Rapid identification of elaiophylin and geldanamycin inStreptomyces fermentation broths using CPC coupled with a photodiode array detector and LC-MS methodologies

During the course of screening microbial broth extracts in various high through-put bioassays (eg receptor binding or enzyme inhibition), several actinomycete cultures were discovered to produce active metabolites. The natural products elaiophylin and/or geldanamycin are produced by severalStreptomyces violaceusniger strains, and the bioactivity of the extracts from these cultures was frequently associated with the fractions containing these metabolites. CPC coupled to a photodiode array detector and LC-MS techniques were applied to these broth extracts to ascertain rapidly when these natural products were present. These methodologies allowed us to identify the metabolites quickly in the crude extract, and the application demonstrated further the utility of CPC-photodiode array detection and LC-MS as powerful, initial analytical tools in analyses of the complex metabolite profiles produced by microorganisms.     

Bioprospecting Chemical Diversity and Bioactivity in a Marine Derived Aspergillus terreus

A comparative metabolomic study of a marine derived fungus (Aspergillus terreus) grown under various culture conditions is presented. The fungus was grown in eleven different culture conditions using solid agar, broth cultures, or grain based media (OSMAC). Multivariate analysis of LC/MS data from the organic extracts revealed drastic differences in the metabolic profiles and guided our subsequent isolation efforts. The compound 7‐desmethylcitreoviridin was isolated and identified, and is fully described for the first time. In addition, 16 known fungal metabolites were also isolated and identified. All compounds were elucidated by detailed spectroscopic analysis and tested for antibacterial activities against five human pathogens and tested for cytotoxicity. This study demonstrates that LC/MS based multivariate analysis provides a simple yet powerful tool to analyze the metabolome of a single fungal strain grown under various conditions. This approach allows environmentally‐induced changes in metabolite expression to be rapidly visualized, and uses these differences to guide the discovery of new bioactive molecules.     

Characterization of peptidyl-nucleoside antifungal antibiotics from fermentation broth

Summary Characterization of sinefungin related antifungal antibiotics from fermentation broth was accomplished by coupling photodiode array (PDA) detection to high performance liquid chromatography (HPLC). From the combined HPLC-PDA evaluation of broth filtrate, we detected five sinefungin related components. Fast atom bombardment (FAB) mass spectroscopic evaluations, mass-analysed ion kinetic energy spectra (MIKES) and collision activated (CA) MIKES of these components confirmed their respective identities. Our findings from the combination of HPLC photodiode array acquisition and FAB-mass spectrometry suggest we have detected the presence of a previously unreported sinefungin analogue.     

Metabolic profiling of saponins in Medicago sativa and Medicago truncatula using HPLC coupled to an electrospray ion-trap mass spectrometer

Triterpene saponins isolated from Medicago sativa (alfalfa) and Medicago truncatula roots were separated, profiled and identified using an optimized, reversed-phase HPLC with on-line photodiode array detection and electrospray ionization mass spectrometry method (HPLC/PDA/ESI/MS). ESI source polarity and solvent conditions were compared. The effects of these parameters on mass spectral attributes were determined. Ion structures were confirmed using tandem mass spectrometry (MS/MS). Fifteen saponins were identified in alfalfa (cultivars Apollo, Radius, and Kleszczewska) based upon negative-ion HPLC/PDA/ESI/MS, HPLC/PDA/ESI/MS/MS and literature data. In addition, the identification of two new malonated saponins in alfalfa are proposed. Negative-ion HPLC/PDA/ESI/MS and HPLC/PDA/ESI/MS/MS spectra were utilized along with HPLC retention times to profile and identify 27 saponins in M. truncatula (cultivar Jemalong, A17). M. truncatula yielded a much more complex mixture of saponins than observed for alfalfa. The authors are not aware of any previous reports identifying saponin glycosides in M. truncatula.     

Simultaneous determination of cytotoxic bufadienolides in the Chinese medicine ChanSu by high-performance liquid chromatography coupled with photodiode array and mass spectrometry detections

ChanSu (toad venom) is a traditional Chinese medicine for the treatment of serious liver and gastric cancers. The major cytotoxic compounds in ChanSu are bufadienolides. In this paper, a strategy combining qualitative LC/MS analysis and quantitative HPLC determination of major bufadienolides was used for global quality control of ChanSu crude drug. Majority of the bufadienolides in methanol extract of ChanSu were unambiguously characterized by high-performance liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS), and by comparing with pure compounds. In addition, eight major bufadienolides were simultaneously determined in one single HPLC run within 30 min with photodiode array detection (DAD). All compounds showed good linearity in a wide concentration range, and their limits of detection (LOD) were around 1 ng. Thus, > 95% of the bufadienolides in ChanSu could be characterized, and > 90% of them were quantitated. The established method is rapid, simple and sensitive, and could be used for the routine analysis of ChanSu crude drug and its preparations. This research sets a good example for the comprehensive quality control of traditional medicine.     

Metabolite profiling of plant carotenoids using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Although modern MS has facilitated the advent of metabolomics, some natural products such as carotenoids are not readily compatible to detection by MS. In the present article, we describe how matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) can be utilized to acquire mass spectra of carotenoids effectively. The procedure is sensitive (pmole range), reduces 'spot to spot' variation and provides high mass accuracy, thus aiding identification. The technique has been applied in vivo to the analysis of carotenoids in isolated plant cells and in vitro to three applications: (i) to show compatibility with purification methods such as LC, TLC and HPLC; (ii) for the rapid identification and quantification (by isotope dilution) of carotenoids present in crude extracts from plant tissues and whole cells; (iii) simultaneous semi-quantitative determination of carotenoids metabolites (m/z values) in crude plant extracts. Multivariate analysis of the recorded m/z values shows the effectiveness of the procedure in distinguishing genotypes from each other. In addition, the utility of the technique has been demonstrated on two mutant tomato populations, to determine alterations in carotenoid content, and a comparison made with traditional HPLC-photodiode array analysis. These data show that MALDI/TOF-MS can be used to rapidly profile, identify and quantify plant carotenoids reproducibly, as well as detecting other metabolites (m/z) in complex biological systems.     

Cis-2′, 3′-Dihydrodiol production on flavone B-Ring by Biphenyl Dioxygenase from Pseudomonas pseudoalcaligenes KF707 expressed in Escherichia coli

Escherichia coli JM109 strains expressing either toluene dioxygenase from Pseudomonas putida F1 or biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 were examined for their ability to catalyze flavones. Biphenyl dioxygenase produced metabolites from flavone and 5,7-dihydroxyflavone which were not found in the control experiments. The absorption maxima of UV-visible spectra for the metabolites from flavone and 5,7-dihydroxyflavone were found at 337 and 348 nm respectively by using a photodiode array detector in the HPLC. Liquid chromatography/mass spectroscopy (LC/MS) showed molecular weights 256 and 288 for the metabolites, respectively. The metabolite of flavone, which was isolated and purified from the bacterial culture, was further subjected to analysis by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. Based on the LC/MS and NMR results, biphenyl dioxygenase inserted oxygen at C2′ and C3′ on the B-ring of flavone, resulting in the formation of flavone cis-2′, 3′-dihydrodiol (2-[3,4-dihydroxy-1.5-cyclohexadienyl]-4H-chromen-4-one). Since this product is not found in Chemical Abstracts, this compound is considered a novel one. In addition, biotransformation of flavones by biphenyl dioxygenase suggested a potential role of bacterial dioxygenase to synthesize novel compounds from plant secondary metabolites. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterisation of a potential biomarker of phospholipidosis from amiodarone-treated rats

A novel and relatively simple analytical method for the separation, characterisation and semi-quantitation of phospholipids (PLs) from extracts of complex biological samples has been developed. This methodology allows PL extracts from cells and tissues to be analysed by liquid chromatography (LC) coupled to electrospray ionisation mass spectrometry (ESI-MS). Complex mixtures of PLs were separated on a high-performance liquid chromatography (HPLC) system using 0.5% ammonium hydroxide in methanol/water/hexane/formate mixture with UV detection at 205 nm. Identification and structural characterisation of molecular species were carried out utilising ESI-MS and MS/MS in the negative ion mode.The abnormal accumulation of PLs (phospholipidosis) was induced in male Sprague-Dawley rats by administration of the cationic amphiphilic drug (CAD), amiodarone. Analysis of the PL profile of liver and lung tissues, lymphocytes and serum from treated rats was carried out using this analytical procedure (LC-ESI/MS/MS). Differences in PL profiles between treated and untreated animals were highlighted by principal component analysis (PCA). This led to the selection of a potential metabolic marker of phospholipidosis (PLD) identified as a lyso-bis-phosphatidic acid (LBPA) derivative, also known as bis(monoglycero)phosphate (BMP). This PL was absent in control animals but was present in quantifiable amounts in all samples from amiodarone-treated rats.     

Narrow-bore sample trapping and chromatography combined with quadrupole/time-of-flight mass spectrometry for ultra-sensitive identification of in vivo and in vitro metabolites

The identification of in vitro and in vivo metabolites is vital to the discovery and development of new pharmaceutical therapies. Analytical strategies to identify metabolites at different stages of this process vary, but all involve the use of liquid chromatography separations combined with detection via mass spectrometry (HPLC/MS). Reported here is the use of narrow-bore column (0.5-1.0 mm i.d.) trapping of metabolites, followed by back-flushing onto a matching analytical column. Separated metabolites were then identified using quadrupole time-of-flight mass spectrometry (MS) and tandem MS. Metabolites in human plasma and from low-level in vitro incubations, that were not identified using standard HPLC/MS approaches, were characterized using the instrumental configuration described here.     

Profiling of phenolic glycosidic conjugates in leaves of Arabidopsis thaliana using LC/MS

Profiling of plant secondary metabolites is still a very difficult task. Liquid chromatography (LC) or capillary electrophoresis hyphenated with different kinds of detectors are methods of choice for analysis of polar, thermo labile compounds with high molecular masses. We demonstrate the applicability of LC combined with UV diode array or/and mass spectrometric detectors for the unambiguous identification and quantification of flavonoid conjugates isolated from Arabidopsis thaliana leaves of different genotypes and grown in different environmental conditions. During LC/UV/MS/MS analyses we were able to identify tetra-, tri-, and di-glycosides of kaempferol, quercetin and isorhamnetin. Based on our results we can conclude that due to the co-elution of different chemical compounds in reversed phase HPLC systems the application of UV detectors does not allow to precisely profile all flavonoid conjugates existing in A. thaliana genotypes. Using MS detection it was possible to unambiguously recognize the glycosylation patterns of the aglycones. However, from the mass spectra we could not conclude neither the anomeric form of the C-1 carbon atoms of sugar moieties in glycosidic bonds between sugars or sugar and aglycone nor the position of the second carbon involved in disaccharides. The applicability of collision induced dissociation techniques (CID MS/MS) for structural analyses of the studied group of plant secondary metabolites with two types of analyzers (triple quadrupole or ion trap) was demonstrated.     

A simple assay for fluorescent siderophores produced by Pseudomonas species and an efficient isolation of pseudobactin

Several iron binding metabolites (siderophores) of Pseudomonas fluorescens B10 (JL-3133) have been detected using C₁₈ reverse phase HPLC coupled with photodiode array detection methods. This analysis utilized a volatile mobile phase of 90% 20 mm NH₄HCO₃/10% MeOH, pH 6.5. It has been shown to be applicable to other P. fluorescens strains for the detection of related metabolites. Direct scale-up of the analytical HPLC conditions allowed for the efficient preparative isolation of pseudobactin, the principle siderophore produced by P. fluorescens B10 (JL-3133).     

Identification of anionic selenium species in Se-rich yeast by electrospray QTOF MS/MS and hybrid linear ion trap/orbitrap MSn

An analytical approach allowing the identification of unknown selenium metabolites in selenium-rich yeast was described. Anion-exchange HPLC of the Se-metabolome fraction co-eluting with salts in size-exclusion chromatography allowed the separation of nine selenium species (excluding isomers and selenate) as monitored by inductively coupled plasma mass spectrometry (ICP MS). The individual fractions were analyzed by electrospray QTOF MS/MS and hybrid linear ion trap/Orbitrap MSn after sample introduction by reversed-phase nanoHPLC and by hydrophilic interaction LC (HILIC), respectively. Out of the nine detected species, eight were identified on the basis of accurate mass measurements and collision induced dissociation/fragmentation information. Seven Se-species (selenohomolanthionine, γ-Glu-selenocystathionine, 2,3-DHP-selenocystathionine, N-acetyl-selenocystathionine, 2,3-DHP-selenohomolanthionine, Se-methyl-selenoglutathione, and 2,3-DHP-Se-methylselenocysteine) were reported for the first time in Se-rich yeast, five of them have never been reported in any biological sample before.     

Role of advances in chromatographic techniques in phytochemistry

Chromatography is the lynchpin of phytochemistry and is the key to obtaining pure compounds for structure elucidation, for pharmacological testing or for development into therapeuticals. It also plays a fundamental role as an analytical technique for quality control and standardisation of phytotherapeuticals. Although liquid chromatography is barely 100 years old, an extraordinary variety of instrumental and ancillary equipment is available, notably in the domain of high-performance liquid chromatography. It is impossible to touch all areas of chromatography in such a review but certain areas are worthy of mention: HPLC, HPTLC, UPLC and countercurrent chromatography. Another important addition has been the development of hyphenated techniques involving HPLC: LC/UV, LC/MS, LC/MS(n) and LC/NMR. These are indispensable nowadays for the early detection and identification of compounds in crude plant extracts.     

Metabolites from the Dark Septate Endophyte Drechslera sp. Evaluation by LC/MS and Principal Component Analysis of Culture Extracts with Histone Deacetylase Inhibitors

Secondary metabolites from the cultures of the dark septate fungal endophyte (DSE) Drechslera sp., isolated from the roots of rye grass (Lollium sp.) and cultured under different experimental conditions, are described here for the first time. The use of suberoylanilidehydroxamic acid (SAHA) and other histone deacetylase inhibitors as epigenetic modifiers in the culture medium was evaluated by LC/MS and LC/MS/MS. Several differences in the metabolite production were detected by means of supervised principal component analysis (PCA) of LC/MS data. The presence of the compounds in the culture medium or in the mycelium was compared. In order to confirm their structure, many of these natural products were isolated from a larger scale culture. These metabolites were characterized as prenylhydroxybenzoic acids and chromans, two compounds, one of each class were previously undescribed, prenylquinoids, diketopiperazines and macrosphelides. Some of the compounds, which were released to the medium, showed good antifungal activity, suggesting that these compounds could protect Lollium from fungal phytopatogens. The use of SAHA as an additive of the cultures also induced the release of hexosylphytosphyngosine to the culture medium. The biotransformation of the inhibitors was observed in addition to the production of antifungal metabolites, showing the ability of this endophytic strain to control xenobiotics.     

Pharmacokinetics of kakkalide and its main metabolites in rat plasma determined by HPLC-DAD and LC-MSn

This study was undertaken to assess the plasma pharmacokinetic profile of kakkalide (KA), the major isoflavone found in extracts from the dried flower of Pueraria lobata. The main metabolites were identified using HPLC-DAD or LC/MS/MS method, and a HPLC-UV method for simultaneous quantification of the metabolites as well as the parent compound in plasma was developed. Rat plasma contained three glucuronide metabolites, irisolidone-7-O-glucuronide (Ir-7G), tectorigenin-7-O-glucuronide (Te-7G) and 6-OH biochanin A-glucuronide (6-OH BiA-G), as well as KA and trace amount of irisolidone (Ir) after oral administration of 200 mg/kg KA. The pharmacokinetics of KA and three glucuronide conjugates in rat plasma was determined for the first time using a simple, selective and accurate HPLC method. The AUC(0-t) values of the glucuronide metabolites are significantly greater than that of KA. They were detectable in rat plasma at different time points, indicating that glucuronidation during KA metabolism in vivo may occur in different sites, first in intestine and then in liver. Moreover, enterohepatic recirculation may result in the slow elimination of these glucuronide metabolites.     

Uptake and metabolism of epicatechin and its access to the brain after oral ingestion

Epicatechin is a flavan-3-ol that is commonly present in green teas, red wine, cocoa products, and many fruits, such as apples. There is considerable interest in the bioavailability of epicatechin after oral ingestion. In vivo studies have shown that low levels of epicatechin are absorbed and found in the circulation as glucuronides, methylated and sulfated forms. Recent research has demonstrated protective effects of epicatechin and one of its in vivo metabolites, 3′-O-methyl epicatechin, against neuronal cell death induced by oxidative stress. Thus, we are interested in the ability of ingested epicatechin to cross the blood brain barrier and target the brain. Rats were administered 100 mg/kg body weight/d epicatechin orally for 1, 5, and 10 d. Plasma and brain extracts were analyzed by HPLC with photodiode array detection and LC-MS/MS. This study reports the presence of the epicatechin glucuronide and 3′-O-methyl epicatechin glucuronide formed after oral ingestion in the rat brain tissue.     

Shotgun proteomics of the haloarchaeon Haloferax volcanii

Haloferax volcanii, an extreme halophile originally isolated from the Dead Sea, is used worldwide as a model organism for furthering our understanding of archaeal cell physiology. In this study, a combination of approaches was used to identify a total of 1296 proteins, representing 32% of the theoretical proteome of this haloarchaeon. This included separation of (phospho)proteins/peptides by 2-dimensional gel electrophoresis (2-D), immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), and Multidimensional Protein Identification Technology (MudPIT) including strong cation exchange (SCX) chromatography coupled with reversed phase (RP) HPLC. Proteins were identified by tandem mass spectrometry (MS/MS) using nanoelectrospray ionization hybrid quadrupole time-of-flight (QSTAR XL Hybrid LC/MS/MS System) and quadrupole ion trap (Thermo LCQ Deca). Results indicate that a SCX RP HPLC fractionation coupled with MS/MS provides the best high-throughput workflow for overall protein identification.     

Simultaneous identification and determination of major taxoids from extracts of Taxus chinensis cell cultures

Liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/ MS) and high-performance liquid chromatography (HPLC) methods have been used to identify and quantify the major taxoids from extracts of Taxus chinensis cell cultures. Chromatography was carried out on a reverse phase C18 column with isocratic-mode elution. By analytically comparing LC/ESI-MS/MS of the extracts with that of the available reference substances and literature, six taxoids were identified as taxuyunnanine C (Tc, 1), yunnanxane (2), 2alpha,5alpha10beta-triacetoxy-14beta-propionyloxytaxa-4(20),11-diene (3), 2alpha,5alpha, 10beta-triacetoxy-14beta-(2-methyl)butyryloxytaxa-4(20),11-diene (4), taxol (5), and baccatin III (B III, 6), respectively. Among them, 2, 3 and 4 were assigned in the absence of the corresponding reference substances, and 3 and 4 were detected in this cell line for the first time. The identification was validated by NMR spectra. The precise quantification of 1 and 5 was made using HPLC. The limit of detection (LOD), 0.5 microg/ml for 5, 1.5 microg/ml for 1, and the linearity and accuracy of the quantitative method were evaluated, indicating a wide linear range and satisfactory accuracy. The amounts of other identified taxoids were calculated on the basis of comparison of the absolute response factors of similar structural substances. The proposed method provides a rapid, conventional and reliable tool to characterize and study cell lines for elucidating the taxane biosynthesis.