinically to treat gastrointestinal disorders and as a vermifuge. 5-Hydroxy-4-methoxycanthin-6-one (CAN), a major canthinone alkaloid found in P. quassioides, has significant pharmacological activities. In the present study, a method using liquid chromatography-quadrupole time-of-flight tandem mass spectrometry together with multiple data processing techniques, including extracted ion chromatogram, multiple mass defect filter, precursor/product ion scanning and neutral loss scanning was developed to screen and characterize the phase I and II metabolites of CAN in plasma, bile, urine and feces of rats after a single oral dose of 20mg/kg. A total of 17 metabolites were tentatively or conclusively identified. Pathways for the metabolism of CAN have been proposed, and include hydroxylation, N-decarbonylation, methylation, oxidation and sequential conjugation. A previously unknown metabolically active site at the C4-C6 position and a novel N-decarbonylation-oxidation metabolic pathway for the prototypical canthinone alkaloid, CAN, were discovered. Our results provide valuable information about the in vivo metabolism of CAN that can also be used as a comprehensive guide for the biotransformation of other canthinone alkaloids.