Laboratory for Pharmaceutical Biotechnology, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium.
This study reports the development and validation of a rapid, sensitive and selective assay for the quantitation of artemisinin, arteannuin B, artemisitene and artemisinic acid in Artemisia annua L. by reversed phase high performance liquid chromatography (HPLC) electrospray (ESI) quadrupole time of flight (Q-TOF) tandem mass spectrometry (MS/MS). A recovery of >97% for all analytes was achieved by immersing one gram of fresh plant material in chloroform for 1min. This result supports the hypothesis that artemisinin and some of its structural analogs present in the leaves A. annua L. are localized entirely in the subcuticular space of the glands on the surface of the leaves. We validated the use of this chloroform extract, without additional sample preparation steps, for quantitative Q-TOF MS/MS. No ion suppression (matrix effect) resulting from interference with other compounds was detected. For every concentration within the range of the standard curve (0.1 to 3.00mug/ml), accuracy was between 85% and 115%. Within- and between-day variations for the analysis of A. annua L. samples were <20%.

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College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea.
The adulteration of dietary supplements with drugs is potentially dangerous for human health. In this study, a method was used to test simultaneously for the presence of three synthetic PDE-5 inhibitors (sildenafil, vardenafil and tadalafil), and sibutramine and its two major metabolites (N-desmethylsibutramine and N-didesmethylsibutramine) using ultra-performance liquid chromatography (UPLC) coupled with quadrupole-time-of-flight mass spectrometry (Q-TOF MS) in dietary supplements. This approach with UPLC/Q-TOF MS uses the high accurate mass of six compounds for identification and has a short run time. The recovery was from 87% to 113%; precision was less than 12.8%. The limit of detection and limit of quantification were from 0.4 to 2.0 µg kg(-1) and from 1.3 to 6.0 µg kg(-1), respectively. This method allows easy and fast analysis and detection of diverse adulterants.
Department of Pharmaceutical Botany, Faculty of Pharmacy, University of Medicine and Pharmacy Gr. T. Popa, 16 Universitatii Street, 700150, Iasi, Romania. biancaivanescu@yahoo.com
Artemisinin, a sesquiterpene lactone from Artemisia annua L., has received considerable attention in the last few decades as a potent antimalarial drug. Artemisinin has rather low toxicity; it is effective against drug-resistant Plasmodium species and against cerebral malaria. This study reports the development of a rapid and sensitive assay for the quantification of artemisinin in A. annua by reversed phase HPLC/MS. In the selected optimal experimental conditions, artemisinin exhibited a well-defined chromatographic peak with a retention time of 2 ± 0.2 min. The chromatographic signal shows a linear dependence with artemisinin concentration, enabling the use of this signal for artemisinin quantification according to the following regression equation: y = 2665.40x - 14697.61. The correlation coefficient (R(2)) was 0.9989. For every concentration within the range of the standard curve (0.1-2 µg mL(-1)), accuracy was between 95 and 104%. Artemisinin content in Romanian A. annua wild plants varies between 0.17 and 0.21%(dry weight basis).
Department of Plant Systems Biology, VIB, Gent, Belgium.
• Biosynthesis of the sesquiterpene lactone and potent antimalarial drug artemisinin occurs in glandular trichomes of Artemisia annua plants and is subjected to a strict network of developmental and other regulatory cues. • The effects of three hormones, jasmonate, gibberellin and cytokinin, were studied at the structural and molecular levels in two different A. annua chemotypes by microscopic analysis of gland development, and by targeted metabolite and transcript profiling. Furthermore, a genome-wide cDNA-amplified fragment length polymorphism (AFLP)-based transcriptome profiling was carried out of jasmonate-elicited leaves at different developmental stages. • Although cytokinin and gibberellin positively affected at least one aspect of gland formation, these two hormones did not stimulate artemisinin biosynthesis. Only jasmonate simultaneously promoted gland formation and coordinated transcriptional activation of biosynthetic gene expression, which ultimately led to increased sesquiterpenoid accumulation with chemotype-dependent effects on the distinct pathway branches. Transcriptome profiling revealed a trichome-specific fatty acyl- coenzyme A reductase, trichome-specific fatty acyl-CoA reductase 1 (TFAR1), the expression of which correlates with trichome development and sesquiterpenoid biosynthesis. • TFAR1 is potentially involved in cuticular wax formation during glandular trichome expansion in leaves and flowers of A. annua plants. Analysis of phytohormone-modulated transcriptional regulons provides clues to dissect the concerted regulation of metabolism and development of plant trichomes.
Division of Pharmacognosy, Section of Metabolomics, Institute of Biology, Leiden University, PO Box 9502, 2300RA Leiden, The Netherlands.
Introduction - Since the discovery of artemisinin in the 1970s, many techniques based on diverse chromatography techniques have been developed to detect and quantify this important antiplasmodial compound. The accurate quantification of this compound in the Artemisia annua plant material is mainly needed for breeding purposes in order to cultivate higher yielding varieties. It is also important for the quality control of herbal preparations containing A. annua plant material.Objective - To evaluate the most common validated quantification techniques (LC-MS, HPLC-ELSD and TLC) and compare the results to quantitative nuclear magnetic resonance spectroscopy (qNMR) in eight different A. annua samples collected from around the world.Methodology - The leaf material were extracted according to standard procedures and analysed with the validated quantification techniques. For the qNMR analysis we did not employ a standard curve but instead used an internal standard (maleid acid) which is not chemically related to artemisinin.Results - We found a significant difference between the results in this study. Compared with the qNMR results the HPLC-ELSD corresponded closely, followed by LC-MS. Quantitation with TLC led to an estimation range of -0.5 to +3.2 mg artemisinin/g of A. annua.Conclusion - These results imply that qNMR, with the addition of an internal standard, can be used to quantify artemisinin in A. annua samples in a rapid and reproducible manner. Copyright (c) 2010 John Wiley & Sons, Ltd.
The compound naphthoquine phosphate is a novel antimalaria drug tablet containing a fixed-dose combination of naphthoquine phosphate and artemisinin in a 1:2.5 ratio. A randomized, open study on the safety and tolerability was conducted in 28 healthy male volunteers using a single oral dose of 350 mg, 700 mg, 1400 mg, or 2100 mg of artemisinin-naphthoquine phosphate. Pharmacokinetics at the last 3 doses were examined in 30 volunteers. Food effects were also determined. Serial blood samples up to 216 hours after single oral dose administration were analyzed for plasma concentrations using a validated high-performance liquid chromatography-tandem mass spectrometry assay. The compound was well tolerated at single doses up to 2100 mg. Increased exposure to naphthoquine phosphate and artemisinin was less than dose proportional and linear. The half-life of naphthoquine phosphate was approximately 255 hours. The combination increased the AUC0-t and Cmax of both artemisinin (by 71% and 49%) and naphthoquine phosphate (by 135% and 104%) compared with mono-therapy. Food intake greatly increased the AUC0-t of artemisinin with a ratio of 77% and reduced that of naphthoquine phosphate from 955 +/- 352 mug.h/L under the fasted state to 446 +/- 231 mug.h/L in the fed condition. The pharmacokinetics and safety profile of the drug support its continued investigation in future clinical studies.
Clinton Foundation, 383 Dorchester Ave., Suite 400, Boston, MA 02127, USA.
A barrier to the development of artemisinin derivative based combination treatment of malaria is the lack of defined specifications and purity test methods for the raw material artemisinin. An HPLC method previously published in the International Pharmacopoeia to evaluate purity of artemisinin as an active pharmaceutical ingredient is adapted for use. Excellent method precision and linearity are demonstrated along with observations of robustness. In support of the development of specifications major impurities are identified using high resolution HPLC-MS, isolation via preparative HPLC followed by NMR. The identified impurities differ from those previously claimed.
Department of Pharmaceutical Biology, GUIDE, University of Groningen, The Netherlands.
Terpenoids belong to the largest class of natural compounds and are produced in all living organisms. The isoprenoid skeleton is based on assembling of C5 building blocks, but the biosynthesis of a great variety of terpenoids ranging from monoterpenoids to polyterpenoids is not fully understood today. Terpenoids play a fundamental role in human nutrition, cosmetics, and medicine. In the past 10 years, many metabolic engineering efforts have been undertaken in plants but also in microorganisms to improve the production of various terpenoids like artemisinin and paclitaxel. Recently, inverse metabolic engineering and combinatorial biosynthesis as main strategies in synthetic biology have been applied to produce high-cost natural products like artemisinin and paclitaxel in heterologous microorganisms. This review describes the recent progresses made in metabolic engineering of the terpenoid pathway with particular focus on fundamental aspects of host selection, vector design, and system biotechnology.
Key Laboratory of Modern Chinese Medicines (China Pharmaceutical University), Ministry of Education, Nanjing 210009, China.
The fact that the effects of herbal medicines (HMs) are brought about by their chemical constituents has created a critical demand for powerful analytical tools performing the chemical analysis to assure their efficacy, safety and quality. Liquid chromatography coupled to mass spectrometry (LC-MS) is an excellent technique to analyze multi-components in complex herbal matrices. Due to its inherent characteristics of accurate mass measurements and high resolution, time-of-flight (TOF) MS is well-suited to this field, especially for qualitative applications. The purpose of this article is to provide an overview on the potential of TOF, including the hybrid quadrupole- and ion trap-TOF (QTOF and IT-TOF), hyphenated to LC for chemical analysis in HMs or HM-treated biological samples. The peculiarities of LC-(Q/IT)TOF-MS for the analysis of HMs are discussed first, including applied stationary phase, mobile-phase selection, accurate mass measurements, fragmentation and selectivity. The final section is devoted to describing the applicability of LC-(Q/IT)TOF-MS to routine analysis of multi-components, including target and non-target (unknown) compounds, in herbal samples, emphasizing both the advantages and limitations of this approach for qualitative and quantitative purposes. The potential and future trends of fast high-performance liquid chromatography (HPLC)(e.g. rapid resolution LC and ultra-performance LC) coupled to (Q)TOF-MS for chemical analysis of HMs are highlighted.
Natural Products Research Center, Hunan Agricultural University, Changsha 410128, People's Republic of China.
Malaria threatens 300-500 million people and kills more than one million people annually. Artemisinin has been widely used as part of the artemisinin-based combination therapies against malaria. However, its supply is seriously short due to very small amounts of production of artemisinin in Artemisia annua. Molecular biologic researches aimed at increasing the artemisinin yield in plant have received more and more attention and therefore corresponding quantification methods for artemisinin analysis are urgently needed. A variety of methods for determination of artemisinin have been developed but they cannot be applied when only very little plant material is available or the material should be kept live, which often occurs in molecular biologic researches. The present work developed a simple, fast and low toxic micro-scale analysis procedure for determination of artemisinin in a single leaf or flower of living Artemisia annua using improved gas chromatography with electron-capture detection. The recovery of >95% was achieved by vortex of a piece of fresh leaf in 1 mL ethyl acetate for 2 min at room temperature. This method provides a powerful tool for biosynthesis study of artemisnin, high-throughput screening high-yield clone in an early stage, or real-time quality control of Artemisia annua crop.
Artemisinin is a polycyclic sesquiterpene lactone that is highly effective against multidrug-resistant strains of Plasmodium falciparum, the etiological agent of the most severe form of malaria. Determination of artemisinin in the source plant, Artemisia annua, is a challenging problem since the compound is present in very low concentrations, is thermolabile and unstable, and lacks chromophoric or fluorophoric groups. The ain of this study was to develop a simple protocol for the quantification of artemisinin in a plant extract using an (1)H-NMR method. Samples were prepared by extraction of leaf material with acetone, treatment with activated charcoal to remove chlorophylls and removal of solvent.(1)H-NMR spectra were measured on samples dissolved in deuterochloroform with tert-butanol as internal standard. Quantification was carried out using the $\d{$\delta$}$ 5.864 signal of artemisinin and the delta 1.276 signal of tert-butanol. The method was optimised and fully validated against a reference standard of artemisinin. The results were compared with those obtained from the same samples quantified using an HPLC-refractive index (RI) method. The (1)H-NMR method gave a linear response for artemisinin within the range 9.85-97.99 mm (r(2)= 0.9968). Using the described method, yields of artemisinin in the range 0.77-1.06% were obtained from leaves of the A. annua hybrid CPQBA x POP, and these values were in agreement with those obtained using an HPLC-RI. Copyright (c) 2008 John Wiley & Sons, Ltd.

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Department of Plant Systems Biology, VIB, Gent, Belgium.
• Biosynthesis of the sesquiterpene lactone and potent antimalarial drug artemisinin occurs in glandular trichomes of Artemisia annua plants and is subjected to a strict network of developmental and other regulatory cues. • The effects of three hormones, jasmonate, gibberellin and cytokinin, were studied at the structural and molecular levels in two different A. annua chemotypes by microscopic analysis of gland development, and by targeted metabolite and transcript profiling. Furthermore, a genome-wide cDNA-amplified fragment length polymorphism (AFLP)-based transcriptome profiling was carried out of jasmonate-elicited leaves at different developmental stages. • Although cytokinin and gibberellin positively affected at least one aspect of gland formation, these two hormones did not stimulate artemisinin biosynthesis. Only jasmonate simultaneously promoted gland formation and coordinated transcriptional activation of biosynthetic gene expression, which ultimately led to increased sesquiterpenoid accumulation with chemotype-dependent effects on the distinct pathway branches. Transcriptome profiling revealed a trichome-specific fatty acyl- coenzyme A reductase, trichome-specific fatty acyl-CoA reductase 1 (TFAR1), the expression of which correlates with trichome development and sesquiterpenoid biosynthesis. • TFAR1 is potentially involved in cuticular wax formation during glandular trichome expansion in leaves and flowers of A. annua plants. Analysis of phytohormone-modulated transcriptional regulons provides clues to dissect the concerted regulation of metabolism and development of plant trichomes.
Laboratory of Protistology and Aquatic Ecology, Department of Biology, Ghent University, 9000 Gent, Belgium.
BACKGROUND Streptophyte green algae share several characteristics of cell growth and cell wall formation with their relatives, the embryophytic land plants. The multilobed cell wall of Micrasterias denticulata that rebuilds symmetrically after cell division and consists of pectin and cellulose, makes this unicellular streptophyte alga an interesting model system to study the molecular controls on cell shape and cell wall formation in green plants. RESULTS Genome-wide transcript expression profiling of synchronously growing cells identified 107 genes of which the expression correlated with the growth phase. Four transcripts showed high similarity to expansins that had not been examined previously in green algae. Phylogenetic analysis suggests that these genes are most closely related to the plant EXPANSIN A family, although their domain organization is very divergent. A GFP-tagged version of the expansin-resembling protein MdEXP2 localized to the cell wall and in Golgi-derived vesicles. Overexpression phenotypes ranged from lobe elongation to loss of growth polarity and planarity. These results indicate that MdEXP2 can alter the cell wall structure and, thus, might have a function related to that of land plant expansins during cell morphogenesis. CONCLUSIONS Our study demonstrates the potential of M. denticulata as a unicellular model system, in which cell growth mechanisms have been discovered similar to those in land plants. Additionally, evidence is provided that the evolutionary origins of many cell wall components and regulatory genes in embryophytes precede the colonization of land.
Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB), Technologiepark 927, B-9052 Gent, Belgium.
Jasmonoyl-isoleucine (JA-Ile) is a plant hormone that regulates a broad array of plant defence and developmental processes. JA-Ile-responsive gene expression is regulated by the transcriptional activator MYC2 that interacts physically with the jasmonate ZIM-domain (JAZ) repressor proteins. On perception of JA-Ile, JAZ proteins are degraded and JA-Ile-dependent gene expression is activated. The molecular mechanisms by which JAZ proteins repress gene expression remain unknown. Here we show that the Arabidopsis JAZ proteins recruit the Groucho/Tup1-type co-repressor TOPLESS (TPL) and TPL-related proteins (TPRs) through a previously uncharacterized adaptor protein, designated Novel Interactor of JAZ (NINJA). NINJA acts as a transcriptional repressor whose activity is mediated by a functional TPL-binding EAR repression motif. Accordingly, both NINJA and TPL proteins function as negative regulators of jasmonate responses. Our results point to TPL proteins as general co-repressors that affect multiple signalling pathways through the interaction with specific adaptor proteins. This new insight reveals how stress-related and growth-related signalling cascades use common molecular mechanisms to regulate gene expression in plants.
The cornerstone of pharmacotherapy for OCD is serotonin reuptake inhibition, either with clomipramine or with selective serotonin reuptake inhibitors (SSRIs). In spite of the success of serotonin reuptake inhibiting drugs, nearly half of OCD patients do not respond to treatment. Treatment response may be affected by genetic polymorphisms of the P450 metabolic system. The four most common enzyme-activity reducing polymorphisms of the P450 CYP2D6 enzyme were determined in 91 outpatients with primary OCD according to DSM-IV criteria, receiving dosages titrated upward to 300 mg/day of venlafaxine or 60 mg/day of paroxetine, using a fixed dosing schedule. Our results show that the investigated CYP2D6 polymorphisms are not a decisive factor in the response to paroxetine and venlafaxine treatment in OCD in spite of their highly significant effect on the blood levels of these medicines.
Department of Plant Systems Biology, Flanders Institute for Biotechnology (VIB) and Department of Plant Biotechnology and Genetics, Ghent University, Gent, Belgium.
Plant trichome initiation is steered by diverse developmental and environmental cues, through molecular mechanisms that remain elusive in most plant species. Using a robust experimental method to investigate the molecular mechanisms by which phytohormones modulate leaf trichome formation, we verified the effect of jasmonates, cytokinins and gibberellins in Arabidopsis (Arabidopsis thaliana). All three phytohormones promoted Arabidopsis trichome initiation, but caused divergent effects on trichome maturation and other leaf parameters. Molecular analysis indicated that the phytohormones mediated trichome initiation by the transcriptional regulation of the components of the TRANSPARENT TESTA GLABRA1 (TTG1) activator/inhibitor complex. In this addendum, we additionally studied the effects of jasmonates, cytokinins and gibberellins on leaf trichome formation in a representative set of plant species, spanning the angiosperm lineage and covering different trichome types. We found that the general ability of the three phytohormones to impinge on trichome initiation is conserved across angiosperms, but that within a particular plant species distinct regulatory networks might be activated to steer the formation of the various trichome types.
The cornerstone of pharmacotherapy for OCD is serotonin reuptake inhibition, either with clomipramine or with selective serotonin reuptake inhibitors (SSRIs). In spite of the success of serotonin reuptake inhibiting drugs, nearly half of OCD patients do not respond to treatment. Treatment response may be affected by genetic polymorphisms of the P450 metabolic system. The four most common enzyme-activity reducing polymorphisms of the P450 CYP2D6 enzyme were determined in 91 outpatients with primary OCD according to DSM-IV criteria, receiving dosages titrated upward to 300 mg/day of venlafaxine or 60 mg/day of paroxetine, using a fixed dosing schedule. Our results show that the investigated CYP2D6 polymorphisms are not a decisive factor in the response to paroxetine and venlafaxine treatment in OCD in spite of their highly significant effect on the blood levels of these medicines.
[1] Department of Plant Systems Biology, Flanders Institute for Biotechnology, Ghent University, Technologiepark 927, B-9052 Ghent, Belgium [2] Department of Plant Biotechnology and Genetics, Ghent University, Technologiepark 927, B-9052 Ghent, Belgium [3] Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium.
Plant hormones have pivotal roles in almost every aspect of plant development. Over the past decades, physiological and genetic studies have revealed that hormone action in plants is determined by complex interactions between hormonal signalling pathways. Evidence is accumulating for the existence of crosstalk between the auxin and jasmonate (JA) signalling pathways. Recently, the JASMONATE ZIM-domain (JAZ) proteins have been identified as the long-sought repressors of JA signalling. Here, we show that expression of JAZ1/TIFY10A is not solely inducible by JA, but that it is also an early auxin-responsive gene. Furthermore, we could show that the auxin-inducible expression of JAZ1/TIFY10A is independent of the JA signalling pathway but is controlled by the auxin/indole-3-acetic acid-auxin response transcription factor signalling pathway. Our results provide evidence for the existence of at least two different input signals regarding JAZ1/TIFY10A expression and thus support the idea of an intimate molecular interplay between auxin and JA signalling.
Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji 611-0011, Japan;
Alkaloids play a key role in plant defense mechanisms against pathogens and herbivores, but the plants themselves need to cope with their toxicity as well. The major alkaloid of the Nicotiana species, nicotine, is translocated via xylem transport from the root tissues where it is biosynthesized to the accumulation sites, the vacuoles of leaves. To unravel the molecular mechanisms behind this membrane transport, we characterized one transporter, the tobacco (Nicotiana tabacum) jasmonate-inducible alkaloid transporter 1 (Nt-JAT1), whose expression was coregulated with that of nicotine biosynthetic genes in methyl jasmonate-treated tobacco cells. Nt-JAT1, belonging to the family of multidrug and toxic compound extrusion transporters, was expressed in roots, stems, and leaves, and localized in the tonoplast of leaf cells. When produced in yeast cells, Nt-JAT1 occurred mainly in the plasma membrane and showed nicotine efflux activity. Biochemical analysis with proteoliposomes reconstituted with purified Nt-JAT1 and bacterial F(0)F(1)-ATPase revealed that Nt-JAT1 functioned as a proton antiporter and recognized endogenous tobacco alkaloids, such as nicotine and anabasine, and other alkaloids, such as hyoscyamine and berberine, but not flavonoids. These findings strongly suggest that Nt-JAT1 plays an important role in the nicotine translocation by acting as a secondary transporter responsible for unloading of alkaloids in the aerial parts and deposition in the vacuoles.
Department of Plant Systems Biology, Flanders Institute for Biotechnology and Department of Plant Biotechnology and Genetics, Ghent University, Technologiepark 927, B-9052 Ghent, Belgium.
The diverse functions of jasmonates (JAs) in plant cells are reflected by the extensive reprogramming of gene expression after JA perception. Here, we review the transcriptome signatures associated with JA signalling in Arabidopsis thaliana and other plant species. Transcript profiling studies clearly highlight the renowned capacity of JAs to elicit evolutionarily distant metabolic pathways across the plant kingdom. However, JA-related transcriptomes show limited overlap and, for most cellular processes, the context in which the JA signal is perceived is crucial in shaping the response. This emphasizes that JA signal transduction is integrated into an elaborate signalling network.
Department of Plant Systems Biology, Flanders Institute for Biotechnology and Department of Molecular Genetics, Ghent University, 9052 Gent, Belgium.
Trichome initiation in Arabidopsis thaliana is controlled by the TRANSPARENT TESTA GLABRA1 (TTG1) network that consists of R2R3- and R1-type MYB-related transcription factors, basic helix-loop-helix (bHLH) proteins, and the WD40 protein TTG1. An experimental method was designed to investigate the molecular mechanisms by which jasmonates, cytokinins, and gibberellins modulate Arabidopsis leaf trichome formation. All three phytohormones provoked a seemingly common effect on cell patterning by promoting trichome initiation, but caused strikingly distinct effects on cell and trichome maturation. The phytohormonal control was mediated by transcriptional regulation of the established TTG1 complex and depended on the R2R3-MYB factor GLABRA1. However, unsuspected degrees of functional specialization of the bHLH factors and a resultant differential molecular regulation of trichome initiation on leaf lamina and leaf margins were revealed. Trichome formation on leaf lamina relied entirely on GLABRA3 and ENHANCER OF GLABRA3. Conversely, TRANSPARENT TESTA8 (TT8) was particularly important for marginal trichome development. This hitherto unknown role for TT8 in trichome formation further underscored the functional redundancy between the three TTG1-dependent bHLH proteins.

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Laboratories devoted to the public health field have to face the analysis of a large number of organic contaminants/residues in many different types of samples. Analytical techniques applied in this field are normally focused on quantification of a limited number of analytes. At present, most of these techniques are based on gas chromatography (GC) or liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). Using these techniques only analyte-specific information is acquired, and many other compounds that might be present in the samples would be ignored. In this paper, we explore the potential of time of flight (TOF) MS hyphenated to GC or LC to provide additional information, highly useful in this field. Thus, all positives reported by standard reference targeted LC-MS/MS methods were unequivocally confirmed by LC-QTOF MS. Only 61% of positives reported by targeted GC-MS/MS could be confirmed by GC-TOF MS, which was due to its lower sensitivity as non-confirmations corresponded to analytes that were present at very low concentrations. In addition, the use of TOF MS allowed searching for additional compounds in large-scope screening methodologies. In this way, different contaminants/residues not included in either LC or GC tandem MS analyses were detected. This was the case of the insecticide thiacloprid, the plant growth regulator paclobutrazol, the fungicide prochloraz or the UV filter benzaphenone, among others. Finally, elucidation of unknowns was another of the possibilities offered by TOF MS thanks to the accurate-mass full-acquisition data available when using this technique.
State Key Lab of New-tech for Chinese Medicine Pharmaceutical Processes, Lianyungang, 222001, China; Jiangsu Kanion Pharmaceutical Co. Ltd., Nanjing, 210017, China.
RATIONALE The Direct Analysis in Real Time (DART) ionization source coupled with a quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) system has the capability to desorb analytes directly from samples from complex Chinese herbal preparations without sample cleanup or chromatographic separation. METHODS In this work, a method based on DART/Q-TOF MS/MS has been developed for rapid determination of geniposide present in 'Re Du Ning Injections', a Chinese herbal preparation. The method has been evaluated for both qualitative and quantitative analysis of geniposide in Re Du Ning Injections. RESULTS Variables including polarity for ion detection, DART gas heater temperature, matrix effect and sample presentation speed were investigated. The quantitative method was validated with respect to linearity, sensitivity, repeatability, precision and accuracy by using both internal and external standards. A comparison of the results obtained using the DART-based method was made with those obtained using a conventional High-Performance Liquid Chromatography/Diode-Array Detector (HPLC/DAD) by analyzing geniposide in four batches of Re Du Ning Injections. CONCLUSIONS The DART/Q-TOF MS/MS-based method provides a rapid, efficient and powerful method to analyze compounds from complex Traditional Chinese Medicines with limited sample preparation thus reducing time and complexity of quality control for those materials. Copyright © 2012 John Wiley & Sons, Ltd.
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China.
Flos Lonicerae, derived from the flower buds of several medicinal Lonicera species, is a commonly used herbal medicine with multiple pharmacological activities, one of the major ones being antioxidant activity. In this study, free radical scavengers in the flower buds of six Lonicera species were screened, identified and quantified by online HPLC-DPPH (1,1-diphenyl-2-picrylhydrazyl) assay coupled with LC quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS). The antioxidants were firstly screened from the complex plant matrix by the online HPLC-DPPH assay. Then the active compounds were identified by LC Q-TOF MS/MS, and the possible fragmentation pathways were proposed. The reactivity of antioxidants available was investigated using an internal standard method by online LC assay. The contents of 12 antioxidants were also determined or estimated by HPLC coupled with diode array detector. The total antioxidant capability determined by the online method was used as the marker to evaluate the quality of Flos Lonicerae. The results were important to clarify the material basis and therapeutic mechanism of Flos Lonicerae.
Institute of Legal Medicine, University Hospital Charité, Turmstraße 21, Building N, D-10559 Berlin, Germany.
Time of flight mass spectrometry provides new possibilities of substance identification by determination of the molecular formula from accurate molecular mass and isotope pattern. However, the huge number of possible isomers requires additional evidence. As a suitable way for routine performance of systematic toxicological analysis, a method for combined use of liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) and high performance liquid chromatography with diode array detector (HPLC-DAD) was developed and applied to blood samples from 77 death cases. The blood samples were prepared by extraction with CH(2)Cl(2) and by protein precipitation with acetonitrile (1:4 (v/v)). The evaporated extracts were reconstituted in 35% acetonitril/0.1% formic acid/H(2)O and aliquots were injected for analysis by LC-QTOF-MS (Agilent 6530) and HPLC-DAD (Agilent 1200). A valve switching system enabled simultaneous operation of both separated chromatographic lines under their respective optimal conditions using the same autosampler. The ESI-QTOF-MS instrument was run in data dependent acquisition mode with switching between MS and MS/MS (cycle time 1.1s) and measuring the full mass spectra and the collision induced dissociation (CID) fragment spectra of all essential [M+H](+) ions. Libraries of accurate mass CID spectra (~2500 substances) and of DAD-UV spectra (~3300 substances) of the authors were used for substance identification. The application of this procedure is demonstrated in detail at four examples with multiple drug intake or administration. In the 77 cases altogether 198 substances were identified (87 by DAD and 195 by QTOF-MS) with a frequency between 1 and 20. In practical application, the sample preparation proved to be suitable for both techniques and for a wide variety of substances with different polarity. The automatic performance of the measurements was efficient and robust. Mutual confirmation, decrease of false positive and false negative identifications, and the semi-quantitative estimation of the concentrations by HPLC-DAD for a first assessment of the toxicological relevance of the qualitative result were shown to be the main advantages of the method combination.
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, NSW, Australia 2109. robyn.peterson@mq.edu.au
Coprophilous fungi inhabit herbivore feces, secreting enzymes to degrade the most recalcitrant parts of plant biomass that have resisted the digestive process. Consequently, the secretomes of coprophilous fungi have high potential to contain novel and efficient plant cell wall degrading enzymes of biotechnological interest. We have used one-dimensional and two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization-time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), and quadrupole time-of-flight liquid chromatography-tandem mass spectrometry (Q-TOF LC-MS/MS) to identify proteins from the secretome of the coprophilous fungus Doratomyces stemonitis C8 (EU551185) isolated from koala feces. As the genome of D. stemonitis has not been sequenced, cross-species identification, de novo sequencing, and zymography formed an integral part of the analysis. A broad range of enzymes involved in the degradation of cellulose, hemicellulose, pectin, lignin, and protein were revealed, dominated by cellobiohydrolase of the glycosyl hydrolase family 7 and endo-1,4-β-xylanase of the glycosyl hydrolase family 10. A high degree of specialization for pectin degradation in the D. stemonitis C8 secretome distinguishes it from the secretomes of some other saprophytic fungi, such as the industrially exploited T. reesei. In the first proteomic analysis of the secretome of a coprophilous fungus reported to date, the identified enzymes provide valuable insight into how coprophilous fungi subsist on herbivore feces, and these findings hold potential for increasing the efficiency of plant biomass degradation in industrial processes such as biofuel production in the future.
Department of Food Science, University of California at Davis, Davis, California 95616, United States.
N-Linked glycans of skim human milk proteins were determined for three mothers. N-Linked glycans are linked to immune defense, cell growth, and cell-cell adhesion, but their functions in human milk are undetermined. Protein-bound N-linked glycans were released with peptidyl N-glycosidase F (PNGase F), enriched by graphitized carbon chromatography, and analyzed with Chip-TOF MS. To be defined as N-glycans, compounds were required, in all three procedural replicates, to match, within 6 ppm, against a theoretical human N-glycan library and be at least 2-fold higher in abundance in PNGase F-treated than in control samples. Fifty-two N-linked glycan compositions were identified, and 24 were confirmed via tandem mass spectra analysis. Twenty-seven compositions have been found previously in human milk, and 25 are novel compositions. By abundance, 84% of N-glycans were fucosylated and 47% were sialylated. The majority (70%) of total N-glycan abundance was composed of N-glycans found in all three milk samples.
Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou, 310029, P. R. China.
The present study developed an improved analytical method for simultaneous quantification of seven neonicotinoids in food by ultraperformance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UPLC-MS/MS) under the multiple reaction monitoring (MRM) mode. The optimization of extraction, cleanup, UPLC separation and MS/MS parameters of analytes were especially focused on. The low limits of quantification (LOQs) of neonicotinoids ranged from 0.1 to 6 mug kg(-1). Meanwhile, reasonable recoveries (65-120%) of seven neonicotinoids for food including apple, cabbage, potato, rice, tea, milk, chicken, pork and egg were demonstrated in different spiked levels within their respective linear range (0.025-150 mug kg(-1)). The developed analytical method would be appropriate for the routine, high throughput, high sensitivity quantification of seven neonicotinoids using simple sample pretreatment.
Key Laboratory of Applied Marine Biotechnology (Ningbo University), Ministry of Education, Ningbo 315211, China.
The photosynthetic glycerolipids composition of algae is crucial for structural and physiological aspects. In this work, a comprehensive characterization of the photosynthetic glycerolipids of the diatom Stephanodiscus sp. was carried out by ultra performance liquid chromatography-electrospray ionization-quadrupole-time of flight mass spectrometry (UPLC-ESI-Q-TOF MS). By use of the MS(E) data collection mode, the Q-TOF instrument offered a very viable alternative to triple quadrupoles for precursor ion scanning of photosynthetic glycerolipids and had the advantage of high efficiency, selectivity, sensitivity and mass accuracy. Characteristic fragment ions were utilized to identify the structures and acyl compositions of photosynthetic glycerolipids. Comparing the abundance of fragment ions, it was possible to determine the position of the sn-glycerol-bound fatty acyl chains. As a result, four classes of photosynthetic glycerolipid in the extract of Stephanodiscus sp. were unambiguously identified, including 16 monogalactosyldiacylglycerols (MGDGs), 9 digalactosyldiacylglycerols (DGDGs), 23 sulfoquinovosyldiacylglycerols (SQDGs) and 8 phosphatidylglycerols (PGs). As far as our knowledge, this is the first report on global identification of photosynthetic glycerolipids, including lipid classes, fatty acyl composition within lipids and the location of fatty acids in lipids (sn-1 vs. sn-2), in the extract of marine microalgae by UPLC-ESI-Q-TOF MS directly.
Department of Chemistry & Biochemistry, Baylor University, Waco, TX, USA.
A variety of chemicals are produced upon pretreatment of lignocellulosic biomass. Aliphatic acids, aromatic acids, aldehydes, and phenolic compounds are of particular interest due to their presumed inhibitory influence on downstream enzymatic or microbial steps in biomass-to-ethanol conversion. Herein, we describe a series of analytical protocols that collectively enable quantitative monitoring of 40 potential fermentation inhibitors in biomass pretreatment samples. Solid samples are accommodated by first employing pressurized fluid extraction to generate an aqueous "wash stream." Sample preparation for liquids involves an initial precipitation-filtration step, followed by liquid-liquid extraction and reconstitution of extracts in water. Samples are analyzed using high-performance liquid chromatography (HPLC) in combination with ultraviolet (UV) absorbance and tandem mass spectrometry (MS/MS) detection. A standard addition approach is utilized for quantitation to alleviate complications arising from co-extracted sample matrix.
Department of Natural Medicinal Chemistry, School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China.
Acetophenones in Cynanchum species, especially cynandione A and its derivatives, whose utilization and toxicity in herbal drugs and folk medicines has caused great interest in the chemical investigation, have extensive biological activities. In this paper, a facile method based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS(n)) was developed for the analysis of cynandione A derivatives in the roots of the Cynanchum wilfordii and C. auriculatum. ESI-MS/MS and ESI-MS(n) analysis of cynandiones A and B in negative ion mode were firstly performed employing two mass spectrometers each equipped with an ion-trap and a quadrupole time-of-flight (Q-TOF) mass analyzer. The results drawn from both instruments were similar to each other. Characteristic fragmentation pathways were proposed by comparing the spectra of two standards acquired in the experiments. The fragment ions at m/z 283 and 268 were obtained, and then were used as diagnostic ions to screen and identify cynandione A derivatives from the roots of above two species, together with an HPLC-MS(n) method. Total of 28 cynandione A derivatives comprising 4 reported and 24 novel components were identified or tentatively identified. Furthermore, breakdown curves were constructed to distinguish two types of isomers among these compounds. To our knowledge, this is the first report on characterization of acetophenones by HPLC-ESI-MS(n), which allows a rapid and complete analysis of cynandione A derivatives in roots of Cynanchum species.

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