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Sample records for detecting tamiflu metabolite

  1. Mixture toxicity of the antiviral drug Tamiflu((R)) (oseltamivir ethylester) and its active metabolite oseltamivir acid.

    PubMed

    Escher, Beate I; Bramaz, Nadine; Lienert, Judit; Neuwoehner, Judith; Straub, Jürg Oliver

    2010-02-18

    Tamiflu (oseltamivir ethylester) is an antiviral agent for the treatment of influenza A and B. The pro-drug Tamiflu is converted in the human body to the pharmacologically active metabolite, oseltamivir acid, with a yield of 75%. Oseltamivir acid is indirectly photodegradable and slowly biodegradable in sewage works and sediment/water systems. A previous environmental risk assessment has concluded that there is no bioaccumulation potential of either of the compounds. However, little was known about the ecotoxicity of the metabolite. Ester hydrolysis typically reduces the hydrophobicity and thus the toxicity of a compound. In this case, a zwitterionic, but overall neutral species is formed from the charged parent compound. If the speciation and predicted partitioning into biological membranes is considered, the metabolite may have a relevant contribution to the overall toxicity. These theoretical considerations triggered a study to investigate the toxicity of oseltamivir acid (OA), alone and in binary mixtures with its parent compound oseltamivir ethylester (OE). OE and OA were found to be baseline toxicants in the bioluminescence inhibition test with Vibrio fischeri. Their mixture effect lay between predictions for concentration addition and independent action for the mixture ratio excreted in urine and nine additional mixture ratios of OE and OA. In contrast, OE was an order of magnitude more toxic than OA towards algae, with a more pronounced effect when the direct inhibition of photosystem II was used as toxicity endpoint opposed to the 24h growth rate endpoint. The binary mixtures in this assay yielded experimental mixture effects that agreed with predictions for independent action. This is consistent with the finding that OE exhibits slightly enhanced toxicity, while OA acts as baseline toxicant. Therefore, with respect to mixture classification, the two compounds can be considered as acting according to different modes of toxic action, although there are

  2. The Tamiflu fiasco and lessons learnt

    PubMed Central

    Gupta, Yogendra Kumar; Meenu, Meenakshi; Mohan, Prafull

    2015-01-01

    Oseltamivir (Tamiflu), a neuraminidase inhibitor, was approved for seasonal flu by US Food and Drug Administration in 1999. A number of randomized controlled trials, systematic reviews, and meta-analysis emphasized a favorable efficacy and safety profile. Majority of them were funded by Roche, which also first marketed and promoted this drug. In 2005 and 2009, the looming fear of pandemic flu led to recommendation by prominent regulatory bodies such as World Health Organization (WHO), Centers for Disease Control and Prevention, European Medicines Agency and others for its use in treatment and prophylaxis of influenza, and it's stockpiling as a measure to tide over the crisis. Serious Adverse Events, especially neuropsychiatric events associated with Tamiflu started getting reported leading to a cascade of questions on clinical utility of this drug. A recent Cochrane review and related articles have questioned the risk-benefit ratio of the drug, besides raising doubts about the regulatory decision of approving it. The recommendations for stockpiling the said drug as given by various international organizations viz WHO have also been put to scrutiny. Although many reviewers have labeled the Tamiflu saga as a “costly mistake,” the episode leaves us with some important lessons. This article takes a comprehensive relook on the subject, and we proceed to suggest some ways and means to avoid a similar situation in the future. PMID:25821304

  3. Detection and characterization of clostebol sulfate metabolites in Caucasian population.

    PubMed

    Balcells, Georgina; Pozo, Oscar J; Garrostas, Lorena; Esquivel, Argitxu; Matabosch, Xavier; Kotronoulas, Aristotelis; Joglar, Jesús; Ventura, Rosa

    2016-06-01

    Anabolic androgenic steroids (AAS) are synthetic testosterone derivatives which undergo extensive metabolism in man. Differences in the excretion of phase II metabolites are strongly associated with inter-individual and inter-ethnic variations. Sulfate metabolites have been described as long-term metabolites for some AAS. Clostebol is the 4-chloro derivative of testosterone and the aim of the present study was the evaluation of clostebol sulfate metabolites in Caucasian population by LC-MS/MS technology. Clostebol was orally administered to four healthy Caucasian male volunteers, and excretion study urines were collected up to 31 days. Several analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) were applied to detect sulfate metabolites in post-administration samples. Sixteen sulfate metabolites were detected, five of them having detectability times above 10 days (S1a, S2a, S3b, S3g and S4b). Interestingly, metabolite S1a could be detected up to the last collected sample of all excretion studies and it was characterized by LC-MS/MS and GC-MS as 4ξ-chloro-5α-androst-3β-ol-17-one 3β-sulfate. Thus, monitoring of S1a improves the detection time of clostebol misuse with respect to the commonly monitored metabolites, excreted in the glucuronide fraction. Importantly, this new metabolite can be incorporated into recently developed LC-MS/MS screening methods base on the direct detection of phase II metabolites.

  4. Meeting Report: Risk Assessment of Tamiflu Use Under Pandemic Conditions

    PubMed Central

    Singer, Andrew C.; Howard, Bruce M.; Johnson, Andrew C.; Knowles, Chris J.; Jackman, Simon; Accinelli, Cesare; Caracciolo, Anna Barra; Bernard, Ian; Bird, Stephen; Boucard, Tatiana; Boxall, Alistair; Brian, Jayne V.; Cartmell, Elise; Chubb, Chris; Churchley, John; Costigan, Sandra; Crane, Mark; Dempsey, Michael J.; Dorrington, Bob; Ellor, Brian; Fick, Jerker; Holmes, John; Hutchinson, Tom; Karcher, Franz; Kelleher, Samuel L.; Marsden, Peter; Noone, Gerald; Nunn, Miles A.; Oxford, John; Rachwal, Tony; Roberts, Noel; Roberts, Mike; Saccà, Maria Ludovica; Sanders, Matthew; Straub, Jürg Oliver; Terry, Adrian; Thomas, Dean; Toovey, Stephen; Townsend, Rodney; Voulvoulis, Nikolaos; Watts, Chris

    2008-01-01

    On 3 October 2007, 40 participants with diverse expertise attended the workshop Tamiflu and the Environment: Implications of Use under Pandemic Conditions to assess the potential human health impact and environmental hazards associated with use of Tamiflu during an influenza pandemic. Based on the identification and risk-ranking of knowledge gaps, the consensus was that oseltamivir ethylester-phosphate (OE-P) and oseltamivir carboxylate (OC) were unlikely to pose an ecotoxicologic hazard to freshwater organisms. OC in river water might hasten the generation of OC-resistance in wildfowl, but this possibility seems less likely than the potential disruption that could be posed by OC and other pharmaceuticals to the operation of sewage treatment plants. The work-group members agreed on the following research priorities: a) available data on the ecotoxicology of OE-P and OC should be published; b) risk should be assessed for OC-contaminated river water generating OC-resistant viruses in wildfowl; c) sewage treatment plant functioning due to microbial inhibition by neuraminidase inhibitors and other antimicrobials used during a pandemic should be investigated; and d) realistic worst-case exposure scenarios should be developed. Additional modeling would be useful to identify localized areas within river catchments that might be prone to high pharmaceutical concentrations in sewage treatment plant effluent. Ongoing seasonal use of Tamiflu in Japan offers opportunities for researchers to assess how much OC enters and persists in the aquatic environment. PMID:19057712

  5. Detection and quantification of boscalid and its metabolites in honeybees.

    PubMed

    Jabot, Claire; Daniele, Gaëlle; Giroud, Barbara; Tchamitchian, Sylvie; Belzunces, Luc P; Casabianca, Hervé; Vulliet, Emmanuelle

    2016-08-01

    Boscalid is a new-generation fungicide that has been detected in several bee matrices. The objective of this work was to characterize boscalid metabolites in honeybees based on in vivo experimentation, and next to verify the presence of theses metabolites into honeybees from colonies presenting troubles. A methodology based on complementary mass spectrometric tools, namely ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-QToF) or triple quadrupole mass spectrometry (UHPLC-QqQ) was implemented. Honeybees were sprayed with boscalid, at field rate (to induce the metabolization process) and the parent compound with its generated metabolites were then extracted using modified EU-QuEChERS method. The mass characteristics including exact mass, isotopic profile and mass fragments allowed assuming the structure of several metabolites. Some of them were unambiguously identified by comparison with synthesized analytical standards. The metabolites were resulted from hydroxylation and dechlorination of the parent compound as well as the substitution of a chlorine atom with an hydroxyl group. The metabolites were then quantified in bee samples collected from various beehives located in France. Boscalid and three of its metabolites were present in some samples at a level ranged between 0.2 and 36.3 ng/g.

  6. Systematic Identification of Protein-Metabolite Interactions in Complex Metabolite Mixtures by Ligand-Detected Nuclear Magnetic Resonance Spectroscopy.

    PubMed

    Nikolaev, Yaroslav V; Kochanowski, Karl; Link, Hannes; Sauer, Uwe; Allain, Frederic H-T

    2016-05-10

    Protein-metabolite interactions play a vital role in the regulation of numerous cellular processes. Consequently, identifying such interactions is a key prerequisite for understanding cellular regulation. However, the noncovalent nature of the binding between proteins and metabolites has so far hampered the development of methods for systematically mapping protein-metabolite interactions. The few available, largely mass spectrometry-based, approaches are restricted to specific metabolite classes, such as lipids. In this study, we address this issue and show the potential of ligand-detected nuclear magnetic resonance (NMR) spectroscopy, which is routinely used in drug development, to systematically identify protein-metabolite interactions. As a proof of concept, we selected four well-characterized bacterial and mammalian proteins (AroG, Eno, PfkA, and bovine serum albumin) and identified metabolite binders in complex mixes of up to 33 metabolites. Ligand-detected NMR captured all of the reported protein-metabolite interactions, spanning a full range of physiologically relevant Kd values (low micromolar to low millimolar). We also detected a number of novel interactions, such as promiscuous binding of the negatively charged metabolites citrate, AMP, and ATP, as well as binding of aromatic amino acids to AroG protein. Using in vitro enzyme activity assays, we assessed the functional relevance of these novel interactions in the case of AroG and show that l-tryptophan, l-tyrosine, and l-histidine act as novel inhibitors of AroG activity. Thus, we conclude that ligand-detected NMR is suitable for the systematic identification of functionally relevant protein-metabolite interactions.

  7. In situ detection of anaerobic alkane metabolites in subsurface environments.

    PubMed

    Agrawal, Akhil; Gieg, Lisa M

    2013-01-01

    Alkanes comprise a substantial fraction of crude oil and refined fuels. As such, they are prevalent within deep subsurface fossil fuel deposits and in shallow subsurface environments such as aquifers that are contaminated with hydrocarbons. These environments are typically anaerobic, and host diverse microbial communities that can potentially use alkanes as substrates. Anaerobic alkane biodegradation has been reported to occur under nitrate-reducing, sulfate-reducing, and methanogenic conditions. Elucidating the pathways of anaerobic alkane metabolism has been of interest in order to understand how microbes can be used to remediate contaminated sites. Alkane activation primarily occurs by addition to fumarate, yielding alkylsuccinates, unique anaerobic metabolites that can be used to indicate in situ anaerobic alkane metabolism. These metabolites have been detected in hydrocarbon-contaminated shallow aquifers, offering strong evidence for intrinsic anaerobic bioremediation. Recently, studies have also revealed that alkylsuccinates are present in oil and coal seam production waters, indicating that anaerobic microbial communities can utilize alkanes in these deeper subsurface environments. In many crude oil reservoirs, the in situ anaerobic metabolism of hydrocarbons such as alkanes may be contributing to modern-day detrimental effects such as oilfield souring, or may lead to more beneficial technologies such as enhanced energy recovery from mature oilfields. In this review, we briefly describe the key metabolic pathways for anaerobic alkane (including n-alkanes, isoalkanes, and cyclic alkanes) metabolism and highlight several field reports wherein alkylsuccinates have provided evidence for anaerobic in situ alkane metabolism in shallow and deep subsurface environments.

  8. In situ detection of anaerobic alkane metabolites in subsurface environments

    PubMed Central

    Agrawal, Akhil; Gieg, Lisa M.

    2013-01-01

    Alkanes comprise a substantial fraction of crude oil and refined fuels. As such, they are prevalent within deep subsurface fossil fuel deposits and in shallow subsurface environments such as aquifers that are contaminated with hydrocarbons. These environments are typically anaerobic, and host diverse microbial communities that can potentially use alkanes as substrates. Anaerobic alkane biodegradation has been reported to occur under nitrate-reducing, sulfate-reducing, and methanogenic conditions. Elucidating the pathways of anaerobic alkane metabolism has been of interest in order to understand how microbes can be used to remediate contaminated sites. Alkane activation primarily occurs by addition to fumarate, yielding alkylsuccinates, unique anaerobic metabolites that can be used to indicate in situ anaerobic alkane metabolism. These metabolites have been detected in hydrocarbon-contaminated shallow aquifers, offering strong evidence for intrinsic anaerobic bioremediation. Recently, studies have also revealed that alkylsuccinates are present in oil and coal seam production waters, indicating that anaerobic microbial communities can utilize alkanes in these deeper subsurface environments. In many crude oil reservoirs, the in situ anaerobic metabolism of hydrocarbons such as alkanes may be contributing to modern-day detrimental effects such as oilfield souring, or may lead to more beneficial technologies such as enhanced energy recovery from mature oilfields. In this review, we briefly describe the key metabolic pathways for anaerobic alkane (including n-alkanes, isoalkanes, and cyclic alkanes) metabolism and highlight several field reports wherein alkylsuccinates have provided evidence for anaerobic in situ alkane metabolism in shallow and deep subsurface environments. PMID:23761789

  9. Detection of a reactive metabolite of misonidazole in human urine

    SciTech Connect

    Varghese, A.J.; Whitmore, G.F.

    1984-08-01

    Chemical studies have indicated that, following reduction of misonidazole to the hydroxylamine derivative, reaction with guanosine leads to the formation of a 2-carbon addition product of guanosine. In this study, the formation of the guanosine product is used to detect the presence of a reactive metabolite of misonidazole in the urine of patients treated with misonidazole. Urine samples were incubated with (/sup 14/C)guanosine and the guanosine product was separated by HPLC analysis. The quantities of product vary as much as 10-fold from patient to patient and it is suggested that the assay be useful as a predictor of patients susceptible to the development of peripheral neuropathy or other effects of misonidazole.

  10. LC-MS/MS detection of unaltered glucuronoconjugated metabolites of metandienone.

    PubMed

    Esquivel, Argitxu; Pozo, Oscar J; Garrostas, Lorena; Balcells, Georgina; Gómez, Cristina; Kotronoulas, Aristotelis; Joglar, Jesús; Ventura, Rosa

    2016-05-30

    The aim of this study was to evaluate the direct detection of glucuronoconjugated metabolites of metandienone (MTD) and their detection times. Metabolites resistant to enzymatic hydrolysis were also evaluated. Based on the common mass spectrometric behaviour of steroid glucuronides, three liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategies were applied for the detection of unpredicted and predicted metabolites: precursor ion scan (PI), neutral loss scan (NL), and theoretical selected reaction monitoring (SRM) methods. Samples from four excretion studies of MTD were analyzed for both the detection of metabolites and the establishment of their detection times. Using PI and NL methods, seven metabolites were observed in post-administration samples. SRM methods allowed for the detection of 13 glucuronide metabolites. The detection times, measured by analysis with an SRM method, were between 1 and 22 days. The metabolite detected for the longest time was 18-nor-17β-hydroxymethyl-17α-methyl-5β-androsta-1,4,13-triene-3-one-17-glucuronide. One metabolite was resistant to hydrolysis with β-glucuronidase; however it was only detected in urine up to four days after administration. The three glucuronide metabolites with the highest retrospectivity were identified by chemical synthesis or mass spectrometric data, and although they were previously reported, this is the first time that analytical data of the intact phase II metabolites are presented for some of them. The LC-MS/MS strategies applied have demonstrated to be useful for detecting glucuronoconjugated metabolites of MTD, including glucuronides resistant to enzymatic hydrolysis which cannot be detected by conventional approaches. Copyright © 2016 John Wiley & Sons, Ltd.

  11. Detection of metabolites of a veterinary counter-irritant in canine urine.

    PubMed

    Dalefield, R R; Oehme, F W

    1998-08-01

    Routine paper chromatographic screening of the urine of racing greyhounds exposed to BIGELOIL, a veterinary counter-irritant, revealed metabolites suggestive of menthol, an ingredient of BIGELOIL. To determine whether BIGELOIL use caused these metabolites, 2 Dalmatian dogs were exposed to BIGELOIL. Thin-layer chromatographic screening of their urine confirmed that exposure to BIGELOIL by either dermal or oral routes causes the same metabolites as those observed in the racing greyhounds. Metabolites suggestive of thymol were also present in some samples. We conclude that, if metabolites suggestive of menthol are detected in urine of animal athletes, further analysis for the other performance-affecting ingredients of BIGELOIL should be undertaken.

  12. Detection of Nitrobenzodiazepines and Their 7-Amino Metabolites in Oral Fluid.

    PubMed

    Vindenes, Vigdis; Strand, Dag Helge; Koksæter, Paul; Gjerde, Hallvard

    2016-05-01

    Clonazepam, nitrazepam and flunitrazepam are frequently used benzodiazepines, both as prescribed medication and as drugs of abuse. Little is, however, known about how these drugs are excreted in oral fluid. It has been claimed that the parent drugs are more likely to be detected in oral fluid than the 7-amino metabolites. The aim of this study was to investigate whether the parent drugs or the 7-amino metabolites of the nitrobenzodiazepines were most frequently detected in authentic oral fluid samples. Oral fluid samples were collected from patients undergoing opioid maintenance treatment. Cases where clonazepam, nitrazepam, flunitrazepam and/or their metabolites were detected were included. The samples were collected using the Intercept Oral Specimen Collection Device. A cutoff concentration of 1 nM (∼0.3 ng/mL) in oral fluid-buffer mixture was applied for all the substances. A total of 1,001 oral fluid samples were positive for clonazepam and/or 7-aminoclonazepam; both substances were detected in 707 samples, only the parent drug in 64 cases and only the metabolite in 230 cases. For nitrazepam, both substances were detected in 139 samples; only the parent drug in 16 cases and only the metabolite in 56 cases. Flunitrazepam only was not detected in any sample; both substances were detected in one of these cases, and only the metabolite in three cases. This study revealed that 7-amino metabolites were more likely to be detected in oral fluid than the parent drugs.

  13. Development of A Concise Synthesis of (−)-Oseltamivir (Tamiflu®)

    PubMed Central

    Trost, Barry M.; Zhang, Ting

    2011-01-01

    We report a full account of our work towards the development of an eight-step synthesis of anti-influenza drug (−)-oseltamivir (Tamiflu®) from commercially available starting material. The final synthetic route proceeds with an overall yield of 30 %. Key transformations include a novel palladium-catalyzed asymmetric allylic alkylation reaction (Pd-AAA) as well as a rhodium-catalyzed chemo-, regio-, and stereoselective aziridination reaction. PMID:21365707

  14. Sulfate metabolites as alternative markers for the detection of 4-chlorometandienone misuse in doping control.

    PubMed

    Balcells, Georgina; Gómez, Cristina; Garrostas, Lorena; Pozo, Óscar J; Ventura, Rosa

    2016-09-30

    Sulfate metabolites have been described as long-term metabolites for some anabolic androgenic steroids (AAS). 4-chlorometandienone (4Cl-MTD) is one of the most frequently detected AAS in sports drug testing and it is commonly detected by monitoring metabolites excreted free or conjugated with glucuronic acid. Sulfation reactions of 4Cl-MTD have not been studied. The aim of this work was to evaluate the sulfate fraction of 4Cl-MTD metabolism by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to establish potential long-term metabolites valuable for doping control purposes. 4Cl-MTD was administered to two healthy male volunteers and urine samples were collected up to 8 days after administration. A theoretical selected reaction monitoring (SRM) method working in negative mode was developed. Ion transitions were based on ionization and fragmentation behaviour of sulfate metabolites as well as specific neutral losses (NL of 15 Da and NL of 36 Da) of compounds with related chemical structure. Six sulfate metabolites were detected after the analysis of excretion study samples. Three of the identified metabolites were characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS). Results showed that five out of the six identified sulfate metabolites were detected in urine up to the last collected samples from both excretion studies. Copyright © 2016 John Wiley & Sons, Ltd.

  15. Detection of Volatile Metabolites of Garlic in Human Breast Milk.

    PubMed

    Scheffler, Laura; Sauermann, Yvonne; Zeh, Gina; Hauf, Katharina; Heinlein, Anja; Sharapa, Constanze; Buettner, Andrea

    2016-06-06

    The odor of human breast milk after ingestion of raw garlic at food-relevant concentrations by breastfeeding mothers was investigated for the first time chemo-analytically using gas chromatography-mass spectrometry/olfactometry (GC-MS/O), as well as sensorially using a trained human sensory panel. Sensory evaluation revealed a clear garlic/cabbage-like odor that appeared in breast milk about 2.5 h after consumption of garlic. GC-MS/O analyses confirmed the occurrence of garlic-derived metabolites in breast milk, namely allyl methyl sulfide (AMS), allyl methyl sulfoxide (AMSO) and allyl methyl sulfone (AMSO₂). Of these, only AMS had a garlic-like odor whereas the other two metabolites were odorless. This demonstrates that the odor change in human milk is not related to a direct transfer of garlic odorants, as is currently believed, but rather derives from a single metabolite. The formation of these metabolites is not fully understood, but AMSO and AMSO₂ are most likely formed by the oxidation of AMS in the human body. The excretion rates of these metabolites into breast milk were strongly time-dependent with large inter-individual differences.

  16. Detection of Volatile Metabolites of Garlic in Human Breast Milk

    PubMed Central

    Scheffler, Laura; Sauermann, Yvonne; Zeh, Gina; Hauf, Katharina; Heinlein, Anja; Sharapa, Constanze; Buettner, Andrea

    2016-01-01

    The odor of human breast milk after ingestion of raw garlic at food-relevant concentrations by breastfeeding mothers was investigated for the first time chemo-analytically using gas chromatography−mass spectrometry/olfactometry (GC-MS/O), as well as sensorially using a trained human sensory panel. Sensory evaluation revealed a clear garlic/cabbage-like odor that appeared in breast milk about 2.5 h after consumption of garlic. GC-MS/O analyses confirmed the occurrence of garlic-derived metabolites in breast milk, namely allyl methyl sulfide (AMS), allyl methyl sulfoxide (AMSO) and allyl methyl sulfone (AMSO2). Of these, only AMS had a garlic-like odor whereas the other two metabolites were odorless. This demonstrates that the odor change in human milk is not related to a direct transfer of garlic odorants, as is currently believed, but rather derives from a single metabolite. The formation of these metabolites is not fully understood, but AMSO and AMSO2 are most likely formed by the oxidation of AMS in the human body. The excretion rates of these metabolites into breast milk were strongly time-dependent with large inter-individual differences. PMID:27275838

  17. Detection of Proton Chemical Exchange between Metabolites and Water in Biological Tissues

    NASA Astrophysics Data System (ADS)

    Guivel-Scharen, V.; Sinnwell, T.; Wolff, S. D.; Balaban, R. S.

    1998-07-01

    Metabolites in proton chemical exchange with water were detected via the water proton signal using saturation transfer techniques in model systems and biological tissues. The metabolites were selectively saturated and the resulting decrease in the much larger water proton pool was used to monitor the metabolite. This indirect detection scheme can result in a several orders of magnitude increase in sensitivity for metabolites over direct detection methods. A control irradiation scheme was devised to compensate for macromolecular/water magnetization transfer. Using this approach, significant chemical exchange regions at ∼1 and 2.5 ppm were detected in kidney medulla. Using a difference imaging technique between a control irradiation above (-1.74 ppm) and below (+1.74 ppm) the water resonance, a chemical exchange image of the kidney was calculated. These data revealed a linear gradient of chemical exchange increasing from the cortex to the medulla. Studies on medullary acid extracts and urine revealed that the exchange observed in the kidney was predominantly with low molecular weight metabolites. Urea (1 ppm) was identified as contributing to the kidney/urine chemical exchange; however, other unidentified metabolites may also contribute to this effect. These studies demonstrate that tissue metabolites can be detected and imaged via the water protons using the signal amplification properties of saturation transfer in the presence of water/macromolecule magnetization transfer.

  18. Detection of hepatotoxicity potential with metabolite profiling (metabolomics) of rat plasma.

    PubMed

    Mattes, W; Davis, K; Fabian, E; Greenhaw, J; Herold, M; Looser, R; Mellert, W; Groeters, S; Marxfeld, H; Moeller, N; Montoya-Parra, G; Prokoudine, A; van Ravenzwaay, B; Strauss, V; Walk, T; Kamp, H

    2014-11-04

    While conventional parameters used to detect hepatotoxicity in drug safety assessment studies are generally informative, the need remains for parameters that can detect the potential for hepatotoxicity at lower doses and/or at earlier time points. Previous work has shown that metabolite profiling (metabonomics/metabolomics) can detect signals of potential hepatotoxicity in rats treated with doxorubicin at doses that do not elicit hepatotoxicity as monitored with conventional parameters. The current study extended this observation to the question of whether such signals could be detected in rats treated with compounds that can elicit hepatotoxicity in humans (i.e., drug-induced liver injury, DILI) but have not been reported to do so in rats. Nine compounds were selected on the basis of their known DILI potential, with six other compounds chosen as negative for DILI potential. A database of rat plasma metabolite profiles, MetaMap(®)Tox (developed by metanomics GmbH and BASF SE) was used for both metabolite profiles and mode of action (MoA) metabolite signatures for a number of known toxicities. Eight of the nine compounds with DILI potential elicited metabolite profiles that matched with MoA patterns of various rat liver toxicities, including cholestasis, oxidative stress, acetaminophen-type toxicity and peroxisome proliferation. By contrast, only one of the six non-DILI compounds showed a weak match with rat liver toxicity. These results suggest that metabolite profiling may indeed have promise to detect signals of hepatotoxicity in rats treated with compounds having DILI potential.

  19. Detection of driver metabolites in the human liver metabolic network using structural controllability analysis

    PubMed Central

    2014-01-01

    Background Abnormal states in human liver metabolism are major causes of human liver diseases ranging from hepatitis to hepatic tumor. The accumulation in relevant data makes it feasible to derive a large-scale human liver metabolic network (HLMN) and to discover important biological principles or drug-targets based on network analysis. Some studies have shown that interesting biological phenomenon and drug-targets could be discovered by applying structural controllability analysis (which is a newly prevailed concept in networks) to biological networks. The exploration on the connections between structural controllability theory and the HLMN could be used to uncover valuable information on the human liver metabolism from a fresh perspective. Results We applied structural controllability analysis to the HLMN and detected driver metabolites. The driver metabolites tend to have strong ability to influence the states of other metabolites and weak susceptibility to be influenced by the states of others. In addition, the metabolites were classified into three classes: critical, high-frequency and low-frequency driver metabolites. Among the identified 36 critical driver metabolites, 27 metabolites were found to be essential; the high-frequency driver metabolites tend to participate in different metabolic pathways, which are important in regulating the whole metabolic systems. Moreover, we explored some other possible connections between the structural controllability theory and the HLMN, and find that transport reactions and the environment play important roles in the human liver metabolism. Conclusion There are interesting connections between the structural controllability theory and the human liver metabolism: driver metabolites have essential biological functions; the crucial role of extracellular metabolites and transport reactions in controlling the HLMN highlights the importance of the environment in the health of human liver metabolism. PMID:24885538

  20. Detection and characterization of a new metabolite of 17alpha-methyltestosterone.

    PubMed

    Pozo, Oscar J; Van Eenoo, Peter; Deventer, Koen; Lootens, Leen; Van Thuyne, Wim; Parr, Maria K; Schänzer, Wilhelm; Sancho, Juan V; Hernández, Felix; Meuleman, Philip; Leroux-Roels, Geert; Delbeke, Frans T

    2009-11-01

    The misuse of the anabolic steroid methyltestosterone is currently routinely monitored in doping control laboratories by gas chromatography-mass spectrometry (GC-MS) of two of its metabolites: 17alpha-methyl-5beta-androstane-3alpha,17beta-diol and 17alpha-methyl-5alpha-androstane-3alpha,17beta-diol. Because of the absence of any easy ionizable moiety, these metabolites are poorly detectable using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI). In this study, the metabolism of methyltestosterone has been reinvestigated by the use of a precursor ion scan method in LC-ESI-MS/MS. Two metabolites have been detected using this method. Both compounds have been confirmed in postadministration urine samples of an urokinase plasminogen activator-severe combined immunodeficiency (uPA-SCID) mouse with humanized liver and were characterized by LC-MS/MS and GC-MS using both quadrupole and time of flight analyzers. From the detailed study of the fragmentation, these metabolites were proposed to be epimethyltestosterone and a dehydrogenated compound. Epimethyltestosterone has previously been described as a minor metabolite, whereas the occurrence of the oxidized metabolite has not been reported. Comparison with the synthesized reference revealed that the structure of the dehydrogenated metabolite is 6-ene-epimethyltestosterone. A selected reaction monitoring method including three transitions for each metabolite has been developed and applied to samples from an excretion study and to samples declared positive after GC-MS analysis. 6-Ene-epimethyltestosterone was found in all samples, showing its applicability in the detection of methyltestosterone misuse.

  1. Detection and characterization of betamethasone metabolites in human urine by LC-MS/MS.

    PubMed

    Matabosch, Xavier; Pozo, Oscar J; Monfort, Núria; Pérez-Mañá, Clara; Farré, Magí; Segura, Jordi; Ventura, Rosa

    2015-08-01

    Glucocorticosteroids are prohibited in sports when administered by systemic routes and allowed using other administrations for therapeutic reasons. Therefore, markers to distinguish between routes of administration through the analysis of urine samples are needed in anti-doping control. As a first step to achieve that goal, the metabolism of betamethasone (BET) was investigated in the present work. Urine samples obtained after BET intramuscular injection were hydrolyzed with β-glucuronidase and subjected to liquid-liquid extraction with ethyl acetate in alkaline conditions. The extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry. Common open screening methods for fluorine containing corticosteroids (precursor ion scan method of m/z 121, 147, 171, and neutral loss (NL) scan methods of 20 and 38 Da in positive ionization, and 46 and 76 Da in negative ionization) were applied to detect BET metabolites. Moreover, an NL method was applied to detect A-ring reduced metabolites of BET, which are ionized as [M+NH4 ](+) (NL of 55, 73, and 91 Da, corresponding to the consecutive losses of NH3 , HF and one, two and three water molecules, respectively). BET and 24 metabolites were detected. Six metabolites were identified by comparison with standards, and for ten, feasible structures were proposed based on mass spectrometric data. Eleven of the characterized metabolites had not been previously reported. Metabolites resulting from 11-oxidation, 6-hydroxylation, C20 or 4-ene-3-one reduction and combination of some of them were detected. Moreover one metabolite resulting from cleavage of the side chain with subsequent oxidation of carbon at C17 was also detected.

  2. Effects of oseltamivir phosphate (Tamiflu) in human sera on results of microneutralization and hemagglutinin-inhibition tests for H5N2 avian influenza virus.

    PubMed

    Yamazaki, Yoshinao; Doy, M; Yamato, S; Kawada, Y; Ogata, T

    2008-01-01

    To determine the influence of oseltamivir phosphate (Tamiflu) on the results of microneutralization and hemagglutinin-inhibition (HI) tests in human sera with H5N2 influenza virus, ten volunteers were administered Tamiflu and blood samples were collected. In the microneutralization test, no consistent effects were observed. However, in the HI test, specimens from all volunteers taken at 4 and 7 h after drug administration showed a higher titer as compared to 0 and 24 h after administration when mammalian cells (horse, guinea pig, and human) were used. These results suggest that the administration of Tamiflu may affect the results of HI tests for H5N2 virus.

  3. Optimization of metabolite detection by quantum mechanics simulations in magnetic resonance spectroscopy.

    PubMed

    Gambarota, Giulio

    2016-09-03

    Magnetic resonance spectroscopy (MRS) is a well established modality for investigating tissue metabolism in vivo. In recent years, many efforts by the scientific community have been directed towards the improvement of metabolite detection and quantitation. Quantum mechanics simulations allow for investigations of the MR signal behaviour of metabolites; thus, they provide an essential tool in the optimization of metabolite detection. In this review, we will examine quantum mechanics simulations based on the density matrix formalism. The density matrix was introduced by von Neumann in 1927 to take into account statistical effects within the theory of quantum mechanics. We will discuss the main steps of the density matrix simulation of an arbitrary spin system and show some examples for the strongly coupled two spin system.

  4. Detection of 191 Taxifolin Metabolites and Their Distribution in Rats Using HPLC-ESI-IT-TOF-MS(n).

    PubMed

    Yang, Ping; Xu, Feng; Li, Hong-Fu; Wang, Yi; Li, Feng-Chun; Shang, Ming-Ying; Liu, Guang-Xue; Wang, Xuan; Cai, Shao-Qing

    2016-09-13

    Taxifolin is a ubiquitous bioactive constituent of foods and herbs. To thoroughly explore its metabolism in vivo, an HPLC-ESI-IT-TOF-MS(n) method combined with specific metabolite detection strategy was used to detect and identify the metabolites of taxifolin in rats. Of the 191 metabolites tentatively identified, 154 were new metabolites, 69 were new compounds and 32 were dimers. This is the first report of the in vivo biotransformation of a single compound into more than 100 metabolites. Furthermore, acetylamination and pyroglutamic acid conjugation were identified as new metabolic reactions. Seventeen metabolites were found to have various taxifolin-related bioactivities. The potential targets of taxifolin and 63 metabolites were predicted using PharmMapper, with results showing that more than 60 metabolites have the same five targets. Metabolites with the same fragment pattern may have the same pharmacophore. Thus these metabolites may exert the same pharmacological effects as taxifolin through an additive effect on the same drug targets. This observation indicates that taxifolin is bioactive not only in the parent form, but also through its metabolites. These findings enhance understanding of the metabolism and effective forms of taxifolin and may provide further insight of the beneficial effects of taxifolin and its derivatives.

  5. Simultaneous detection of four nitrofuran metabolites in honey using a multiplexing biochip screening assay.

    PubMed

    O'Mahony, John; Moloney, Mary; McConnell, Robert I; Benchikh, El O; Lowry, Philip; Furey, Ambrose; Danaher, Martin

    2011-06-15

    A chemiluminescence-based biochip array sensing technique has been developed and applied to the screening of honey samples for residues of banned nitrofuran antibiotics. Using a multiplex approach, metabolites of the four main nitrofuran antibiotics could be simultaneously detected. Individual antibodies specific towards the metabolites were spotted onto biochips. A competitive assay format, with chemiluminescent response, was employed. The method was validated in accordance with EU legislation (2002/657/EC, 2002), and assessed by comparison with UHPLC-MS/MS testing of 134 honey samples of worldwide origin. A similar extraction method, based on extraction of the analytes on Oasis™ SPE cartridges, followed by derivatisation with nitrobenzaldehyde and partition into ethyl acetate, was used for both screening and LC-MS/MS methods. The biochip array method was capable of detecting all four metabolites below the reference point for action of 1 μg kg(-1). The detection capability was below 0.5 μg kg(-1) for the metabolites AHD, AOZ and AMOZ; it was below 0.9 μg kg(-1) for SEM. IC(50) values ranged from 0.14 μg kg(-1) (AMOZ) to 2.19 μg kg(-1) (SEM). This biosensor method possesses the potential to be a fit-for-purpose screening technique in the arena of food safety technology.

  6. Chromatographic tandam mass spectrometric detection of papaverine and its major metabolites in rat urine

    NASA Astrophysics Data System (ADS)

    Peng, Zhihong; Song, Wei; Han, Fengmei; Chen, Huaixia; Zhu, Mingming; Chen, Yong

    2007-10-01

    A rapid, sensitive and specific liquid chromatographic-electrospray ionization (ESI) tandem ion trap mass spectrometric method has been developed for identification of papaverine and its metabolites in rat urine. Six healthy rats were administrated a single dose (100 mg/kg) of papaverine by oral gavage. The urine were sampled for 0-24 h and purified by using a C18 solid-phase extraction cartridge, then the purified urine samples were separated on a reversed-phase C18 column using methanol/2 mmol/L ammonium acetate (70:30, v/v, adjusted to pH 3.5 with formic acid) as mobile phase and detected by an on-line MS detector. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular mass ([Delta]m) and full scan MSn spectra with those of the parent drug. The results indicated that there were 14 metabolites in rat urine, such as de-methoxyl, hydroxyl, glucuronide and sulfate conjugated metabolites and so on. All these metabolites were reported for the first time.

  7. Graphene nano-ink biosensor arrays on a microfluidic paper for multiplexed detection of metabolites.

    PubMed

    Labroo, Pratima; Cui, Yue

    2014-02-27

    The development of a miniaturized and low-cost platform for the highly sensitive, selective and rapid detection of multiplexed metabolites is of great interest for healthcare, pharmaceuticals, food science, and environmental monitoring. Graphene is a delicate single-layer, two-dimensional network of carbon atoms with extraordinary electrical sensing capability. Microfluidic paper with printing technique is a low cost matrix. Here, we demonstrated the development of graphene-ink based biosensor arrays on a microfluidic paper for the multiplexed detection of different metabolites, such as glucose, lactate, xanthine and cholesterol. Our results show that the graphene biosensor arrays can detect multiple metabolites on a microfluidic paper sensitively, rapidly and simultaneously. The device exhibits a fast measuring time of less than 2 min, a low detection limit of 0.3 μM, and a dynamic detection range of 0.3-15 μM. The process is simple and inexpensive to operate and requires a low consumption of sample volume. We anticipate that these results could open exciting opportunities for a variety of applications.

  8. Separation and detection of amino acid metabolites of Escherichia coli in microbial fuel cell with CE.

    PubMed

    Wang, Wei; Ma, Lihong; Lin, Ping; Xu, Kaixuan

    2016-07-01

    In this work, CE-LIF was employed to investigate the amino acid metabolites produced by Escherichia coli (E. coli) in microbial fuel cell (MFC). Two peptides, l-carnosine and l-alanyl-glycine, together with six amino acids, cystine, alanine, lysine, methionine, tyrosine, arginine were separated and detected in advance by a CE-LIF system coupled with a homemade spontaneous injection device. The injection device was devised to alleviate the effect of electrical discrimination for analytes during sample injection. All analytes could be completely separated within 8 min with detection limits of 20-300 nmol/L. Then this method was applied to analyze the substrate solution containing amino acid metabolites produced by E. coli. l-carnosine, l-alanyl-glycine, and cystine were used as the carbon, nitrogen, and sulfur source for the E. coli culture in the MFC to investigate the amino acid metabolites during metabolism. Two MFCs were used to compare the activity of metabolism of the bacteria. In the sample collected at the running time 200 h of MFC, the amino acid methionine was discovered as the metabolite with the concentrations 23.3 μg/L.

  9. Methods to Detect Nitric Oxide and its Metabolites in Biological Samples

    PubMed Central

    Bryan, Nathan S.; Grisham, Matthew B.

    2007-01-01

    Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. NO is involved in many physiological processes including regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification is critical to understanding health and disease. Due to the extremely short physiological half life of this gaseous free radical, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of NO metabolites in biological samples provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The methods described in this review is not an exhaustive or comprehensive discussion of all methods available for the detection of NO but rather a description of the most commonly used and practical methods which allow accurate and sensitive quantification of NO products/metabolites in multiple biological matrices under normal physiological conditions. PMID:17664129

  10. Detection and identification of plasma progesterone metabolites in the female Florida manatee (Trichechus manatus latirostris) using GC/MS/MS.

    PubMed

    Tripp, K M; Dubois, M; Delahaut, P; Verstegen, J P

    2009-08-01

    Florida manatees (Trichechus manatus latirostris) have relatively low peripheral concentrations of progesterone (P4). The objective of this study was to determine if these relatively low P4 concentrations are associated with a high ratio of progestin metabolites and to document metabolite concentrations from individual blood samples obtained from manatees during diestrus or pregnancy. Metabolites known to exist in elephants-terrestrial manatee relatives-were targeted. These included 5alpha-reduced progestins (5alpha-pregnane-3,20-dione [5alpha-DHP] and 3alpha-hydroxy-5alpha-pregnan-20-one [5alpha-P3-OH]) and 17alpha-hydroxyprogesterone (17alpha-OHP), which occurs in Asian elephants. An additional, inactive metabolite, 20alpha-hydroxyprogesterone (20alpha-OHP), indicative of P4 overproduction, was also targeted. Progesterone itself was the predominant progestin detected in pregnant and nonpregnant manatee plasma (n = 10) using gas chromatography-mass spectrometry with tandem quadrupole detectors (GC/MS/MS). Progesterone concentrations in pregnant females varied from early (moderate to high) through mid and late (low) pregnancy. Progesterone concentrations ranged from low to high in nonpregnant, nonlactating females. The most commonly detected metabolite was 5alpha-P3-OH (n = 7), which occurred in pregnant (lower limit of detection [LLOD] to high) and nonpregnant (trace to high) females. The 5alpha-DHP metabolite was also detected in pregnant (LLOD to moderate) and nonpregnant (low) females. The 17alpha-OHP metabolite was not detected in any tested female. The 20alpha-OHP metabolite was detected in one nonpregnant, nonlactating, captive female (LLOD). Metabolites were most prevalent during early pregnancy, concurrent with maximum P4 concentrations. Based on their concentrations in peripheral circulation, we inferred that these metabolites may have, opposite to elephants, a limited physiologic role during luteal, pregnant, and nonpregnant phases in the manatee.

  11. Detection of Pesticides and Pesticide Metabolites Using the Cross Reactivity of Enzyme Immunoassays

    USGS Publications Warehouse

    Thurman, E.M.; Aga, D.S.

    2001-01-01

    Enzyme immunoassay is an important environmental analysis method that may be used to identify many pesticide analytes in water samples. Because of similarities in chemical structure between various members of a pesticide class, there often may be an unwanted response that is characterized by a percentage of cross reactivity. Also, there may be cross reactivity caused by degradation products of the target analyte that may be present in the sample. In this paper, the concept of cross reactivity caused by degradation products or by nontarget analytes is explored as a tool for identification of metabolites or structurally similar compounds not previously known to be present in water samples. Two examples are examined in this paper from various water quality studies. They are alachlor and its metabolite, alachlor ethane sulfonic acid, and atrazine and its class members, prometryn and propazine. A method for using cross reactivity for the detection of these compounds is explained in this paper.

  12. Detection of pesticides and pesticide metabolites using the cross reactivity of enzyme immunoassays.

    PubMed

    Thurman, E M; Aga, D S

    2001-01-01

    Enzyme immunoassay is an important environmental analysis method that may be used to identify many pesticide analytes in water samples. Because of similarities in chemical structure between various members of a pesticide class, there often may be an unwanted response that is characterized by a percentage of cross reactivity. Also, there may be cross reactivity caused by degradation products of the target analyte that may be present in the sample. In this paper, the concept of cross reactivity caused by degradation products or by nontarget analytes is explored as a tool for identification of metabolites or structurally similar compounds not previously known to be present in water samples. Two examples are examined in this paper from various water quality studies. They are alachlor and its metabolite, alachlor ethane sulfonic acid, and atrazine and its class members, prometryn and propazine. A method for using cross reactivity for the detection of these compounds is explained in this paper.

  13. Detection of stanozolol O- and N-sulfate metabolites and their evaluation as additional markers in doping control.

    PubMed

    Balcells, Georgina; Matabosch, Xavier; Ventura, Rosa

    2016-10-07

    Stanozolol (STAN) is one of the most frequently detected anabolic androgenic steroids in sports drug testing. STAN misuse is commonly detected by monitoring metabolites excreted conjugated with glucuronic acid after enzymatic hydrolysis or using direct detection by liquid chromatography-tandem mass spectrometry (LC-MS/MS). It is well known that some of the previously described metabolites are the result of the formation of sulfate conjugates in C17, which are converted to their 17-epimers in urine. Therefore, sulfation is an important phase II metabolic pathway of STAN that has not been comprehensively studied. The aim of this work was to evaluate the sulfate fraction of STAN metabolism by LC-MS/MS to establish potential long-term metabolites valuable for doping control purposes. STAN was administered to six healthy male volunteers involving oral or intramuscular administration and urine samples were collected up to 31 days after administration. Sulfation of the phase I metabolites commercially available as standards was performed in order to obtain MS data useful to develop analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) to detect potential sulfate metabolites. Eleven sulfate metabolites (M-I to M-XI) were detected and characterized by LC-MS/MS. This paper provides valuable data on the ionization and fragmentation of O-sulfates and N-sulfates. For STAN, results showed that sulfates do not improve the retrospectivity of the detection compared to the previously described long-term metabolite (epistanozolol-N-glucuronide). However, sulfate metabolites could be additional markers for the detection of STAN misuse.

  14. Aflatoxin metabolism in humans: detection of metabolites and nucleic acid adducts in urine by affinity chromatography

    SciTech Connect

    Groopman, J.D.; Donahue, P.R.; Zhu, J.Q.; Chen, J.S.; Wogan, G.N.

    1985-10-01

    A high-affinity IgM monoclonal antibody specific for aflatoxins was covalently bound to Sepharose 4B and used as a preparative column to isolate aflatoxin derivatives from the urine of people and experimental animals who had been exposed to the carcinogen environmentally or under laboratory conditions. Aflatoxin levels were quantified by radioimmunoassay and high-performance liquid chromatography after elution from the affinity column. In studies on rats injected with ( UC)aflatoxin B1, the authors identified the major aflatoxin-DNA adduct, 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-aflatoxin B1 (AFB1-N7-Gua), and the oxidative metabolites M1 and P1 as the major aflatoxin species present in the urine. When this methodology was applied to human urine samples obtained from people from the Guangxi Province of China exposed to aflatoxin B1 through dietary contamination, the aflatoxin metabolites detected were also AFB1-N7-Gua and aflatoxins M1 and P1. Therefore, affinity chromatography using a monoclonal antibody represents a useful and rapid technique with which to isolate this carcinogen and its metabolites in biochemical epidemiology and for subsequent quantitative measurements, providing exposure information that can be used for risk assessment.

  15. Development of a Biosensor Concept to Detect the Production of Cluster-Specific Secondary Metabolites.

    PubMed

    Sun, Yi-Qian; Busche, Tobias; Rückert, Christian; Paulus, Constanze; Rebets, Yuriy; Novakova, Renata; Kalinowski, Jörn; Luzhetskyy, Andriy; Kormanec, Jan; Sekurova, Olga N; Zotchev, Sergey B

    2017-03-03

    Genome mining of actinomycete bacteria aims at the discovery of novel bioactive secondary metabolites that can be developed into drugs. A new repressor-based biosensor to detect activated secondary metabolite biosynthesis gene clusters in Streptomyces was developed. Biosynthetic gene clusters for undecylprodigiosin and coelimycin in the genome of Streptomyces lividans TK24, which encoded TetR-like repressors and appeared to be almost "silent" based on the RNA-seq data, were chosen for the proof-of-principle studies. The bpsA reporter gene for indigoidine synthetase was placed under control of the promotor/operator regions presumed to be controlled by the cluster-associated TetR-like repressors. While the biosensor for undecylprodigiosin turned out to be nonfunctional, the coelimycin biosensor was shown to perform as expected, turning on biosynthesis of indigoidine in response to the concomitant production of coelimycin. The developed reporter system concept can be applied to those cryptic gene clusters that encode metabolite-sensing repressors to speed up discovery of novel bioactive compounds in Streptomyces.

  16. In vivo detection of the new psychoactive substance AM-694 and its metabolites.

    PubMed

    Bertol, Elisabetta; Vaiano, Fabio; Di Milia, Maria Grazia; Mari, Francesco

    2015-11-01

    AM-694 or 1-(5-fluoropentyl)-3-(2-iodobenzoyl)indole is a synthetic cannabinoid that acts as a selective and a powerful agonist for CB1 receptor, inducing cannabinoid-like effects (euphoria, sedation, hallucinations and anxiety). Its spread, like for other synthetic cannabinoids, has increased in recent years and many web sources freely supply these kinds of new drugs. It can be taken by smoking or through oral consumption. A 25-years-old man was hospitalized at the local hospital following a major trauma after ingestion of alcohol and an unknown pill. Urine and blood samples were sent to our Forensic Toxicology Division to investigate on possible substance abuse. A general unknown screening of biological samples, extracted by liquid-liquid extraction (ethylacetate and dichloromethane) in basic, acidic and neutral conditions, was achieved to verify the presence of drugs of abuse and/or their metabolites, both in gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). For the quantification of AM-694, urine was extracted by solid phase extraction (SPE) on a Bond Elut Certify cartridge; an acidic hydrolysis (HCl 30%, 95°C, 60min) was necessary before liquid-liquid extraction of metabolites. For the detection of benzodiazepines and their metabolites, an enzymatic hydrolysis was applied (β-glucuronidase, pH 4.5, 50°C, 18h). Quantification of AM-694 (internal standard AM-2201), midazolam and α-hydroxymidazolam (internal standard halazepam) were performed by LC-MS/MS analysis in multiple reaction monitoring ([M+H](+): m/z 436→190, 272, AM-694; m/z 360→155, 127, AM-2201; m/z 326→291, 223, midazolam; m/z 342→168, 203, α-hydroxymidazolam; m/z 353→241, 222, halazepam). The general unknown screening revealed the presence of AM-694 (urine sample) and benzodiazepines (urine and blood). The concentration of AM-694, obtained by LC-MS/MS, was 0.084μg/L. Midazolam and α-hydroxymidazolam were detected in urine (0

  17. Removal of stigmasterol from Kraft mill effluent by aerobic biological treatment with steroidal metabolite detection.

    PubMed

    Chamorro, Soledad; Vergara, Juan P; Jarpa, Mayra; Hernandez, Victor; Becerra, Jose; Vidal, Gladys

    2016-10-14

    Stigmasterol is a phytosterol contained in Kraft mill effluent that is able to increase over 100% after aerobic biological treatment. This compound can act as an endocrine disrupter as its structure is similar to that of cholesterol. The aim of this study was to evaluate the removal of stigmasterol from Kraft mill effluents treated by a moving bed biofilm reactor (MBBR) with steroidal metabolite detection. The MBBR was operated for 145 days, with a hydraulic retention time of 2 days. Stigmasterol and steroidal metabolites were detected by gas chromatography with a flame ionization detector during MBBR operation. The results show that the MBBR removed 87.4% of biological oxygen demand (BOD5), 61.5% of chemical oxygen demand (COD), 24.5% of phenol and 31.5% of lignin, expressed in average values. The MBBR system successfully removed 100% of the stigmasterol contained in the influent (33 µg L(-1)) after 5 weeks of operation. In that case, the organic load rate was 0.343 kg COD m(-3) d(-1). Furthermore, different steroidal compounds (e.g., testosterone propionate, stigmast-4-en-3-one, 5α-pregnan-12-one-20α-hydroxy, 5α-pregnane-3,11,20-trione and 3α-hydroxy-5α-androstane-11,17-dione were detected in the Kraft mill effluent as potential products of phytosterol biotransformation.

  18. Detection times of marijuana metabolites in urine by immunoassay and GC-MS.

    PubMed

    Huestis, M A; Mitchell, J M; Cone, E J

    1995-10-01

    Reports of prolonged drug excretion have provided the basis for the common assumption that cannabinoid metabolites may he detected in urine for a week or longer. The accuracy, sensitivity, and specificity of immunoassays for the detection of cannabinoids and metabolites are unique for a specific assay and may change overtime. it is important that individuals who select assays and those who interpret test results be aware of qualitative and quantitative changes that occur. In the present study, detection times of cannabinoids in urine were determined using cannabinoid immunoassays with 20-, 50-, and 100-ng/mL cutoffs and using gas chromatography-mass spectrometry (GC-MS). Six subjects each smoked a single marijuana cigarette (placebo, 1.75, or 3.55% delta9-tetrahydrocannabinol [THC]) each week while residing on the clinical ward of the Addiction Research Center. Each urine specimen was analyzed under blind conditions by immunoassay according to the manufacturer's instructions. The following cannabinoid reagents were evaluated: EMIT d.a.u. 100, EMIT d.a.u. 50, EMIT d.a.u. 20, EMIT II 100, EMIT II 50, Abuscreen OnLine, and Abuscreen RIA, DRI, and ADx. All urine specimens were also analyzed for 11-nor-9-carboxy-delta9-THC by GC-MS using a 15-ng/mL cutoff. Urinary cannabinoid detection times varied substantially across assays, subjects, doses, and cutoff concentrations. Detection times were shorter than previously assumed. Mean detection times increased from a maximum of 0.5 days after the low dose to 1.5 days after the high dose using the 100-ng/mL cutoff. Mean detection times were less than 1 day following the low dose and less than 2 days following high-dose exposure using the 50-ng/mL cutoff. Mean detection times ranged from 1 to 5 days after the low dose and from 3 to 6 days after the high dose using the 20-ng/mL cutoff immunoassay. GC-MS detection times were approximately twice as long as mean detection times using an immunoassay with a cutoff of 50 ng

  19. Detection of the novel metabolite ethylphenidate after methylphenidate overdose with alcohol coingestion.

    PubMed

    Markowitz, J S; Logan, B K; Diamond, F; Patrick, K S

    1999-08-01

    Methylphenidate is the most commonly prescribed psychostimulant in clinical use today. Known methylphenidate metabolites include ritalinic acid, corresponding lactams, and p-hydroxymethylphenidate. Recent in vitro work using rat liver preparations has indicated that the methylphenidate ethyl ester, ethylphenidate, is formed upon incubation with ethanol. This report describes the first detection of ethylphenidate in human blood and liver samples obtained from two suicide victims who had overdosed on methylphenidate and coingested ethanol. Amounts of ethylphenidate detected in whole blood specimens in these two cases (8 ng/mL and 1 ng/mL, respectively) were small relative to methylphenidate and ritalinic acid concentrations. Nonetheless, given the high likelihood that methylphenidate and ethanol coingestion frequently occurs, the detection of ethylphenidate in humans warrants further investigation into the extent of its formation as well as into any associated toxicity in nonoverdose situations.

  20. Gene probes for the detection of 6-deoxyhexose metabolism in secondary metabolite-producing streptomycetes.

    PubMed

    Stockmann, M; Piepersberg, W

    1992-01-01

    DNA probes were designed from the streptomycin production genes strDELM of Streptomyces griseus involved in the biosynthesis of the 6-deoxyhexose (6DOH) dihydrostreptose which could detect the genomic fragments coding for 6DOH formation in other actinomycetes strains. In about 70% of the 43 strains tested at least one signal could be detected with strD-, strE- or strLM-specific probes. Evidence is presented that the hybridizing genes are mostly clustered and probably engaged in the formation of secondary metabolites. Because of the wide-spread use of 6DOH constituents in natural products these probes should allow to detect a vast array of different secondary metabolic gene clusters in actinomycetes.

  1. Meat, the metabolites: an integrated metabolite profiling and lipidomics approach for the detection of the adulteration of beef with pork.

    PubMed

    Trivedi, Drupad K; Hollywood, Katherine A; Rattray, Nicholas J W; Ward, Holli; Trivedi, Dakshat K; Greenwood, Joseph; Ellis, David I; Goodacre, Royston

    2016-04-07

    Adulteration of high quality food products with sub-standard and cheaper grades is a world-wide problem taxing the global economy. Currently, many traditional tests suffer from poor specificity, highly complex outputs and a lack of high-throughput processing. Metabolomics has been successfully used as an accurate discriminatory technique in a number of applications including microbiology, cancer research and environmental studies and certain types of food fraud. In this study, we have developed metabolomics as a technique to assess the adulteration of meat as an improvement on current methods. Different grades of beef mince and pork mince, purchased from a national retail outlet were combined in a number of percentage ratios and analysed using GC-MS and UHPLC-MS. These techniques were chosen because GC-MS enables investigations of metabolites involved in primary metabolism whilst UHPLC-MS using reversed phase chromatography provides information on lipophilic species. With the application of chemometrics and statistical analyses, a panel of differential metabolites were found for identification of each of the two meat types. Additionally, correlation was observed between metabolite content and percentage of fat declared on meat products' labelling.

  2. Meat, the metabolites: an integrated metabolite profiling and lipidomics approach for the detection of the adulteration of beef with pork

    PubMed Central

    Trivedi, Drupad K.; Hollywood, Katherine A.; Rattray, Nicholas J. W.; Ward, Holli; Trivedi, Dakshat K.; Greenwood, Joseph; Ellis, David I.

    2016-01-01

    Adulteration of high quality food products with sub-standard and cheaper grades is a world-wide problem taxing the global economy. Currently, many traditional tests suffer from poor specificity, highly complex outputs and a lack of high-throughput processing. Metabolomics has been successfully used as an accurate discriminatory technique in a number of applications including microbiology, cancer research and environmental studies and certain types of food fraud. In this study, we have developed metabolomics as a technique to assess the adulteration of meat as an improvement on current methods. Different grades of beef mince and pork mince, purchased from a national retail outlet were combined in a number of percentage ratios and analysed using GC-MS and UHPLC-MS. These techniques were chosen because GC-MS enables investigations of metabolites involved in primary metabolism whilst UHPLC-MS using reversed phase chromatography provides information on lipophilic species. With the application of chemometrics and statistical analyses, a panel of differential metabolites were found for identification of each of the two meat types. Additionally, correlation was observed between metabolite content and percentage of fat declared on meat products’ labelling. PMID:26911805

  3. Gas and liquid chromatography-mass spectrometry detection of the urinary metabolites of UR-144 and its major pyrolysis product.

    PubMed

    Grigoryev, Andrej; Kavanagh, Pierce; Melnik, Alexandra; Savchuk, Sergey; Simonov, Anton

    2013-06-01

    Studies on the pyrolysis of the synthetic cannabinoid agonist UR-144 ((1-pentyl-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone) have shown that its major pyrolysis product is a tetramethylcyclopropane ring-opened alkene. Considering that smoking is a common way of ingesting synthetic cannabimimetics, the presence of the metabolites of this pyrolysis product would be expected in biological fluids. Using GC-MS and LC-MS-MS methods, a series of phase I metabolites of UR-144 and its pyrolysis product were detected in the urine samples from patients admitted to hospital with suspected drug intoxication. The metabolites were tentatively identified as the products of mono-hydroxylation, di-hydroxylation, mono-hydroxylation with formation of the carbonyl group on the N-alkyl chain, carboxylation and N-dealkylation with mono-hydroxylation. In the case of the UR-144 pyrolysis product, metabolites with hydration of the aliphatic double bond were also identified. The parent compounds were detected as trace amounts in some urine samples, and the hydrated derivative of the UR-144 pyrolysis product was detected in the majority of samples. The detection of mono-hydroxylated metabolites of UR-144 (LC-MS-MS) and mono-hydroxylated/with hydration metabolites of the UR-144 pyrolysis product (GC-MS) was found to be the most useful method of establishing UR-144 ingestion.

  4. Development and in silico evaluation of large-scale metabolite identification methods using functional group detection for metabolomics

    PubMed Central

    Mitchell, Joshua M.; Fan, Teresa W.-M.; Lane, Andrew N.; Moseley, Hunter N. B.

    2014-01-01

    Large-scale identification of metabolites is key to elucidating and modeling metabolism at the systems level. Advances in metabolomics technologies, particularly ultra-high resolution mass spectrometry (MS) enable comprehensive and rapid analysis of metabolites. However, a significant barrier to meaningful data interpretation is the identification of a wide range of metabolites including unknowns and the determination of their role(s) in various metabolic networks. Chemoselective (CS) probes to tag metabolite functional groups combined with high mass accuracy provide additional structural constraints for metabolite identification and quantification. We have developed a novel algorithm, Chemically Aware Substructure Search (CASS) that efficiently detects functional groups within existing metabolite databases, allowing for combined molecular formula and functional group (from CS tagging) queries to aid in metabolite identification without a priori knowledge. Analysis of the isomeric compounds in both Human Metabolome Database (HMDB) and KEGG Ligand demonstrated a high percentage of isomeric molecular formulae (43 and 28%, respectively), indicating the necessity for techniques such as CS-tagging. Furthermore, these two databases have only moderate overlap in molecular formulae. Thus, it is prudent to use multiple databases in metabolite assignment, since each major metabolite database represents different portions of metabolism within the biosphere. In silico analysis of various CS-tagging strategies under different conditions for adduct formation demonstrate that combined FT-MS derived molecular formulae and CS-tagging can uniquely identify up to 71% of KEGG and 37% of the combined KEGG/HMDB database vs. 41 and 17%, respectively without adduct formation. This difference between database isomer disambiguation highlights the strength of CS-tagging for non-lipid metabolite identification. However, unique identification of complex lipids still needs additional

  5. Development and in silico evaluation of large-scale metabolite identification methods using functional group detection for metabolomics.

    PubMed

    Mitchell, Joshua M; Fan, Teresa W-M; Lane, Andrew N; Moseley, Hunter N B

    2014-01-01

    Large-scale identification of metabolites is key to elucidating and modeling metabolism at the systems level. Advances in metabolomics technologies, particularly ultra-high resolution mass spectrometry (MS) enable comprehensive and rapid analysis of metabolites. However, a significant barrier to meaningful data interpretation is the identification of a wide range of metabolites including unknowns and the determination of their role(s) in various metabolic networks. Chemoselective (CS) probes to tag metabolite functional groups combined with high mass accuracy provide additional structural constraints for metabolite identification and quantification. We have developed a novel algorithm, Chemically Aware Substructure Search (CASS) that efficiently detects functional groups within existing metabolite databases, allowing for combined molecular formula and functional group (from CS tagging) queries to aid in metabolite identification without a priori knowledge. Analysis of the isomeric compounds in both Human Metabolome Database (HMDB) and KEGG Ligand demonstrated a high percentage of isomeric molecular formulae (43 and 28%, respectively), indicating the necessity for techniques such as CS-tagging. Furthermore, these two databases have only moderate overlap in molecular formulae. Thus, it is prudent to use multiple databases in metabolite assignment, since each major metabolite database represents different portions of metabolism within the biosphere. In silico analysis of various CS-tagging strategies under different conditions for adduct formation demonstrate that combined FT-MS derived molecular formulae and CS-tagging can uniquely identify up to 71% of KEGG and 37% of the combined KEGG/HMDB database vs. 41 and 17%, respectively without adduct formation. This difference between database isomer disambiguation highlights the strength of CS-tagging for non-lipid metabolite identification. However, unique identification of complex lipids still needs additional

  6. Detection of "Bath Salt" Synthetic Cathinones and Metabolites in Urine via DART-MS and Solid Phase Microextraction

    NASA Astrophysics Data System (ADS)

    LaPointe, Joseph; Musselman, Brian; O'Neill, Teresa; Shepard, Jason R. E.

    2015-01-01

    A rapid and sensitive method, direct analysis in real time mass spectrometry (DART-MS) was applied to the characterization and semiquantitative analysis of synthetic cathinones and their metabolites in urine. DART-MS was capable of detecting three different cathinones and three metabolites down to sub-clinical levels directly without any sample preparations. The process produced a spectrum within seconds because no extraction or derivatization was required for analysis and the high mass accuracy of the instrumentation allowed analysis without the need for lengthy chromatographic separations. The use of solid phase microextration demonstrated a relative increase in the detectability of both drugs and metabolites, improving the detection signal on average more than an order of magnitude over direct detection, while providing cleaner spectra devoid of the major peaks associated with urine that oftentimes dominate such samples.

  7. Detection of "bath salt" synthetic cathinones and metabolites in urine via DART-MS and solid phase microextraction.

    PubMed

    LaPointe, Joseph; Musselman, Brian; O'Neill, Teresa; Shepard, Jason R E

    2015-01-01

    A rapid and sensitive method, direct analysis in real time mass spectrometry (DART-MS) was applied to the characterization and semiquantitative analysis of synthetic cathinones and their metabolites in urine. DART-MS was capable of detecting three different cathinones and three metabolites down to sub-clinical levels directly without any sample preparations. The process produced a spectrum within seconds because no extraction or derivatization was required for analysis and the high mass accuracy of the instrumentation allowed analysis without the need for lengthy chromatographic separations. The use of solid phase microextration demonstrated a relative increase in the detectability of both drugs and metabolites, improving the detection signal on average more than an order of magnitude over direct detection, while providing cleaner spectra devoid of the major peaks associated with urine that oftentimes dominate such samples.

  8. Chemical-shift-selective filter for the in vivo detection of J-coupled metabolites at 3T.

    PubMed

    Schulte, Rolf F; Trabesinger, Andreas H; Boesiger, Peter

    2005-02-01

    A chemical-shift-selective filter (CSSF) was applied to the detection of J-coupled metabolites in the human brain. This filter is an acquisition-based technique that requires the chemical shifts (CS's) of different metabolites, but not their whole multiplet structures, to be resolved. The sequence is based on the 2D constant-time spin-echo experiment, which yields pure CS spectra in the indirect dimension. Localization is achieved through point-resolved spectroscopy (PRESS). The method enables unequivocal detection of glutamate and myo-inositol, both in vitro and in vivo in the human brain, at 3T.

  9. Detection of cannabigerol and its presumptive metabolite in human urine after Cannabis consumption.

    PubMed

    Hidvégi, E; Somogyi, G P

    2010-06-01

    Kemp et al. (1995) could detect delta9-tetrahydrocannabinol (delta9-THC), cannabinol and cannabidiol, three neutral cannabinoids, and the metabolites of delta9-THC in urine samples of Cannabis consumers. In this study we aimed to identify cannabigerol (CBG), which in its acid form is one of the main intermediate compounds of the biosynthesis of cannabinoids in hemp, in authority urine samples of proved Cannabis consumers. For this reason we applied the modified method of Kemp et al. to test for CBG, since enzymatic hydrolysis seems to be necessary for the formation of free neutral cannabinoids from conjugates. After extraction, derivatisation with N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and GC/MS analysis, peaks of characteristic fragment ions (m/z 337, 391, 377 and 460) of bis-trimethylsilyl derivative of CBG appeared at 12.48 minutes in both real sample and the urine spiked with CBG. It shows that CBG enters the body during Cannabis smoking and is excreted with urine in a conjugated form, like other neutral cannabinoids. Analysing the chromatograms of hydrolysed and trimethylsilylated extracts we checked for the presence of CBG-metabolites based on the study of Harvey and Brown (1990). We detected a compound in the Cannabis consumers' urine extracts, having fragment ions at m/z 425, 465 and 479 at the retention time of 14.19 min which is presumed to be the 4"-hydroxy-CBG or 5"-hydroxy-CBG. However, it could not be identified completely by GC/MS. This peak was absent in non-hydrolysed urine samples, indicating that it is also excreted in glucuronated form.

  10. Mutation-induced loop opening and energetics for binding of tamiflu to influenza N8 neuraminidase.

    PubMed

    Kar, Parimal; Knecht, Volker

    2012-05-31

    Tamiflu, also known as oseltamivir (OTV), binds to influenza A neuraminidase (H5N1) with very high affinity (0.32 nM). However, this inhibitor binds to other neuraminidases as well. In the present work, a systematic computational study is performed to investigate the mechanism underlying the binding of oseltamivir to N8 neuraminidase (NA) in "open" and "closed" conformations of the 150-loop through molecular dynamics simulations and the popular and well established molecular mechanics Poisson-Boltzmann (MM-PBSA) free energy calculation method. Whereas the closed conformation is stable for wild type N8, it transforms into the open conformation for the mutants Y252H, H274Y, and R292K, indicating that bound to oseltamivir these mutants are preferentially in the open conformation. Our calculations show that the binding of wild type oseltamivir to the closed conformation of N8 neuraminidase is energetically favored compared to the binding to the open conformation. We observe water mediated binding of oseltamivir to the N8 neuraminidase in both conformations which is not seen in the case of binding of the same drug to the H5N1 neuraminidase. The decomposition of the binding free energy reveals the mechanisms underlying the binding and changes in affinity due to mutations. Considering the mutant N8 variants in the open conformation adopted during the simulations, we observe a significant loss in the size of the total binding free energy for the N8(Y252H)-OTV, N8(H274Y)-OTV, and N8(R292K)-OTV complexes compared to N8(WT)-OTV, mainly due to the decrease in the size of the intermolecular electrostatic energy. For R292K, an unfavorable shift in the van der Waals interactions also contributes to the drug resistance. The mutations cause a significant expansion in the active site cavity, increasing its solvent accessible surface compared to the crystal structures of both the open and closed conformations. Our study underscores the need to consider dynamics in rationalizing the

  11. Analysis of the cyanolichen Lichina pygmaea metabolites using in situ DART-MS: from detection to thermochemistry of mycosporine serinol.

    PubMed

    Le Pogam, Pierre; Legouin, Béatrice; Le Lamer, Anne-Cécile; Boustie, Joël; Rondeau, David

    2015-03-01

    Direct Analysis in Real Time DART-HRMS is here first applied to the detection of molecules from a lichen, Lichina pygmaea. The aim was to propose an innovative method of in situ detection of lichen secondary metabolites using the possibilities of elemental composition determination available when a DART source is interfaced with a TOF analyzer. Three kinds of samples have been submitted to DART ionization, i.e. an intact thallus, a powder obtained from the crushed lichen and an aqueous extract. In situ analysis of crushed lichen, yields an extensive chemical profile, comparable to what is obtained from the aqueous extract, comprising both major polar metabolites described in literature along with some other signals that could correspond to potentially unknown metabolites. One of the detected secondary metabolites, mycosporine serinol, underwent a dehydration reaction prior to its transfer in the gas-phase by DART ionization. The consideration of the thermal transfers involved in the DART ionization process and the possibility to record time-dependent mass spectra through the use of the TOF analyzer allowed establishing Arrhenius plots of this water molecule loss to obtain associated thermodynamic quantities. The low values of corresponding activation enthalpy (Δr‡Hm° of the order of 25 kJ mol(-1)) enabled formulating some assumption regarding a possible role of such metabolites in the lichen.

  12. Analysis of glycolysis metabolites by capillary zone electrophoresis with indirect UV detection.

    PubMed

    Shen, P; Hauri, D; Ross, J; Oefner, P J

    1996-01-01

    The glycolysis metabolites glucose 6-phosphate (G6-P), fructose 6-phosphate (F6-P), fructose 1,6-bisphosphate (F1,6-BP), fructose 2,6-bisphosphate (F2,6-BP), glyceraldehyde phosphate (GAP), dihydroxyacetone phosphate (DHAP), phosphoenolpyruvate (PEP), pyruvate, and lactate were analyzed by capillary zone electrophoresis (CZE) with indirect UV detection. The chromophores phthalic acid, sorbic acid, and 4-hydroxybenzoic acid were studied as background electrolytes. Both detection sensitivity and resolution were found to depend on the pH and the concentration of the carrier electrolyte. Optimum separation and detection of the phosphate compounds were accomplished upon reversal of electroendosmotic flow (EOF) with OFM Anion-BT (Waters Corp., Milford, MA) at a concentration of 4-6 mM 4-hydroxybenzoic acid, pH 11.6-12.0, with the detection wave-length set at 280 nm. The highly alkaline pH allowed the successful separation of the isomers F6-P and G6-P, as well as F1,6-BP and F2,6-BP, respectively. The effect of sample ionic strength on the detection limits of G6-P, F6-P, F1,6-BP, and F2,6-BP was also investigated: These limits ranged from 1 to 3 microM in both low- and high-ionic-strength samples. However, high Mg2+ concentrations in the sample led to a progressive loss of resolution between F1,6-BP and F2,6-BP, unless the inlet reservoir was replenished with fresh electrolyte after every injection. Linearity of detection was observed over one to two orders of magnitude.

  13. Structural characterization of plasma metabolites detected via LC-electrochemical coulometric array using LC-UV fractionation, MS, and NMR.

    PubMed

    Bird, Susan S; Sheldon, Diane P; Gathungu, Rose M; Vouros, Paul; Kautz, Roger; Matson, Wayne R; Kristal, Bruce S

    2012-11-20

    Liquid chromatography (LC) separation combined with electrochemical coulometric array detection (EC) is a sensitive, reproducible, and robust technique that can detect hundreds of redox-active metabolites down to the level of femtograms on column, making it ideal for metabolomics profiling. EC detection cannot, however, structurally characterize unknown metabolites that comprise these profiles. Several aspects of LC-EC methods prevent a direct transfer to other structurally informative analytical methods, such as LC-MS and NMR. These include system limits of detection, buffer requirements, and detection mechanisms. To address these limitations, we developed a workflow based on the concentration of plasma, metabolite extraction, and offline LC-UV fractionation. Pooled human plasma was used to provide sufficient material necessary for multiple sample concentrations and platform analyses. Offline parallel LC-EC and LC-MS methods were established that correlated standard metabolites between the LC-EC profiling method and the mass spectrometer. Peak retention times (RT) from the LC-MS and LC-EC system were linearly related (r(2) = 0.99); thus, LC-MS RTs could be directly predicted from the LC-EC signals. Subsequent offline microcoil-NMR analysis of these collected fractions was used to confirm LC-MS characterizations by providing complementary, structural data. This work provides a validated workflow that is transferrable across multiple platforms and provides the unambiguous structural identifications necessary to move primary mathematically driven LC-EC biomarker discovery into biological and clinical utility.

  14. Insights into the Anaerobic Biodegradation Pathway of n-Alkanes in Oil Reservoirs by Detection of Signature Metabolites

    PubMed Central

    Bian, Xin-Yu; Maurice Mbadinga, Serge; Liu, Yi-Fan; Yang, Shi-Zhong; Liu, Jin-Feng; Ye, Ru-Qiang; Gu, Ji-Dong; Mu, Bo-Zhong

    2015-01-01

    Anaerobic degradation of alkanes in hydrocarbon-rich environments has been documented and different degradation strategies proposed, of which the most encountered one is fumarate addition mechanism, generating alkylsuccinates as specific biomarkers. However, little is known about the mechanisms of anaerobic degradation of alkanes in oil reservoirs, due to low concentrations of signature metabolites and lack of mass spectral characteristics to allow identification. In this work, we used a multidisciplinary approach combining metabolite profiling and selective gene assays to establish the biodegradation mechanism of alkanes in oil reservoirs. A total of twelve production fluids from three different oil reservoirs were collected and treated with alkali; organic acids were extracted, derivatized with ethanol to form ethyl esters and determined using GC-MS analysis. Collectively, signature metabolite alkylsuccinates of parent compounds from C1 to C8 together with their (putative) downstream metabolites were detected from these samples. Additionally, metabolites indicative of the anaerobic degradation of mono- and poly-aromatic hydrocarbons (2-benzylsuccinate, naphthoate, 5,6,7,8-tetrahydro-naphthoate) were also observed. The detection of alkylsuccinates and genes encoding for alkylsuccinate synthase shows that anaerobic degradation of alkanes via fumarate addition occurs in oil reservoirs. This work provides strong evidence on the in situ anaerobic biodegradation mechanisms of hydrocarbons by fumarate addition. PMID:25966798

  15. Insights into the Anaerobic Biodegradation Pathway of n-Alkanes in Oil Reservoirs by Detection of Signature Metabolites

    NASA Astrophysics Data System (ADS)

    Bian, Xin-Yu; Maurice Mbadinga, Serge; Liu, Yi-Fan; Yang, Shi-Zhong; Liu, Jin-Feng; Ye, Ru-Qiang; Gu, Ji-Dong; Mu, Bo-Zhong

    2015-05-01

    Anaerobic degradation of alkanes in hydrocarbon-rich environments has been documented and different degradation strategies proposed, of which the most encountered one is fumarate addition mechanism, generating alkylsuccinates as specific biomarkers. However, little is known about the mechanisms of anaerobic degradation of alkanes in oil reservoirs, due to low concentrations of signature metabolites and lack of mass spectral characteristics to allow identification. In this work, we used a multidisciplinary approach combining metabolite profiling and selective gene assays to establish the biodegradation mechanism of alkanes in oil reservoirs. A total of twelve production fluids from three different oil reservoirs were collected and treated with alkali; organic acids were extracted, derivatized with ethanol to form ethyl esters and determined using GC-MS analysis. Collectively, signature metabolite alkylsuccinates of parent compounds from C1 to C8 together with their (putative) downstream metabolites were detected from these samples. Additionally, metabolites indicative of the anaerobic degradation of mono- and poly-aromatic hydrocarbons (2-benzylsuccinate, naphthoate, 5,6,7,8-tetrahydro-naphthoate) were also observed. The detection of alkylsuccinates and genes encoding for alkylsuccinate synthase shows that anaerobic degradation of alkanes via fumarate addition occurs in oil reservoirs. This work provides strong evidence on the in situ anaerobic biodegradation mechanisms of hydrocarbons by fumarate addition.

  16. Competitive FRET-aptamer-based detection of methylphosphonic acid, a common nerve agent metabolite.

    PubMed

    Bruno, John G; Carrillo, Maria P; Phillips, Taylor; Vail, Neal K; Hanson, Douglas

    2008-09-01

    Competitive fluorescence resonance energy transfer (FRET)-aptamer-based assay formats are described for one-step detection of methylphosphonic acid (MPA; a metabolite of several organophosphorus (OP) nerve agents). AminoMPA was attached to tosyl-magnetic beads and used for DNA aptamer selection from which one dominant aptamer sequence emerged. Two different FRET approaches were attempted. In one approach, the complementary DNA sequence was used as a template for labeling the aptamer with Alexa Fluor 546 (AF 546)-14-dUTP by asymmetric PCR. Following 3-dimensional (3-D), molecular modeling of the aptamer-MPA complex, a series of three fluoresceinated aptamers labeled at positions 50, 51, and 52 in the putative optimal binding pocket were synthesized. In both FRET formats, aminoMPA was linked to Black Hole Quencher (BHQ-1 or BHQ-2)-succinimides and allowed to bind the fluorescein or AF 546-labeled MPA aptamer. Following gel filtration to purify the labeled MPA aptamer-BHQ-aminoMPA FRET complexes, the complexes were competed against various concentrations of unlabeled MPA, MPA derivatives, and unrelated compounds in titration and cross-reactivity studies. Both approaches yielded low microgram per milliliter detection limits for MPA with generally low levels of cross-reactivity for unrelated compounds. However, the data suggest a pattern of traits that may effect the direction (lights on or off) and intensity of the FRET.

  17. Detection of methamphetamine and its main metabolite in fingermarks by liquid chromatography-mass spectrometry.

    PubMed

    Zhang, Ting; Chen, Xueguo; Yang, Ruiqin; Xu, Yingjian

    2015-03-01

    A sensitive and efficient method applying liquid chromatography-mass spectrometry for the analysis of methamphetamine and its main metabolite in fingermark deposits was described. Using this method, good linear relationship of methamphetamine was obtained in the range of 0.005μg to 0.5μg per cotton swab, the limit of detection was 1.5ng per cotton swab, the limit of quantitation was 5.0ng per cotton swab and the average values of recovery ratios were above 70.1%. Moreover, the influence factors for the detection of methamphetamine in fingermarks, such as kinds of substrates, development methods and extraction methods, were all discussed in details. The results showed that good recovery ratios could be obtained on painted wood and smooth substrates surfaces. Development methods in commercial powder could not influence the quality of examination of exogenous drug in latent fingermark. Furthermore, the results indicated that the method mentioned here could be applied in the analysis of forensic trace evidences and samples obtained in clinically addicted cases.

  18. Quantitative bioluminescence imaging--a method for the detection of metabolite distributions in frozen tissues

    NASA Astrophysics Data System (ADS)

    Mueller-Klieser, Wolfgang; Walenta, Stefan; Schwickert, Georg

    1994-02-01

    A novel technique allows for measurement of metabolite distributions in tissue cryosections at a microscopic level using bioluminescence, single photon imaging, and computerized image analysis. Metabolites, such as ATP, glucose and lactate are registered in absolute concentration units, and the respective images can be correlated with each other and with histological structures by specific algorithms. One striking difference between malignant tumors and normal tissue is the pronounced heterogeneity of metabolite distributions in malignancies contrasted by rather homogeneous patterns obtained in many normal organs. The heterogeneous distribution of metabolites in solid tumors reflects the chaotic organization of the histological architecture and of the microvascular supply in cancerous tissue. Pixel-to-pixel comparison of metabolite distributions measured in cervix cancers of patients revealed a negative linear correlation between glucose and ATP concentrations at identical locations. In contrast, local lactate concentration was positively correlated with ATP.

  19. Detection of metabolites of lysergic acid diethylamide (LSD) in human urine specimens: 2-oxo-3-hydroxy-LSD, a prevalent metabolite of LSD.

    PubMed

    Poch, G K; Klette, K L; Hallare, D A; Manglicmot, M G; Czarny, R J; McWhorter, L K; Anderson, C J

    1999-03-05

    Seventy-four urine specimens previously found to contain lysergic acid diethylamide (LSD) by gas chromatography-mass spectrometry (GC-MS) were analyzed by a new procedure for the LSD metabolite 2-oxo-3-hydroxy-LSD (O-H-LSD) using a Finnigan LC-MS-MS system. This procedure proved to be less complex, shorter to perform and provides cleaner chromatographic characteristics than the method currently utilized by the Navy Drug Screening Laboratories for the extraction of LSD from urine by GC-MS. All of the specimens used in the study screened positive for LSD by radioimmunoassay (Roche Abuscreen). Analysis by GC-MS revealed detectable amounts of LSD in all of the specimens. In addition, isolysergic diethylamide (iso-LSD), a byproduct of LSD synthesis, was quantitated in 64 of the specimens. Utilizing the new LC-MS-MS method, low levels of N-desmethyl-LSD (nor-LSD), another identified LSD metabolite, were detected in some of the specimens. However, all 74 specimens contained O-H-LSD at significantly higher concentrations than LSD, iso-LSD, or nor-LSD alone. The O-H-LSD concentration ranged from 732 to 112 831 pg/ml (mean, 16340 pg/ml) by quantification with an internal standard. The ratio of O-H-LSD to LSD ranged from 1.1 to 778.1 (mean, 42.9). The presence of O-H-LSD at substantially higher concentrations than LSD suggests that the analysis for O-H-LSD as the target analyte by employing LC-MS-MS will provide a much longer window of detection for the use of LSD than the analysis of the parent compound, LSD.

  20. Detection of urinary metabolites common to structurally related 17alpha-alkyl anabolic steroids in horses and application to doping tests in racehorses: methandienone, methandriol, and oxymetholone.

    PubMed

    Yamada, Masayuki; Aramaki, Sugako; Kurosawa, Masahiko; Saito, Koichi; Nakazawa, Hiroyuki

    2008-06-01

    Methandienone, methandriol, and oxymetholone, which are anabolic steroids possessing 17alpha-methyl and 17beta-hydroxy groups, were developed as oral formulations for therapeutic purposes. However, they have been used in racehorses to enhance racing performance. In humans, it has been reported that structurally related anabolic steroids having the 17alpha-methyl and 17beta-hydroxy groups, including 17alpha-methyltestosterone, mestanolone, methandienone, methandriol, and oxymetholone, have metabolites in common. In this study, we found that metabolites common to those of 17alpha-methyltestosterone and mestanolone were detected in horse urine after the administration of oxymetholone, methandienone, and methandriol. Based on analytical data, we confirmed these to be the common metabolites of five structurally related steroids, 17alpha-methyltestosterone, mestanolone, oxymetholone, methandienone, and methandriol. Furthermore, we detected hitherto unknown urinary metabolites of methandriol and oxymetholone in horses. The parent steroid itself was detected in horse urine after the administration of methandriol, other than metabolites common to 17alpha-methyltestosterone and mestanolone. On the other hand, the major metabolite of oxymetholone was mestanolone, aside from metabolites presumed to be the stereoisomers of 2-hydroxymethyl-17alpha-methyl-5alpha-androstan-3,17beta-diol and 2,17alpha-di(hydroxymethyl)-5alpha-androstan-3,17beta-diol. The simultaneous detection of common metabolites and other main metabolites would help us narrow down the candidate-administered steroid for the doping tests in racehorses.

  1. Oxidative stress/reactive metabolite gene expression signature in rat liver detects idiosyncratic hepatotoxicants

    SciTech Connect

    Leone, Angelique; Nie, Alex; Brandon Parker, J.; Sawant, Sharmilee; Piechta, Leigh-Anne; Kelley, Michael F. Mark Kao, L.; Jim Proctor, S.; Verheyen, Geert; Johnson, Mark D.; Lord, Peter G.; McMillian, Michael K.

    2014-03-15

    Previously we reported a gene expression signature in rat liver for detecting a specific type of oxidative stress (OS) related to reactive metabolites (RM). High doses of the drugs disulfiram, ethinyl estradiol and nimesulide were used with another dozen paradigm OS/RM compounds, and three other drugs flutamide, phenacetin and sulindac were identified by this signature. In a second study, antiepileptic drugs were compared for covalent binding and their effects on OS/RM; felbamate, carbamazepine, and phenobarbital produced robust OS/RM gene expression. In the present study, liver RNA samples from drug-treated rats from more recent experiments were examined for statistical fit to the OS/RM signature. Of all 97 drugs examined, in addition to the nine drugs noted above, 19 more were identified as OS/RM-producing compounds—chlorpromazine, clozapine, cyproterone acetate, dantrolene, dipyridamole, glibenclamide, isoniazid, ketoconazole, methapyrilene, naltrexone, nifedipine, sulfamethoxazole, tamoxifen, coumarin, ritonavir, amitriptyline, valproic acid, enalapril, and chloramphenicol. Importantly, all of the OS/RM drugs listed above have been linked to idiosyncratic hepatotoxicity, excepting chloramphenicol, which does not have a package label for hepatotoxicity, but does have a black box warning for idiosyncratic bone marrow suppression. Most of these drugs are not acutely toxic in the rat. The OS/RM signature should be useful to avoid idiosyncratic hepatotoxicity of drug candidates. - Highlights: • 28 of 97 drugs gave a positive OS/RM gene expression signature in rat liver. • The specificity of the signature for human idiosyncratic hepatotoxicants was 98%. • The sensitivity of the signature for human idiosyncratic hepatotoxicants was 75%. • The signature can help eliminate hepatotoxicants from drug development.

  2. Detection and characterization of metabolites in biological matrices using mass defect filtering of liquid chromatography/high resolution mass spectrometry data.

    PubMed

    Zhu, Mingshe; Ma, Li; Zhang, Donglu; Ray, Kenneth; Zhao, Weiping; Humphreys, W Griffith; Skiles, Gary; Sanders, Mark; Zhang, Haiying

    2006-10-01

    An improved mass defect filter (MDF) method employing both drug and core structure filter templates was applied to the processing of high resolution liquid chromatography/mass spectrometry (LC/MS) data for the detection and structural characterization of oxidative metabolites with mass defects similar to or significantly different from those of the parent drugs. The effectiveness of this approach was investigated using nefazodone as a model compound, which is known to undergo multiple common and uncommon oxidative reactions. Through the selective removal of all ions that fall outside of the preset filter windows, the MDF process facilitated the detection of all 14 nefazodone metabolites presented in human liver microsomes in the MDF-filtered chromatograms. The capability of the MDF approach to remove endogenous interferences from more complex biological matrices was examined by analyzing omeprazole metabolites in human plasma. The unprocessed mass chromatogram showed no distinct indication of metabolite peaks; however, after MDF processing, the metabolite peaks were easily identified in the chromatogram. Compared with precursor ion scan and neutral loss scan techniques, the MDF approach was shown to be more effective for the detection of metabolites in a complex matrix. The comprehensive metabolite detection capability of the MDF approach, together with accurate mass determination, makes high resolution LC/MS a useful tool for the screening and identification of both common and uncommon drug metabolites.

  3. Integrated circuit-based electrochemical sensor for spatially resolved detection of redox-active metabolites in biofilms.

    PubMed

    Bellin, Daniel L; Sakhtah, Hassan; Rosenstein, Jacob K; Levine, Peter M; Thimot, Jordan; Emmett, Kevin; Dietrich, Lars E P; Shepard, Kenneth L

    2014-01-01

    Despite advances in monitoring spatiotemporal expression patterns of genes and proteins with fluorescent probes, direct detection of metabolites and small molecules remains challenging. A technique for spatially resolved detection of small molecules would benefit the study of redox-active metabolites that are produced by microbial biofilms and can affect their development. Here we present an integrated circuit-based electrochemical sensing platform featuring an array of working electrodes and parallel potentiostat channels. 'Images' over a 3.25 × 0.9 mm(2) area can be captured with a diffusion-limited spatial resolution of 750 μm. We demonstrate that square wave voltammetry can be used to detect, identify and quantify (for concentrations as low as 2.6 μM) four distinct redox-active metabolites called phenazines. We characterize phenazine production in both wild-type and mutant Pseudomonas aeruginosa PA14 colony biofilms, and find correlations with fluorescent reporter imaging of phenazine biosynthetic gene expression.

  4. Integrated circuit-based electrochemical sensor for spatially resolved detection of redox-active metabolites in biofilms

    PubMed Central

    Bellin, Daniel L.; Sakhtah, Hassan; Rosenstein, Jacob K.; Levine, Peter M.; Thimot, Jordan; Emmett, Kevin; Dietrich, Lars E. P.; Shepard, Kenneth L.

    2014-01-01

    Despite advances in monitoring spatiotemporal expression patterns of genes and proteins with fluorescent probes, direct detection of metabolites and small molecules remains challenging. A technique for spatially resolved detection of small molecules would benefit the study of redox-active metabolites produced by microbial biofilms, which can drastically affect colony development. Here we present an integrated circuit-based electrochemical sensing platform featuring an array of working electrodes and parallel potentiostat channels. “Images” over a 3.25 × 0.9 mm area can be captured with a diffusion-limited spatial resolution of 750 μm. We demonstrate that square wave voltammetry can be used to detect, identify, and quantify (for concentrations as low as 2.6 μM) four distinct redox-active metabolites called phenazines. We characterize phenazine production in both wild-type and mutant Pseudomonas aeruginosa PA14 colony biofilms, and find correlations with fluorescent reporter imaging of phenazine biosynthetic gene expression. PMID:24510163

  5. Abortion after deliberate Arthrotec® addition to food. Mass spectrometric detection of diclofenac, misoprostol acid, and their urinary metabolites.

    PubMed

    Watzer, Bernhard; Lusthof, Klaas J; Schweer, Horst

    2015-07-01

    Arthrotec(®) (AT) is a combination of diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), and misoprostol (MP), a synthetic analogue of prostaglandin E1 (PGE1). MP is a lipophilic methyl ester prodrug. It is readily metabolized to the biologically active misoprostol acid (MPA). During the last few years, medical studies exhibited MP to be an excellent abortive. In this paper, we describe a rare criminal case of MP abortion, initiated by the expectant father. After the abortion, samples of vomit and urine were collected. Systemic exposure to MP is difficult to prove, because both MP and the active metabolite MPA are hardly excreted in urine. Therefore, in addition to routine toxicological analysis, we used slightly modified, well-established liquid and gas chromatographic/tandem mass spectrometric (LC/MS/MS and GC/MS/MS) methods, for the direct and the indirect detection of MPA and its metabolites. In this case, we were able to demonstrate the presence of the major MP metabolites 2,3-dinor-MPA and 2,3,4,5-tetranor-MPA in the urine of the victim. We also detected paracetamol, 3-methoxyparacetamol and diclofenac-glucuronide in the urine. In the vomit of the victim, we detected diclofenac and MPA. These results, combined with the criminal investigations, showed that the accused had mixed MP into the food of his pregnant girlfriend. Finally, these investigations contributed to a confession of the accused.

  6. Integrated circuit-based electrochemical sensor for spatially resolved detection of redox-active metabolites in biofilms

    NASA Astrophysics Data System (ADS)

    Bellin, Daniel L.; Sakhtah, Hassan; Rosenstein, Jacob K.; Levine, Peter M.; Thimot, Jordan; Emmett, Kevin; Dietrich, Lars E. P.; Shepard, Kenneth L.

    2014-02-01

    Despite advances in monitoring spatiotemporal expression patterns of genes and proteins with fluorescent probes, direct detection of metabolites and small molecules remains challenging. A technique for spatially resolved detection of small molecules would benefit the study of redox-active metabolites that are produced by microbial biofilms and can affect their development. Here we present an integrated circuit-based electrochemical sensing platform featuring an array of working electrodes and parallel potentiostat channels. ‘Images’ over a 3.25 × 0.9 mm2 area can be captured with a diffusion-limited spatial resolution of 750 μm. We demonstrate that square wave voltammetry can be used to detect, identify and quantify (for concentrations as low as 2.6 μM) four distinct redox-active metabolites called phenazines. We characterize phenazine production in both wild-type and mutant Pseudomonas aeruginosa PA14 colony biofilms, and find correlations with fluorescent reporter imaging of phenazine biosynthetic gene expression.

  7. Can an immunoassay become a standard technique in detecting oxycodone and its metabolites?

    PubMed

    Abadie, Jude M; Allison, Kim H; Black, David A; Garbin, James; Saxon, Andrew J; Bankson, Daniel D

    2005-01-01

    Opiate toxicology testing is routinely performed in the hospital setting to identify abusers and/or to determine those patients who are not taking prescribed opiate analgesics such as oxycodone. Commercially available assays for opiate detection in urine have decreased sensitivity for oxycodone, which contributes to a high false-negative rate. Functioning as a beta site, our Veterans Affairs hospital evaluated a new enzyme immunoassay, DRI Oxycodone Assay, for its use in the qualitative and semiquantitative detection of oxycodone in urine. We hypothesize that an immunoassay for oxycodone with superior sensitivity and specificity, when compared to the traditional opiate assays, would reduce the need for more expensive and time-consuming confirmatory testing. We used the new liquid homogenous enzyme immunoassay to determine oxycodone results in a total of 148 urine samples from 4 different sample groups. Gas chromatography-mass spectroscopy was subsequently used to confirm the presence or absence of oxycodone (or its primary metabolite, noroxycodone). We also evaluated within-run, between-run, and linearity studies and conducted a crossover study to establish a cutoff value for oxycodone. In our patient population, we used the new DRI immunoassay to evaluate 17,069 urine samples to estimate oxycodone misuse profiles (patients not taking prescribed oxycodone or taking oxycodone without a prescription) during a 4-month period. The sensitivity and specificity of the new oxycodone immunoassay were 97.7% and 100%, respectively, at the cutoff concentration of 300 ng/mL. The assay linearity was 1,250 ng/mL, and the sensitivity was 10 ng/mL. Within-run precision and between-run coefficient of variation were 2.3% and 1.8%, respectively. None of the 15 compounds that we evaluated for interference had crossover significant enough to produce a positive oxycodone result when using 300 ng/mL as the cutoff value. None of the 17,069 oxycodone immunoassays was followed with a request

  8. Detection of nandrolone metabolites in urine after a football game in professional and amateur players: a Bayesian comparison.

    PubMed

    Robinson, N; Taroni, F; Saugy, M; Ayotte, C; Mangin, P; Dvorak, J

    2001-11-01

    Nandrolone (19-nortestosterone) is a widely used anabolic steroid in sports where strength plays an essential role. Once nandrolone has been metabolised, two major metabolites are excreted in urine, 19-norandrosterone (NA) and 19-noretiocholanolone (NE). In 1997, in France, quite a few sportsmen had concentrations of 19-norandrosterone very close to the IOC cut off limit (2ng/ml). At that time, a debate took place about the capability of the human male body to produce by itself these metabolites without any intake of nandrolone or related compounds. The International Football Federation (FIFA) was very concerned with this problematic, especially because the World Cup was about to start in France. In this respect, a statistical study was held with all football players from the first and second divisions of the Swiss Football National League. All players gave a urine sample after effort and around 6% of them showed traces of 19-norandrosterone. These results were compared with amateur football players (control group) and around 6% of them had very small amounts of 19-norandrosterone and/or 19-noretiocholanolone in urine after effort, whereas none of them had detectable traces of one or the other metabolite before effort. The origin of these compounds in urine after a strenuous physical activity is still unknown, but three hypotheses can be put forward. First, an endogenous production of nandrolone metabolites takes place. Second, nandrolone metabolites are released from the fatty tissues after an intake of nandrolone, some related compounds or some contaminated nutritive supplements. Finally, the sportsmen may have taken something during or just before the football game.

  9. Detoxification of Atrazine by Endophytic Streptomyces sp. Isolated from Sugarcane and Detection of Nontoxic Metabolite.

    PubMed

    Mesquini, Josiane A; Sawaya, Alexandra C H F; López, Begonã G C; Oliveira, Valéria M; Miyasaka, Natalia R S

    2015-12-01

    Atrazine is still one of the most used agricultural pesticides worldwide and it has been recognized as a major contaminant of surface and ground water. The aims of this research were to isolate an endophytic microorganism from leaves of sugarcane, evaluate its ability to degrade atrazine, and investigate the formation of metabolites. By sequencing of the 16S rRNA gene, the endophytic isolate atz2 was identified as Streptomyces sp. The reduction in atrazine concentration by Streptomyces sp. atz2 was 98 % and UHPLC-MS/MS analyses showed the appearance of an unknown metabolite observed as m/z 311. Ecotoxicity tests with an aquatic organism, Daphnia similis, confirmed that this metabolite was nontoxic. This mechanism of detoxification of atrazine is different from the ones of other free-living microorganisms that inhabit the soil or rhizosphere. The results show new aspects of atrazine detoxification, highlighting a new role of endophytic bacteria in plants.

  10. An integrated approach for profiling oxidative metabolites and glutathione adducts using liquid chromatography coupled with ultraviolet detection and triple quadrupole-linear ion trap mass spectrometry.

    PubMed

    Chen, Guiying; Cheng, Zhongzhe; Zhang, Kerong; Jiang, Hongliang; Zhu, Mingshe

    2016-09-10

    The use of liquid chromatography (LC) coupled with triple quadrupole linear ion trap (Qtrap) mass spectrometry (MS) for both quantitative and qualitative analysis in drug metabolism and pharmacokinetic studies is of great interest. Here, a new Qtrap-based analytical methodology for simultaneous detection, structural characterization and semi-quantitation of in vitro oxidative metabolites and glutathione trapped reactive metabolites was reported. In the current study, combined multiple ion monitoring and multiple reaction monitoring were served as surveying scans to trigger product ion spectral acquisition of oxidative metabolites and glutathione adduct, respectively. Then, detection of metabolites and recovery of their MS/MS spectra were accomplished using multiple data mining approaches. Additionally, on-line ultraviolet (UV) detection was employed to determine relative concentrations of major metabolites. Analyses of metabolites of clozapine and nomifensine in rat liver microsomes not only revealed multiple oxidative metabolites and glutathione adducts, but also identified their major oxidative metabolism and bioactivation pathways. The results demonstrated that the LC/UV/MS method enabled Qtrap to perform the comprehensive profiling of oxidative metabolites and glutathione adducts in vitro.

  11. Isoflavone metabolism in domestic cats (Felis catus): comparison of plasma metabolites detected after ingestion of two different dietary forms of genistein and daidzein.

    PubMed

    Whitehouse-Tedd, K M; Cave, N J; Ugarte, C E; Waldron, L A; Prasain, J K; Arabshahi, A; Barnes, S; Hendriks, W H; Thomas, D G

    2013-03-01

    Some felid diets contain isoflavones but the metabolic capacity of cats toward isoflavones is relatively unknown, despite the understanding that isoflavones have divergent biological potential according to their metabolite end products. The objective of this study was to determine the plasma metabolites detectable in domestic cats after exposure to 2 different dietary forms of isoflavones, either as a soy extract tablet (n = 6) or as part of a dietary matrix (n = 4). Serial blood samples were collected after isoflavone exposure to identify the plasma metabolites of each cat. Genistein was detected in its unconjugated form or as a monosulfate. Daidzein was detected as both a mono- and disulfate as well as in its unconjugated form. Other daidzein metabolites detected included equol mono- and disulfate, dihydrodaidzein, and O-desmethylangolensin. No β-glucuronide metabolites of either isoflavone were detected. Equol was produced in markedly fewer cats after ingestion of a soy extract tablet as a single oral bolus compared with cats consuming an isoflavone-containing diet. The detectable metabolites of the isoflavones, genistein and daidzein, in domestic cat plasma after dietary ingestion has been described in the present study for the first time. The metabolic capacity for isoflavones by domestic cats appears to be efficient, with only minimal proportions of the ingested amount detected in their unconjugated forms. This has implications for the potential of isoflavones to exert physiological activity in the domestic cat when consumed at concentrations representative of typical dietary intake.

  12. Improved genome annotation through untargeted detection of pathway-specific metabolites

    PubMed Central

    2011-01-01

    Background Mass spectrometry-based metabolomics analyses have the potential to complement sequence-based methods of genome annotation, but only if raw mass spectral data can be linked to specific metabolic pathways. In untargeted metabolomics, the measured mass of a detected compound is used to define the location of the compound in chemical space, but uncertainties in mass measurements lead to "degeneracies" in chemical space since multiple chemical formulae correspond to the same measured mass. We compare two methods to eliminate these degeneracies. One method relies on natural isotopic abundances, and the other relies on the use of stable-isotope labeling (SIL) to directly determine C and N atom counts. Both depend on combinatorial explorations of the "chemical space" comprised of all possible chemical formulae comprised of biologically relevant chemical elements. Results Of 1532 metabolic pathways curated in the MetaCyc database, 412 contain a metabolite having a chemical formula unique to that metabolic pathway. Thus, chemical formulae alone can suffice to infer the presence of some metabolic pathways. Of 248,928 unique chemical formulae selected from the PubChem database, more than 95% had at least one degeneracy on the basis of accurate mass information alone. Consideration of natural isotopic abundance reduced degeneracy to 64%, but mainly for formulae less than 500 Da in molecular weight, and only if the error in the relative isotopic peak intensity was less than 10%. Knowledge of exact C and N atom counts as determined by SIL enabled reduced degeneracy, allowing for determination of unique chemical formula for 55% of the PubChem formulae. Conclusions To facilitate the assignment of chemical formulae to unknown mass-spectral features, profiling can be performed on cultures uniformly labeled with stable isotopes of nitrogen (15N) or carbon (13C). This makes it possible to accurately count the number of carbon and nitrogen atoms in each molecule, providing a

  13. Showing Its Colors. Thin-Layer Chromatographic Detection of Cannabinoid Metabolites.

    ERIC Educational Resources Information Center

    Bonicamp, Judith M.

    1986-01-01

    Describes a chemistry laboratory experiment in which thin-layer chromatography (TLC) is used to analyze urine specimens containing metabolites of the drug tetrahydro-cannabinol, which comes from the marijuana plant. The materials needed to conduct the experiment are listed, and the procedure and expected results are outlined. (TW)

  14. Detection of Volatile Metabolites Derived from Garlic (Allium sativum) in Human Urine.

    PubMed

    Scheffler, Laura; Sauermann, Yvonne; Heinlein, Anja; Sharapa, Constanze; Buettner, Andrea

    2016-12-01

    The metabolism and excretion of flavor constituents of garlic, a common plant used in flavoring foods and attributed with several health benefits, in humans is not fully understood. Likewise, the physiologically active principles of garlic have not been fully clarified to date. It is possible that not only the parent compounds present in garlic but also its metabolites are responsible for the specific physiological properties of garlic, including its influence on the characteristic body odor signature of humans after garlic consumption. Accordingly, the aim of this study was to investigate potential garlic-derived metabolites in human urine. To this aim, 14 sets of urine samples were obtained from 12 volunteers, whereby each set comprised one sample that was collected prior to consumption of food-relevant concentrations of garlic, followed by five to eight subsequent samples after garlic consumption that covered a time interval of up to 26 h. The samples were analyzed chemo-analytically using gas chromatography-mass spectrometry/olfactometry (GC-MS/O), as well as sensorially by a trained human panel. The analyses revealed three different garlic-derived metabolites in urine, namely allyl methyl sulfide (AMS), allyl methyl sulfoxide (AMSO) and allyl methyl sulfone (AMSO₂), confirming our previous findings on human milk metabolite composition. The excretion rates of these metabolites into urine were strongly time-dependent with distinct inter-individual differences. These findings indicate that the volatile odorant fraction of garlic is heavily biotransformed in humans, opening up a window into substance circulation within the human body with potential wider ramifications in view of physiological effects of this aromatic plant that is appreciated by humans in their daily diet.

  15. Detection of Volatile Metabolites Derived from Garlic (Allium sativum) in Human Urine

    PubMed Central

    Scheffler, Laura; Sauermann, Yvonne; Heinlein, Anja; Sharapa, Constanze; Buettner, Andrea

    2016-01-01

    The metabolism and excretion of flavor constituents of garlic, a common plant used in flavoring foods and attributed with several health benefits, in humans is not fully understood. Likewise, the physiologically active principles of garlic have not been fully clarified to date. It is possible that not only the parent compounds present in garlic but also its metabolites are responsible for the specific physiological properties of garlic, including its influence on the characteristic body odor signature of humans after garlic consumption. Accordingly, the aim of this study was to investigate potential garlic-derived metabolites in human urine. To this aim, 14 sets of urine samples were obtained from 12 volunteers, whereby each set comprised one sample that was collected prior to consumption of food-relevant concentrations of garlic, followed by five to eight subsequent samples after garlic consumption that covered a time interval of up to 26 h. The samples were analyzed chemo-analytically using gas chromatography-mass spectrometry/olfactometry (GC-MS/O), as well as sensorially by a trained human panel. The analyses revealed three different garlic-derived metabolites in urine, namely allyl methyl sulfide (AMS), allyl methyl sulfoxide (AMSO) and allyl methyl sulfone (AMSO2), confirming our previous findings on human milk metabolite composition. The excretion rates of these metabolites into urine were strongly time-dependent with distinct inter-individual differences. These findings indicate that the volatile odorant fraction of garlic is heavily biotransformed in humans, opening up a window into substance circulation within the human body with potential wider ramifications in view of physiological effects of this aromatic plant that is appreciated by humans in their daily diet. PMID:27916960

  16. Removal of oseltamivir (Tamiflu) and other selected pharmaceuticals from wastewater using a granular bioplastic formulation entrapping propagules of Phanerochaete chrysosporium.

    PubMed

    Accinelli, Cesare; Saccà, Maria Ludovica; Batisson, Isabelle; Fick, Jerker; Mencarelli, Mariangela; Grabic, Roman

    2010-09-01

    The capacity of the ligninolytic fungus Phanerochaete chrysosporium to degrade a wide variety of environmentally persistent xenobiotics has been largely reported in the literature. Beside other factors, one barrier to a wider use of this bioremediation fungus is the availability of effective formulations that ensure easy preparation, handling and application. In this series of laboratory experiments, we evaluated the efficiency of a granular bioplastic formulation entrapping propagules of P. chrysosporium for removal of four selected pharmaceuticals from wastewater samples. Addition of inoculated granules to samples of the wastewater treatment plant of Bologna significantly increased the removal of the antiviral drug oseltamivir (Tamiflu), and the antibiotics, erythromycin, sulfamethoxazol, and ciprofloxacin. Similar effects were also observed in effluent water. Oseltamivir was the most persistent of the four active substances. After 30d of incubation, approximately two times more oseltamivir was removed in bioremediated wastewater than controls. The highest removal efficiency of the bioplastic formulation was observed with the antibiotic ciprofloxacin. Microbiological DNA-based analysis showed that the bioplastic matrix supported the growth of P. chrysosporium, thus facilitating its adaptation to unusual environment such as wastewater.

  17. Advances in Electronic-Nose Technologies for the Detection of Volatile Biomarker Metabolites in the Human Breath

    PubMed Central

    Wilson, Alphus D.

    2015-01-01

    Recent advancements in the use of electronic-nose (e-nose) devices to analyze human breath profiles for the presence of specific volatile metabolites, known as biomarkers or chemical bio-indicators of specific human diseases, metabolic disorders and the overall health status of individuals, are providing the potential for new noninvasive tools and techniques useful to point-of-care clinical disease diagnoses. This exciting new area of electronic disease detection and diagnosis promises to yield much faster and earlier detection of human diseases and disorders, allowing earlier, more effective treatments, resulting in more rapid patient recovery from various afflictions. E-nose devices are particularly suited for the field of disease diagnostics, because they are sensitive to a wide range of volatile organic compounds (VOCs) and can effectively distinguish between different complex gaseous mixtures via analysis of electronic aroma sensor-array output profiles of volatile metabolites present in the human breath. This review provides a summary of some recent developments of electronic-nose technologies, particularly involving breath analysis, with the potential for providing many new diagnostic applications for the detection of specific human diseases associated with different organs in the body, detectable from e-nose analyses of aberrant disease-associated VOCs present in air expired from the lungs. PMID:25738426

  18. Advances in electronic-nose technologies for the detection of volatile biomarker metabolites in the human breath.

    PubMed

    Wilson, Alphus D

    2015-03-02

    Recent advancements in the use of electronic-nose (e-nose) devices to analyze human breath profiles for the presence of specific volatile metabolites, known as biomarkers or chemical bio-indicators of specific human diseases, metabolic disorders and the overall health status of individuals, are providing the potential for new noninvasive tools and techniques useful to point-of-care clinical disease diagnoses. This exciting new area of electronic disease detection and diagnosis promises to yield much faster and earlier detection of human diseases and disorders, allowing earlier, more effective treatments, resulting in more rapid patient recovery from various afflictions. E-nose devices are particularly suited for the field of disease diagnostics, because they are sensitive to a wide range of volatile organic compounds (VOCs) and can effectively distinguish between different complex gaseous mixtures via analysis of electronic aroma sensor-array output profiles of volatile metabolites present in the human breath. This review provides a summary of some recent developments of electronic-nose technologies, particularly involving breath analysis, with the potential for providing many new diagnostic applications for the detection of specific human diseases associated with different organs in the body, detectable from e-nose analyses of aberrant disease-associated VOCs present in air expired from the lungs.

  19. Detection and mapping of illicit drugs and their metabolites in fingermarks by MALDI MS and compatibility with forensic techniques

    PubMed Central

    Groeneveld, G.; de Puit, M.; Bleay, S.; Bradshaw, R.; Francese, S.

    2015-01-01

    Despite the proven capabilities of Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS) in laboratory settings, research is still needed to integrate this technique into current forensic fingerprinting practice. Optimised protocols enabling the compatible application of MALDI to developed fingermarks will allow additional intelligence to be gathered around a suspect’s lifestyle and activities prior to the deposition of their fingermarks while committing a crime. The detection and mapping of illicit drugs and metabolites in latent fingermarks would provide intelligence that is beneficial for both police investigations and court cases. This study investigated MALDI MS detection and mapping capabilities for a large range of drugs of abuse and their metabolites in fingermarks; the detection and mapping of a mixture of these drugs in marks, with and without prior development with cyanoacrylate fuming or Vacuum Metal Deposition, was also examined. Our findings indicate the versatility of MALDI technology and its ability to retrieve chemical intelligence either by detecting the compounds investigated or by using their ion signals to reconstruct 2D maps of fingermark ridge details. PMID:26118853

  20. Detection and mapping of illicit drugs and their metabolites in fingermarks by MALDI MS and compatibility with forensic techniques

    NASA Astrophysics Data System (ADS)

    Groeneveld, G.; de Puit, M.; Bleay, S.; Bradshaw, R.; Francese, S.

    2015-06-01

    Despite the proven capabilities of Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS) in laboratory settings, research is still needed to integrate this technique into current forensic fingerprinting practice. Optimised protocols enabling the compatible application of MALDI to developed fingermarks will allow additional intelligence to be gathered around a suspect’s lifestyle and activities prior to the deposition of their fingermarks while committing a crime. The detection and mapping of illicit drugs and metabolites in latent fingermarks would provide intelligence that is beneficial for both police investigations and court cases. This study investigated MALDI MS detection and mapping capabilities for a large range of drugs of abuse and their metabolites in fingermarks; the detection and mapping of a mixture of these drugs in marks, with and without prior development with cyanoacrylate fuming or Vacuum Metal Deposition, was also examined. Our findings indicate the versatility of MALDI technology and its ability to retrieve chemical intelligence either by detecting the compounds investigated or by using their ion signals to reconstruct 2D maps of fingermark ridge details.

  1. Efficient approach for the detection and identification of new androgenic metabolites by applying SRM GC-CI-MS/MS: a methandienone case study.

    PubMed

    Polet, Michael; Van Gansbeke, Wim; Van Eenoo, Peter; Deventer, Koen

    2016-07-01

    Identification of anabolic androgenic steroids (AAS) is a vital issue in doping control and toxicology, and searching for metabolites with longer detection times remains an important task. Recently, a gas chromatography chemical ionization triple quadrupole mass spectrometry (GC-CI-MS/MS) method was introduced, and CI, in comparison with electron ionization (EI), proved to be capable of increasing the sensitivity significantly. In addition, correlations between AAS structure and fragmentation behavior could be revealed. This enables the search for previously unknown but expected metabolites by selection of their predicted transitions. The combination of both factors allows the setup of an efficient approach to search for new metabolites. The approach uses selected reaction monitoring which is inherently more sensitive than full scan or precursor ion scan. Additionally, structural information obtained from the structure specific CI fragmentation pattern facilitates metabolite identification. The procedure was demonstrated by a methandienone case study. Its metabolites have been studied extensively in the past, and this allowed an adequate evaluation of the efficiency of the approach. Thirty three metabolites were detected, including all relevant previously discovered metabolites. In our study, the previously reported long-term metabolite (18-nor-17β-hydroxymethyl,17α-methyl-androst-1,4,13-trien-3-one) could be detected up to 26 days by using GC-CI-MS/MS. The study proves the validity of the approach to search for metabolites of new synthetic AAS and new long-term metabolites of less studied AAS and illustrates the increase in sensitivity by using CI. Copyright © 2016 John Wiley & Sons, Ltd.

  2. Detection of in Vitro Metabolite Formation of Leflunomide: A Fluorescence Dynamics and Electronic Structure Study.

    PubMed

    Sharma, Poornima; Gangopadhyay, Debraj; Mishra, Phool Chand; Mishra, Hirdyesh; Singh, Ranjan K

    2016-04-14

    The metabolic transformation of antirheumatic fluorescent drug leflunomide into its active metabolite teriflunomide through isoxazole ring opening has been monitored in vitro using steady state and time domain fluorescence spectroscopy and density functional theory. During metabolic reaction, absorption of leflunomide split into two bands resembling absorption spectra of teriflunomide. The fluorescence spectra reveal slow conversion of leflunomide to E and Z forms of teriflunomide in aqueous medium, which becomes faster at basic pH. The E form, which is more potent as a drug, becomes more stable with an increase in the basicity of the medium. Both molecules are associated with charge transfer due to twisting in the lowest singlet excited state. Excited state charge transfer followed by proton transfer was also observed in the Z form during the ring opening of leflunomide. Quantum yield and radiative decay rates have been observed to decrease for the metabolite because of an increase in nonradiative decay channels.

  3. Urine metabolomic analysis to detect metabolites associated with the development of contrast induced nephropathy

    PubMed Central

    Diercks, Deborah B.; Owen, Kelly P.; Kline, Jeffrey A.; Sutter, Mark E.

    2016-01-01

    Objective Contrast induced nephropathy (CIN) is a result of injury to the proximal tubules. The incidence of CIN is around 11% for imaging done in the acute care setting. We aim to analyze the metabolic patterns in the urine, before and after dosing with intravenous contrast for computed tomography (CT) imaging of the chest, to determine if metabolomic changes exist in patients who develop CIN. Methods A convenience sample of high risk patients undergoing a chest CT with intravenous contrast were eligible for enrollment. Urine samples were collected prior to imaging and 4 to 6 hours post imaging. Samples underwent gas chromatography/mass spectrometry profiling. Peak metabolite values were measured and data was log transformed. Significance analysis of microarrays and partial least squares was used to determine the most significant metabolites prior to CT imaging and within subject. Analysis of variance was used to rank metabolites associated with temporal change and CIN. CIN was defined as an increase in serum creatinine level of ≥ 0.5 mg/dL or ≥ 25% above baseline within 48 hours after contrast administration. Results We sampled paired urine samples from 63 subjects. The incidence of CIN was 6/63 (9.5%). Patients without CIN had elevated urinary citric acid and taurine concentrations in the pre-CT urine. Xylulose increased in the post CT sample in patients who developed CIN. Conclusion Differences in metabolomics patterns in patients who do and do not develop CIN exist. Metabolites may be potential early identifiers of CIN and identify patients at high-risk for developing this condition prior to imaging. PMID:28168227

  4. Simultaneous detection of five one-carbon metabolites in plasma using stable isotope dilution liquid chromatography tandem mass spectrometry.

    PubMed

    Adaikalakoteswari, Antonysunil; Webster, Craig; Goljan, Ilona; Saravanan, Ponnusamy

    2016-02-15

    Disturbance in one-carbon (1-C) cycle occurs due to nutritional deficiencies (vitamin B12/folate) or specific genetic polymorphisms. This leads to altered levels of key 1-C metabolites such as SAM (s-adenosyl methionine), SAH (s-adenosyl homocysteine), methionine, homocysteine and MMA (methyl malonic acid). These 1-C metabolites are determinants of cellular methylation potential and epigenetic modifications of DNA which impairs metabolic pathways in several pathological diseases and developmental programming. Though methods were able to measure these analytes only independently, none of the methods detect simultaneously. Therefore we developed a method to measure these five 1-C metabolites in a single run using liquid chromatography tandem mass spectrometry (LC-MS/MS). We used stable isotopes dilution LC-MS/MS to measure the 1-C metabolites in human plasma. Blood samples were collected from pregnant women (n=30) at early gestation in the ongoing, multicentre, prospective PRiDE study. Linearity exhibited across the calibration range for all the analytes with the limit of detection (LOD) of 1.005nmol/l for SAM, 0.081nmol/l for SAH, 0.002μmol/l for methionine, 0.046μmol/l for homocysteine and 3.920nmol/l for MMA. The average recovery for SAM was 108%, SAH-110%, methionine-97%, homocysteine-91% and MMA-102%. The inter-assay CV for SAM was 7.3, SAH-5.6%, methionine-3.5%, homocysteine-7.0% and MMA-4.0%. The intra-assay CV for SAM was 8.7%, SAH-4.7%, methionine-5.4%, homocysteine-8.1% and MMA-6.1%. Pregnant women at early gestation with low B12 levels had significantly higher homocysteine, MMA, lower levels of methionine, SAM and SAM:SAH ratio and higher triglycerides. We developed a simple and rapid method to simultaneously quantify 1-C metabolites such as SAM, SAH, methionine, homocysteine and MMA in plasma by stable isotope dilution LC-MS/MS which would be useful to elucidate the epigenetic mechanisms related in the gene-nutrient interactions.

  5. Prototype Systems Containing Human Cytochrome P450 for High-Throughput Real-Time Detection of DNA Damage by Compounds That Form DNA-Reactive Metabolites.

    PubMed

    Brito Palma, Bernardo; Fisher, Charles W; Rueff, José; Kranendonk, Michel

    2016-05-16

    The formation of reactive metabolites through biotransformation is the suspected cause of many adverse drug reactions. Testing for the propensity of a drug to form reactive metabolites has increasingly become an integral part of lead-optimization strategy in drug discovery. DNA reactivity is one undesirable facet of a drug or its metabolites and can lead to increased risk of cancer and reproductive toxicity. Many drugs are metabolized by cytochromes P450 in the liver and other tissues, and these reactions can generate hard electrophiles. These hard electrophilic reactive metabolites may react with DNA and may be detected in standard in vitro genotoxicity assays; however, the majority of these assays fall short due to the use of animal-derived organ extracts that inadequately represent human metabolism. The current study describes the development of bacterial systems that efficiently detect DNA-damaging electrophilic reactive metabolites generated by human P450 biotransformation. These assays use a GFP reporter system that detects DNA damage through induction of the SOS response and a GFP reporter to control for cytotoxicity. Two human CYP1A2-competent prototypes presented here have appropriate characteristics for the detection of DNA-damaging reactive metabolites in a high-throughput manner. The advantages of this approach include a short assay time (120-180 min) with real-time measurement, sensitivity to small amounts of compound, and adaptability to a microplate format. These systems are suitable for high-throughput assays and can serve as prototypes for the development of future enhanced versions.

  6. Differential radial capillary action of ligand assay for high-throughput detection of protein-metabolite interactions.

    PubMed

    Roelofs, Kevin G; Wang, Jingxin; Sintim, Herman O; Lee, Vincent T

    2011-09-13

    Interactions of proteins with low-molecular-weight ligands, such as metabolites, cofactors, and allosteric regulators, are important determinants of metabolism, gene regulation, and cellular homeostasis. Pharmaceuticals often target these interactions to interfere with regulatory pathways. We have developed a rapid, precise, and high-throughput method for quantitatively measuring protein-ligand interactions without the need to purify the protein when performed in cells with low background activity. This method, differential radial capillary action of ligand assay (DRaCALA), is based on the ability of dry nitrocellulose to separate the free ligand from bound protein-ligand complexes. Nitrocellulose sequesters proteins and bound ligand at the site of application, whereas free ligand is mobilized by bulk movement of the solvent through capillary action. We show here that DRaCALA allows detection of specific interactions between three nucleotides and their cognate binding proteins. DRaCALA allows quantitative measurement of the dissociation constant and the dissociation rate. Furthermore, DRaCALA can detect the expression of a cyclic-di-GMP (cdiGMP)-binding protein in whole-cell lysates of Escherichia coli, demonstrating the power of the method to bypass the prerequisite for protein purification. We have used DRaCALA to investigate cdiGMP signaling in 54 bacterial species from 37 genera and 7 eukaryotic species. These studies revealed the presence of potential cdiGMP-binding proteins in 21 species of bacteria, including 4 unsequenced species. The ease of obtaining metabolite-protein interaction data using the DRaCALA assay will facilitate rapid identification of protein-metabolite and protein-pharmaceutical interactions in a systematic and comprehensive approach.

  7. [Cross-reactivity of Instant-View M-1 for detection of benzodiazepine-related drugs and their metabolites in urine].

    PubMed

    Torikoshi, Aiko; Namera, Akira; Arima, Yousuke; Toubou, Hirokazu; Tajima, Takashi; Shiraishi, Hiroaki; Nagao, Masataka

    2014-03-01

    Immunoassays are useful methods for the determination of regulated drugs in clinical and forensic laboratories. Although the Instant-View M-1 (IV M-1) immunoassay kit is frequently used to screen drugs in laboratories in Japan, basic information about the IV M-1 such as its specificity and reactivity is not available. In this study, we determined the specificity and cross-reactivity of IV M-1 for the detection of benzodiazepine-related drugs and their metabolites in urine. The IV M-1 could detect triazolobenzodiazepines such as triazolam in urine at concentrations > or = 300 ng/mL. However, thienodiazepines such as etizolam could not be detected because of lack of cross reactivity. A correlation was observed between the structure of the metabolites and the reactivity of the kit; 4-hydroxy metabolites of alprazolam and triazolam were detectable, whereas a-hydroxy metabolites were not. Furthermore, 7-amino metabolites such as nitrazepam could not be detected at any concentration, including high concentrations. The specificity and reactivity of various kits used for detection of drugs in urine are different. Therefore, it is necessary to consider the basic features of the kit used while assessing the results obtained.

  8. Detection and quantification of (R) and (S)-dechloroethylifosfamide metabolites in plasma from children by enantioselective LC/MS/MS.

    PubMed

    Aleksa, Katarina; Nava-Ocampo, Alejandro; Koren, Gideon

    2009-07-01

    Ifosfamide (IF), a potent chemotherapeutic agent for solid tumors, is known to cause high rates of nephrotoxicity in children with cancer, which is most likely due to the renal production of the metabolite chloroacetaldehyde. Using plasma samples obtained from pediatric oncology patients, we developed a simple nonderivatizing enantioselective liquid chromatography mass spectrometry method to detect the (R) and (S)-2- and 3-dechloroethylifosfamide metabolites. The (R) and (S)-enantiomers of the 2- and 3-DCEIF (N-3-dechlroethylifosfamide) were detectable in all 22 patients' samples with levels ranging from 9.9 to 238.7 ng/ml for (R)-2-DCEIF, 15.8 to 663.0 ng/ml for (S)-2-DCEIF, 20.8 to 852.8 ng/l for (R)-3-DCEIF and 28.0 to 862.0 ng/ml for (S)-3-DCEIF. In addition, the lower limit of quantification for this method is 1 ng/ml. Future studies should concentrate on (R) or (S) production of the 2-DCEIF and 3-DCEIF and subsequently chloroacetaldehyde formation with the aim of considering the administration of only the (R)-IF as its metabolism results in a lower production of chloroacetaldehyde.

  9. A validated HPLC method with electrochemical detection for simultaneous assay of 5-aminosalicylic acid and its metabolite in human plasma.

    PubMed

    Palumbo, Giancarlo; Bacchi, Simona; Primavera, Luisa; Palumbo, Paola; Carlucci, Giuseppe

    2005-06-01

    A high-performance liquid chromatographic method was developed, validated and applied to the simultaneous determination of 5-aminosalicylic acid (5-ASA) and its acetylated metabolite (acetyl-5-ASA) in human plasma. The method involves liquid-liquid extraction with methanol followed by isocratic reversed-phase chromatography on a Kromasil KR100 C(18) column with electrochemical detection. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from spiked human plasma samples. The effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers on retention of 5-ASA, acetyl 5-ASA and internal standard were investigated. Limits' of detection were 5 ng/mL for 5-ASA and 10 ng/mL for acetyl-5-ASA, respectively. The method can be used for supporting therapeutical drug monitoring and pharmacokinetic studies.

  10. Approach for detecting mutagenicity of biodegraded and ozonated pharmaceuticals, metabolites and transformation products from a drinking water perspective.

    PubMed

    Gartiser, Stefan; Hafner, Christoph; Kronenberger-Schäfer, Kerstin; Happel, Oliver; Trautwein, Christoph; Kümmerer, Klaus

    2012-09-01

    Many pharmaceuticals and related metabolites are not efficiently removed in sewage treatment plants and enter into surface water. There, they might be subject of drinking water abstraction and treatment by ozonation. In this study, a systematic approach for producing and effect-based testing of transformation products (TPs) during the drinking water ozonation process is proposed. For this, two pharmaceutical parent substances, three metabolites and one environmental degradation product were investigated with respect to their biodegradability and fate during drinking water ozonation. The Ames test (TA98, TA100) was used for the identification of mutagenic activity present in the solutions after testing inherent biodegradability and/or after ozonation of the samples. Suspicious results were complemented with the umu test. Due to the low substrate concentration required for ozonation, all ozonated samples were concentrated via solid phase extraction (SPE) before performing the Ames test. With the exception of piracetam, all substances were only incompletely biodegradable, suggesting the formation of stable TPs. Metformin, piracetam and guanylurea could not be removed completely by the ozonation process. We received some evidence that technical TPs are formed by ozonation of metformin and piracetam, whereas all tested metabolites were not detectable by analytical means after ozonation. In the case of guanylurea, one ozonation TP was identified by LC/MS. None of the experiments showed an increase of mutagenic effects in the Ames test. However, the SPE concentration procedure might lead to false-positive results due to the generation of mutagenic artefacts or might lead to false-negative results by missing adequate recovery efficiency. Thus, these investigations should always be accompanied by process blank controls that are carried out along the whole ozonation and SPE procedure. The study presented here is a first attempt to investigate the significance of

  11. A novel quartz crystal microbalance sensor array based on molecular imprinted polymers for simultaneous detection of clenbuterol and its metabolites.

    PubMed

    Feng, Fan; Zheng, Jianwu; Qin, Peng; Han, Tao; Zhao, Dayun

    2017-05-15

    For the rapid and robust detection of both parent clenbuterol (CLB) and its metabolites in swine urine samples, a novel quartz crystal microbalance (QCM) sensor array for CLB detection based on molecularly imprinted polymers (MIPs) was developed in this investigation. At first, clenbuterol and the structural analogs of its metabolites, 4-Aminohippuric acid (AHA) and 4-hydroxymandelic acid (HMA), were chosen as molecular templates. Through computational molecular modeling, the optimum ratio between the functional monomer and molecular template was selected. The surface imprinting method was applied to modify QCM electrode surface to graft a thin MIP film. The grafting polymer was characterized by Fourier-transformed infrared spectrometry (FTIR) and atomic force microscopy (AFM), respectively. After then, an array system composed of three sensors was employed to test the responses with different solutions and the principal component analysis (PCA) was adopted to analyze the corresponding data. As a result, for the designed sensor to clenbuterol, a linear equation y=100.07x-722.96 (R(2)=0.9928) was found between the sensor frequency shift ΔF and negative logarithm of clenbuterol concentration (-lgC). The limitation of detection (LOD) was 3.0ng/mL, which is lower than the Codex Alimentarius Commission regulations residue limit 10μg/L. The corresponding data of the three template solutions were analyzed by PCA, obtaining 100% recognition. The result demonstrated the feasibility that the developed method could be applied to detect whether the livestock was feed with CLB nutrient redistribution agent by checking the urine samples.

  12. System-wide detection of protein-small molecule complexes suggests extensive metabolite regulation in plants

    PubMed Central

    Veyel, Daniel; Kierszniowska, Sylwia; Kosmacz, Monika; Sokolowska, Ewelina Maria; Michaelis, Aenne; Luzarowski, Marcin; Szlachetko, Jagoda; Willmitzer, Lothar; Skirycz, Aleksandra

    2017-01-01

    Protein small molecule interactions are at the core of cell regulation controlling metabolism and development. We reasoned that due to the lack of system wide approaches only a minority of those regulatory molecules are known. In order to see whether or not this assumption is true we developed an effective approach for the identification of small molecules having potential regulatory role that obviates the need of protein or small molecule baits. At the core of this approach is a simple biochemical co-fractionation taking advantage of size differences between proteins and small molecules. Metabolomics based analysis of small molecules co-fractionating with proteins identified a multitude of small molecules in Arabidopsis suggesting the existence of numerous, small molecules/metabolites bound to proteins representing potential regulatory molecules. The approach presented here uses Arabidopsis cell cultures, but is generic and hence applicable to all biological systems. PMID:28205532

  13. Liquid Microjunction Surface Sampling Probe Electrospray Mass Spectrometry for Detection of Drugs and Metabolites in Thin Tissue Sections

    SciTech Connect

    Van Berkel, Gary J; Kertesz, Vilmos; Koeplinger, Kenneth A.; Vavek, Marissa; Kong, Ah-Ng Tony

    2008-01-01

    A self-aspirating, liquid micro-junction surface sampling probe/electrospray emitter mass spectrometry system was demonstrated for use in the direct analysis of spotted and dosed drugs and their metabolites in thin tissue sections. Proof-of-principle sampling and analysis directly from tissue without the need for sample preparation was demonstrated first by raster scanning a region on a section of rat liver onto which reserpine was spotted. The mass spectral signal from selected reaction monitoring was used to develop a chemical image of the spotted drug on the tissue. The probe was also used to selectively spot sample areas of sagittal whole mouse body tissue sections that had been dosed orally (90 mg/kg) with R,S-sulforaphane 3 hrs prior to sacrifice. Sulforaphane and its glutathione and N-acetyl cysteine conjugates were monitored with selected reaction monitoring and detected in the stomach and various other tissues from the dosed mouse. No signal for these species was observed in the tissue from a control mouse. The same dosed tissue section was used to illustrate the possibility of obtaining a line scan across the whole body section. In total these results illustrate the potential for rapid screening of the distribution of drugs and metabolites in tissue sections with the micro-liquid junction surface sampling probe/electrospray mass spectrometry approach.

  14. Ibuprofen exposure in Lemna gibba L.: Evaluation of growth and phytotoxic indicators, detection of ibuprofen and identification of its metabolites in plant and in the medium.

    PubMed

    Pietrini, F; Di Baccio, D; Aceña, J; Pérez, S; Barceló, D; Zacchini, M

    2015-12-30

    Ibuprofen (IBU) is detected worldwide in water bodies due to the incomplete removal by wastewater treatments. Contrasting results have been reported on the toxicity of IBU on aquatic biomonitor plants such as duckweed, and no data about IBU detection and metabolism in plants has been reported. In this work, the effects of 1 mg L(-1) IBU on Lemna gibba L. were monitored in an 8-day laboratory test. In particular, an increase in frond number (+12%) and multiplication rate (+10%) while no variations in photosynthetic pigment content were observed. Moreover, UPLC-HRMS analysis of the presence of IBU and its metabolites in plants and in the growth medium was performed. The results showed that, besides IBU, 11 IBU metabolites were detected in plants. Among the IBU metabolites, hydroxyl- and dihydroxyl-IBU were found, whereas carboxyl-IBU was undetectable. Interestingly, some IBU metabolites were detected in the plant growth solution at the end of the IBU treatment, while no IBU products were found in the IBU solution without plants, suggesting a role for L. gibba in IBU metabolism. The findings of this work represent an important step for a better evaluation of the effects of IBU and its metabolites in duckweed, with notable implications for the eco-toxicological assessment of IBU in the aquatic ecosystem.

  15. Direct Analysis in Real Time-Mass Spectrometry for the Rapid Detection of Metabolites of Aconite Alkaloids in Intestinal Bacteria

    NASA Astrophysics Data System (ADS)

    Li, Xue; Hou, Guangyue; Xing, Junpeng; Song, Fengrui; Liu, Zhiqiang; Liu, Shuying

    2014-12-01

    In the present work, direct analysis of real time ionization combined with multi-stage tandem mass spectrometry (DART-MSn) was used to investigate the metabolic profile of aconite alkaloids in rat intestinal bacteria. A total of 36 metabolites from three aconite alkaloids were identified by using DART-MSn, and the feasibility of quantitative analysis of these analytes was examined. Key parameters of the DART ion source, such as helium gas temperature and pressure, the source-to-MS distance, and the speed of the autosampler, were optimized to achieve high sensitivity, enhance reproducibility, and reduce the occurrence of fragmentation. The instrument analysis time for one sample can be less than 10 s for this method. Compared with ESI-MS and UPLC-MS, the DART-MS is more efficient for directly detecting metabolic samples, and has the advantage of being a simple, high-speed, high-throughput method.

  16. Detection of benzimidazole carbamates and amino metabolites in liver by surface plasmon resonance-biosensor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two surface plasmon resonance (SPR) biosensor screening assays were developed and validated to detect 11 benzimidazole carbamate (BZT) and four amino-benzimidazole veterinary drug residues in liver tissue. The assays used polyclonal antibodies, raised in sheep, to detect BZTs and amino-benzimidazole...

  17. Detection and characterization of DNA adducts formed from metabolites of the fungicide ortho-phenylphenol.

    PubMed

    Zhao, Shouxun; Narang, Amarjit; Gierthy, John; Eadon, George

    2002-05-22

    The significance of DNA adduction in ortho-phenylphenol-induced carcinogenesis remains unclear. Establishing adduct structures may contribute to resolving this issue. The chemical structures of the DNA adduction products resulting from the in vitro reaction of phenylbenzoquinone, the putative ultimate carcinogenic metabolite of the fungicide/disinfectant ortho-phenylphenol, are reported here. Three isomeric adducts that resulted from reaction of deoxyguanosine were characterized by UV, LC-ESI-MS, and MS/MS, and 1D and 2D COSY-NMR spectroscopy. The proposed mechanism of product formation is nucleophilic attack by the deoxyguanosine exocyclic amine nitrogen on an electrophilic quinone carbon, followed by stabilization through enolization. Another nucleophilic attack forms a five-membered ring, which aromatizes by dehydration to form the final product. Adducts were also characterized from deoxyadenosine and deoxycytidine, although conversions were at least 10 times lower. Structures are also proposed for these products. Cell culture studies confirmed that HepG2 cells incubated with phenylbenzoquinone at concentrations associated with cytotoxicity form the same DNA adducts.

  18. Old meets new: using interspecies interactions to detect secondary metabolite production in actinomycetes.

    PubMed

    Seyedsayamdost, Mohammad R; Traxler, Matthew F; Clardy, Jon; Kolter, Roberto

    2012-01-01

    Actinomycetes, a group of filamentous, Gram-positive bacteria, have long been a remarkable source of useful therapeutics. Recent genome sequencing and transcriptomic studies have shown that these bacteria, responsible for half of the clinically used antibiotics, also harbor a large reservoir of gene clusters, which have the potential to produce novel secreted small molecules. Yet, many of these clusters are not expressed under common culture conditions. One reason why these clusters have not been linked to a secreted small molecule lies in the way that actinomycetes have typically been studied: as pure cultures in nutrient-rich media that do not mimic the complex environments in which these bacteria evolved. New methods based on multispecies culture conditions provide an alternative approach to investigating the products of these gene clusters. We have recently implemented binary interspecies interaction assays to mine for new secondary metabolites and to study the underlying biology of interactinomycete interactions. Here, we describe the detailed biological and chemical methods comprising these studies.

  19. Determination of mesotrione residues and metabolites in crops, soil, and water by liquid chromatography with fluorescence detection.

    PubMed

    Alferness, Philip; Wiebe, Lawrence

    2002-07-03

    A method for the determination of residues of mesotrione and two metabolites in a variety of environmental matrixes has been developed. Mesotrione, a new selective herbicide for use in corn, is 2-(4-methylsulfonyl-2-nitrobenzoyl)-1,3-cyclohexanedione. The metabolite 4-methylsulfonyl-2-nitrobenzoic acid (MNBA) is determined with the parent compound in crops, whereas two metabolites, 2-amino-4-methylsulfonyl-benzoic acid (AMBA) and MNBA are determined with parent in soil and water. Crop samples are macerated with an acetonitrile/water mixture, and an aliquot is evaporated and acidified then centrifuged. Soil is shaken with an ammonium hydroxide solution, and an aliquot is acidified then centrifuged. For water analysis, an aliquot is acidified. Crop and soil extracts, and water, are cleaned up using reverse-phase high-performance liquid chromatography (RPHPLC) with mesotrione and MNBA isolated using a fraction collector. During this clean up, AMBA is determined in soil and water samples using fluorescence detection. The collected mesotrione and MNBA fractions are converted into AMBA via oxidation followed by reduction in the case of mesotrione, or by reduction alone in the case of MNBA. Both fractions are analyzed by RPHPLC with fluorescence detection using an AMBA external reference standard. The method was tested on corn grain, fodder, and forage, as well as on sugar cane. The limits of quantitation (LOQ) for each analyte are 0.01 mg/kg for crops, 0.005 mg/kg for soil, and 0.10 microg/L for water. Method fortification recoveries from all crop commodities averaged 79% (CV = 7%, n = 37 and 82% (CV = 5%, n = 37) for mesotrione and MNBA, respectively. Soil was fortified at 0.005 and 0.05 mg/kg. Recoveries were 79% (CV = 4%, n = 12), 96% (CV = 2%, n = 12), and 89% (CV = 2%, n = 12) for mesotrione, MNBA, and AMBA, respectively. Groundwater, drinking water, seawater, and river water were fortified at 0.1 and 1.0 microg/L. Recoveries for all waters were 80% (CV = 7%, n = 51

  20. Simultaneous detection and quantification of amphetamines, diazepam and its metabolites, cocaine and its metabolites, and opiates in hair by LC-ESI-MS-MS using a single extraction method.

    PubMed

    Miller, Eleanor I; Wylie, Fiona M; Oliver, John S

    2008-09-01

    A liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous identification and quantification of amphetamines, diazepam and its metabolites, cocaine and its metabolites, and opiates from hair using a single extraction method. As part of the method development, Gemini C18, Synergi Hydro RP, and Zorbax Stablebond-Phenyl LC columns were tested with three different mobile phases. Analyte recovery and limit of detection were evaluated for two different solid-phase extraction methods that used Bond Elut Certify and Clean Screen cartridges. Phosphate buffer (pH 5.0) was chosen as the optimum hair incubation medium because of the high stability of cocaine and 6-monoacetylmorphine using this method and faster sample preparation. The optimized method was fully validated. Linearity was established over the concentration range 0.2-10 ng/mg hair, and the correlation coefficients were all greater than 0.99. Total extraction recoveries were greater than 76%, detection limits were between 0.02 and 0.09 ng/mg, and the intra- and interday imprecisions were generally less than 20% in spiked hair. The intra- and interbatch imprecision of the method for a pooled authentic hair sample ranged from 1.4 to 23.4% relative standard deviation (RSD) and 8.3 to 25.4% RSD, respectively, for representative analytes from the different drug groups. The percent matrix effect ranged from 63.5 to 135.6%, with most analytes demonstrating ion suppression. Sixteen postmortem samples collected from suspected drug-related deaths were analyzed for the 17 drugs of abuse and metabolites included in the method. The method was sufficiently sensitive and specific for the analysis of drugs and metabolites in postmortem hair samples. There is scope for the inclusion of other target drugs and metabolites in the method.

  1. Separation and determination of L-tryptophan and its metabolites by capillary micellar electrokinetic chromatography with amperometric detection.

    PubMed

    Wang, Wei; Qiu, Bin; Xu, Xueqin; Zhang, Lan; Chen, Guonan

    2005-02-01

    A high-performance method of capillary micellar electrokinetic chromatography (CMEKC) with amperometric detection (AD), using a newly designed pre-aligned electrochemical cell, has been developed for the separation and determination of L-tryptophan (Trp) and its eight metabolites including 3-hydroxy-L-kynurenine (3-HK), 5-hydroxy-L-tryptophan (5-HTP), L-kynurenine (KN), 5-hydroxyindole-3-acetic acid (5-HIAA), xanthurenic acid (XA), indole-3-pyruvic acid (IPA), 5-hydroxytryptamine (5-HT), and tryptamine (Tryp). A carbon disk electrode was used as the working electrode and the optimal detection potential was 0.85 V (versus Ag/AgCl). At 24 kV of applied voltage, the nine compounds were completely separated, within 23 min, in a 10 mol/L Na(2)HPO(4)-NaOH buffer (pH 11.0) containing 40 mmol/L sodium dodecyl sulfate (SDS) and 3% methanol (v/v). A good linear relationship was obtained for all analytes in this paper and the detection limits of 3-HK, 5-HTP, KN, Trp, 5-HIAA, XA, IPA, 5-HT, and Tryp were 7.42, 5.18, 34.6, 3.99, 15.1, 12.7, 260, 6.72, and 8.01 nmol/L, respectively. This method has been applied to analyze the metabolism of Trp in rabbit urine.

  2. The quantitative analysis of heroin, methadone and their metabolites and the simultaneous detection of cocaine, acetylcodeine and their metabolites in human plasma by high-performance liquid chromatography coupled with tandem mass spectrometry.

    PubMed

    Rook, Elisabeth J; Hillebrand, Michel J X; Rosing, Hilde; van Ree, Jan M; Beijnen, Jos H

    2005-09-25

    For a pharmacokinetic-pharmacodynamic study in opioid tolerant patients, who were treated with heroin in combination with methadone, a liquid chromatographic assay with tandem mass spectrometry detection (LC-MS/MS) was developed for the simultaneous determination of heroin, methadone, heroin metabolites 6-monoacetylmorphine, morphine, and morphine-6 and 3-glucuronide and methadone metabolite EMDP. To detect any abuse of substances besides the prescribed opioids the assay was extended with the detection of cocaine, its metabolites benzoylecgonine and norcocaine and illicit heroin adulterants acetylcodeine and codeine. Heroin-d6, morphine-d3, morphine-3-glucuronide-d3 and methadone-d9 were used as internal standards. The sample pre-treatment consisted of solid phase extraction using mixed mode sorbent columns (MCX Oasis). Chromatographic separation was performed at 25 degrees C on a reversed phase Zorbax column with a gradient mobile phase consisting of ammonium formate (pH 4.0) and acetonitrile. The run time was 15 min. MS with relatively mild electrospray ionisation under atmospheric pressure was applied. The triple quadrupole MS was operating in the positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over a concentration range of 5-500 ng/mL for all analytes. The total recovery of heroin varied between 86 and 96% and of the heroin metabolites between 76 and 101%. Intra-assay and inter-assay accuracy and precision of all analytes were always within the designated limits (< or =20% at lower limit of quantification (LLQ) and < or =15% for other samples). This specific and sensitive assay was successfully applied in pharmacokinetic studies with medically prescribed heroin and toxicological cases.

  3. Detection of the metabolites of human plasma and follicular fluid in IVF-ET with microextraction and LC-TOF-MS.

    PubMed

    Wang, Gang; Yao, Shun; Liang, Xin; Zuo, Tao; Zhu, Minghui

    2015-01-01

    The metabolites from human plasma and follicular fluid in IVF-ET are related with the metabolic activity of follicular cells and further with oocyte quality. With liquid chromatography-time-of-flight-mass spectrometry (LC-TOF-MS) and organic extraction from plasma and follicular fluid, a fast and efficient method was established for detecting the metabolites of human plasma and follicular fluid. Certain metabolites were closely related with human physiological activities, such as hormone, amino acid, estrogen and fatty acid, and estrogen fatty acyllipid lipid were successfully detected in human plasma and follicular fluid. The metabolites in follicular fluid and plasma on the day of oocyte retrieval were found to be similar. But those in the latter and basal plasma were partially different. Methyl (3α ,5β ,7α ,12α)-3,7,12-trihydroxycholan-24-oate, androst-4-ene-3,17-dione and tryptophan were identified and might be potential biomarkers on oocyte quality. It demonstrates that LC-TOF-MS combined with organic extraction could be an efficient tool to investigate the metabolites of human plasma and follicular fluid. Based on this study, it is expected to provide the basis for the further steps to explore possible influential factors on oocyte quality.

  4. Detection and Characterization of the Effect of AB-FUBINACA and Its Metabolites in a Rat Model.

    PubMed

    Hsin-Hung Chen, Michael; Dip, Aybike; Ahmed, Mostafa; Tan, Michael L; Walterscheid, Jeffrey P; Sun, Hua; Teng, Ba-Bie; Mozayani, Ashraf

    2016-04-01

    Synthetic cannabinoids were originally developed by academic and pharmaceutical laboratories with the hope of providing therapeutic relief from the pain of inflammatory and degenerative diseases. However, recreational drug enthusiasts have flushed the market with new strains of these potent drugs that evade detection yet endanger public health and safety. Although many of these drug derivatives were published in the medical literature, others were merely patented without further characterization. AB-FUBINACA is an example of one of the new indazole-carboxamide synthetic cannabinoids introduced in the past year. Even though AB-FUBINACA has become increasingly prominent in forensic drug and toxicology specimens analyses, little is known about the pharmacology of this substance. To study its metabolic fate, we utilized Wistar rats to study the oxidative products of AB-FUBINACA in urine and its effect on gene expressions in liver and heart. Rats were injected with 5 mg/kg of AB-FUBINACA each day for 5 days. Urine samples were collected every day at the same time. On day 5 after treatment, we collected the organs such as liver and heart. The urine samples were analyzed by mass spectrometry, which revealed several putative metabolites and positioning of the hydroxyl addition on the molecule. We used quantitative PCR gene expression array to analyze the hepatotoxicity and cardiotoxicity on these rats and confirmed by real-time quantitative RT-PCR. We identified three genes significantly associated with dysfunction of oxidation and inflammation. Our study reports in vivo metabolites of AB-FUBINACA in urine and its effect on the gene expressions in liver and heart.

  5. Detection and Characterization of the Effect of AB‐FUBINACA and Its Metabolites in a Rat Model

    PubMed Central

    Hsin‐Hung Chen, Michael; Dip, Aybike; Ahmed, Mostafa; Tan, Michael L.; Walterscheid, Jeffrey P.; Sun, Hua

    2015-01-01

    ABSTRACT Synthetic cannabinoids were originally developed by academic and pharmaceutical laboratories with the hope of providing therapeutic relief from the pain of inflammatory and degenerative diseases. However, recreational drug enthusiasts have flushed the market with new strains of these potent drugs that evade detection yet endanger public health and safety. Although many of these drug derivatives were published in the medical literature, others were merely patented without further characterization. AB‐FUBINACA is an example of one of the new indazole‐carboxamide synthetic cannabinoids introduced in the past year. Even though AB‐FUBINACA has become increasingly prominent in forensic drug and toxicology specimens analyses, little is known about the pharmacology of this substance. To study its metabolic fate, we utilized Wistar rats to study the oxidative products of AB‐FUBINACA in urine and its effect on gene expressions in liver and heart. Rats were injected with 5 mg/kg of AB‐FUBINACA each day for 5 days. Urine samples were collected every day at the same time. On day 5 after treatment, we collected the organs such as liver and heart. The urine samples were analyzed by mass spectrometry, which revealed several putative metabolites and positioning of the hydroxyl addition on the molecule. We used quantitative PCR gene expression array to analyze the hepatotoxicity and cardiotoxicity on these rats and confirmed by real‐time quantitative RT‐PCR. We identified three genes significantly associated with dysfunction of oxidation and inflammation. Our study reports in vivo metabolites of AB‐FUBINACA in urine and its effect on the gene expressions in liver and heart. J. Cell. Biochem. 117: 1033–1043, 2016. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals. Inc. PMID:26517302

  6. Comparison of three fecal steroid metabolites for pregnancy detection used with single sampling in bighorn sheep (Ovis canadensis).

    PubMed

    Schoenecker, Kathryn A; Lyda, Robin O; Kirkpatrick, Jay

    2004-04-01

    We conpared three fecal steroid metabolite assays for their usefulness in detecting pregnalcy among free-ranging Rocky Mountain bighorn sheep (Ovis canadensis canadensis) from Bighorn Canyon National Recreation Area, Wyoming and Montana (USA) and captive bighorn ewes at ZooMontana in Billings, Montana. Fecal samples were collected from 11 free-ranging, radio-collared bighorn ewes in late January-May 2001 and from 20 free-ranging, radio-collared ewes in late March to mid-May 2002. Free-ranging ewes were monitored the following spring to determine whether or not they lambed. In addition, two captive ewes were studied at ZooMontana. With three exceptions, free-ranging bighorn ewes that produced lambs had nonspecific progesterone metabolite (iPdG) levels of >1800 ng/g feces and iPdG levels >7000 ng/gm feces when samples were collected between early March and mid-May. Samples collected earlier in the year were inconclusive. One false negative was suspected to be the result of sample collection error. Of the captive ewes, nonspecific pregnanediol-3alpha-glucuronide (PdG) and iPdG followed a predictable curve over the course of the 180-day pregnancies. We conclude that estrone conjugates are not useful in diagnosing pregnancy; however, fecal steroid analysis of PdG and iPdG can be used to accurately determine pregnancy and reproductive function in bighorn sheep. This holds great potential as a noninvasive technique for understanding the role of reproductive disease in wild bighom sheep.

  7. High matrix interference affecting detection of PAH metabolites in bile of Atlantic hagfish (Myxine glutinosa) used for biomonitoring of deep-water oil production.

    PubMed

    Sundt, Rolf C; Beyer, Jonny; Vingen, Sjur; Sydnes, Magne O

    2011-06-01

    The characteristic biology and wide distribution of hagfish species makes them relevant for use in pollution biomonitoring at great water depths, particularly in regions where deep-water oil production may take place. The exposure of fish to petrogenic contaminants can normally be detected from the level of polycyclic aromatic hydrocarbon (PAH) metabolites in bile fluid. Some of these metabolites are strong fluorophores, allowing analytical detection by means of simple fluorometric techniques such as fixed wavelength fluorescence (FF) and synchronous fluorescence scanning (SFS). In the present study bile from Atlantic hagfish (Myxine glutinosa) collected in pristine areas (Barents Sea and southwestern Norway) displayed strong bile fluorescence levels, suggesting the presence of PAH contaminants. However, gas-chromatography-mass spectrometry (GC-MS) analyses ruled out PAHs as the origin for this fluorescence signal. Rather, the bile of Myxine contains components resulting in unusually strong background fluorescence interfering at the wavelength pairs used for detection of PAH metabolites. Possible background for the observed matrix interference and implications for detection of PAH metabolites in hagfish is discussed.

  8. Chemometrics-enhanced high performance liquid chromatography-ultraviolet detection of bioactive metabolites from phytochemically unknown plants.

    PubMed

    Alvarez-Zapata, Radamés; Sánchez-Medina, Alberto; Chan-Bacab, Manuel; García-Sosa, Karlina; Escalante-Erosa, Fabiola; García-Rodríguez, Rosa Virginia; Peña-Rodríguez, Luis Manuel

    2015-11-27

    This work describes the use of Colubrina greggii as a model to investigate the use of chemometric analysis combined with data from a leishmanicidal bioassay, using Principal Component Analysis (PCA) and Orthogonal Projections to Latent Structures (O-PLS), to detect biologically active natural products in crude extracts from plants having little or no phytochemical information. A first analysis of the HPLC-UV profiles of the extract and its semi-purified fractions using both Principal Component Analysis (PCA) and Orthogonal Partial Least Squares (O-PLS) indicated that the components at tR 48.2, 48.7, 51.8min correlated with the variation in bioactivity. However, a further O-PLS analysis of the HPLC-UV profiles of fractions obtained through a final semi-preparative HPLC purification showed two components at tR 48.7 and 49.5min which correlated with the variation of the bioactivity in a high performance predictive model, with high determination coefficient, high correlation coefficient values (R(2) and Q(2)=0.99) and a low root mean square error (RMSE=0.018). This study demonstrates that the association of chemometric analysis with bioassay results can be an excellent strategy for the detection and isolation of bioactive metabolites from phytochemically unknown plant crude extracts.

  9. INNATE IMMUNITY. Cytosolic detection of the bacterial metabolite HBP activates TIFA-dependent innate immunity.

    PubMed

    Gaudet, Ryan G; Sintsova, Anna; Buckwalter, Carolyn M; Leung, Nelly; Cochrane, Alan; Li, Jianjun; Cox, Andrew D; Moffat, Jason; Gray-Owen, Scott D

    2015-06-12

    Host recognition of pathogen-associated molecular patterns (PAMPs) initiates an innate immune response that is critical for pathogen elimination and engagement of adaptive immunity. Here we show that mammalian cells can detect and respond to the bacterial-derived monosaccharide heptose-1,7-bisphosphate (HBP). A metabolic intermediate in lipopolysaccharide biosynthesis, HBP is highly conserved in Gram-negative bacteria, yet absent from eukaryotic cells. Detection of HBP within the host cytosol activated the nuclear facto κB pathway in vitro and induced innate and adaptive immune responses in vivo. Moreover, we used a genome-wide RNA interference screen to uncover an innate immune signaling axis, mediated by phosphorylation-dependent oligomerization of the TRAF-interacting protein with forkhead-associated domain (TIFA) that is triggered by HBP. Thus, HBP is a PAMP that activates TIFA-dependent immunity to Gram-negative bacteria.

  10. Real-time bilinear rotation decoupling in absorptive mode J-spectroscopy: Detecting low-intensity metabolite peak close to high-intensity metabolite peak with convenience

    NASA Astrophysics Data System (ADS)

    Verma, Ajay; Baishya, Bikash

    2016-05-01

    "Pure shift" NMR spectra display singlet peak per chemical site. Thus, high resolution is offered at the cost of valuable J-coupling information. In the present work, real-time BIRD (BIlinear Rotation Decoupling) is applied to the absorptive-mode 2D J-spectroscopy to provide pure shift spectrum in the direct dimension and J-coupling information in the indirect dimension. Quite often in metabolomics, proton NMR spectra from complex bio-fluids display tremendous signal overlap. Although conventional J-spectroscopy in principle overcomes this problem by separating the multiplet information from chemical shift information, however, only magnitude mode of the experiment is practical, sacrificing much of the potential high resolution that could be achieved. Few J-spectroscopy methods have been reported so far that produce high-resolution pure shift spectrum along with J-coupling information for crowded spectral regions. In the present work, high-quality J-resolved spectrum from important metabolomic mixture such as tissue extract from rat cortex is demonstrated. Many low-intensity metabolite peaks which are obscured by the broad dispersive tails from high-intensity metabolite peaks in regular magnitude mode J-spectrum can be clearly identified in real-time BIRD J-resolved spectrum. The general practice of removing such spectral overlap is tedious and time-consuming as it involves repeated sample preparation to change the pH of the tissue extract sample and subsequent spectra recording.

  11. Real-time bilinear rotation decoupling in absorptive mode J-spectroscopy: Detecting low-intensity metabolite peak close to high-intensity metabolite peak with convenience.

    PubMed

    Verma, Ajay; Baishya, Bikash

    2016-05-01

    "Pure shift" NMR spectra display singlet peak per chemical site. Thus, high resolution is offered at the cost of valuable J-coupling information. In the present work, real-time BIRD (BIlinear Rotation Decoupling) is applied to the absorptive-mode 2D J-spectroscopy to provide pure shift spectrum in the direct dimension and J-coupling information in the indirect dimension. Quite often in metabolomics, proton NMR spectra from complex bio-fluids display tremendous signal overlap. Although conventional J-spectroscopy in principle overcomes this problem by separating the multiplet information from chemical shift information, however, only magnitude mode of the experiment is practical, sacrificing much of the potential high resolution that could be achieved. Few J-spectroscopy methods have been reported so far that produce high-resolution pure shift spectrum along with J-coupling information for crowded spectral regions. In the present work, high-quality J-resolved spectrum from important metabolomic mixture such as tissue extract from rat cortex is demonstrated. Many low-intensity metabolite peaks which are obscured by the broad dispersive tails from high-intensity metabolite peaks in regular magnitude mode J-spectrum can be clearly identified in real-time BIRD J-resolved spectrum. The general practice of removing such spectral overlap is tedious and time-consuming as it involves repeated sample preparation to change the pH of the tissue extract sample and subsequent spectra recording.

  12. The Tamiflu saga continues: will our conduct change after the publication of the latest systematic review on benefits and harms of oseltamivir?

    PubMed

    Bachelet, Vivienne C

    2014-05-20

    In 2013, we wrote about the harm, waste and deception stemming from conducts adopted by the pharmaceutical industry, by concealing raw data and Clinical Study Reports (CSRs) from the regulator’s view when requesting the marketing patent. We described the case of Tamiflu (Roche), a drug that has been widely used in our population and profusely prescribed by physicians. Health authorities, entailing a great cost for the countries in the region, have also purchased it. In this editorial, we will show how the idea of using antivirals for prophylaxis and treatment of influenza took hold, starting from the first enthusiastic recommendations up to the systematic review published last month in the BMJ.

  13. SARM-S4 and metabolites detection in sports drug testing: a case report.

    PubMed

    Grata, Elia; Perrenoud, Laurent; Saugy, Martial; Baume, Norbert

    2011-12-10

    Recently, pharmaceutical industry developed a new class of therapeutics called Selective Androgen Receptor Modulator (SARM) to substitute the synthetic anabolic drugs used in medical treatments. Since the beginning of the anti-doping testing in sports in the 1970s, steroids have been the most frequently detected drugs mainly used for their anabolic properties. The major advantage of SARMs is the reduced androgenic activities which are the main source of side effects following anabolic agents' administration. In 2010, the Swiss laboratory for doping analyses reported the first case of SARMs abuse during in-competition testing. The analytical steps leading to this finding are described in this paper. Screening and confirmation results were obtained based on liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses. Additional information regarding the SARM S-4 metabolism was investigated by ultra high-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS).

  14. Determination of chloroacetanilide herbicide metabolites in water using high-performance liquid chromatography-diode array detection and high-performance liquid chromatography/mass spectrometry

    USGS Publications Warehouse

    Hostetler, K.A.; Thurman, E.M.

    2000-01-01

    Analytical methods using high-performance liquid chromatography-diode array detection (HPLC-DAD) and high-performance liquid chromatography/mass spectrometry (HPLC/MS) were developed for the analysis of the following chloroacetanilide herbicide metabolites in water: alachlor ethanesulfonic acid (ESA); alachlor oxanilic acid; acetochlor ESA; acetochlor oxanilic acid; metolachlor ESA; and metolachlor oxanilic acid. Good precision and accuracy were demonstrated for both the HPLC-DAD and HPLC/MS methods in reagent water, surface water, and ground water. The average HPLC-DAD recoveries of the chloroacetanilide herbicide metabolites from water samples spiked at 0.25, 0.5 and 2.0 ??g/l ranged from 84 to 112%, with relative standard deviations of 18% or less. The average HPLC/MS recoveries of the metabolites from water samples spiked at 0.05, 0.2 and 2.0 ??g/l ranged from 81 to 118%, with relative standard deviations of 20% or less. The limit of quantitation (LOQ) for all metabolites using the HPLC-DAD method was 0.20 ??g/l, whereas the LOQ using the HPLC/MS method was at 0.05 ??g/l. These metabolite-determination methods are valuable for acquiring information about water quality and the fate and transport of the parent chloroacetanilide herbicides in water. Copyright (C) 2000 Elsevier Science B.V.

  15. Quantitation of l-alpha-acetylmethadol and its metabolites in human serum by capillary gas-liquid chromatography and nitrogen detection.

    PubMed

    Verebey, K; DePace, A; Mulé, S J

    1985-10-11

    A procedure is described for the simultaneous measurement of l-alpha-acetylmethadol and its two pharmacologically active metabolites: noracetylmethadol and dinoracetylmethadol. In the method an intramolecular conversion reaction of the two metabolites to their amide configuration is utilized. The reaction is performed while the metabolites are still in the serum. Following solvent extraction the samples are analyzed by capillary gas-liquid chromatography coupled with nitrogen detection. Quantitation is achieved by internal standardization. The lower limit of sensitivity is 5 ng/ml in serum. Absolute sensitivity is 0.1 ng for all three compounds. The advantages over other procedures are: speed due to the single extraction step; increased recovery of noracetylmethadol and dinoracetylmethadol due to decreased polarity of the amides; greater stability of the metabolites in the amide configuration; better chromatographic quantitation and separation because detector response for the amides is greater than it is for the original configuration of the metabolites and the area of the chromatographic tracing is free of interfering substances.

  16. Therapeutic targeting of Neu1 sialidase with oseltamivir phosphate (Tamiflu®) disables cancer cell survival in human pancreatic cancer with acquired chemoresistance

    PubMed Central

    O’Shea, Leah K; Abdulkhalek, Samar; Allison, Stephanie; Neufeld, Ronald J; Szewczuk, Myron R

    2014-01-01

    Background Resistance to drug therapy, along with high rates of metastasis, contributes to the low survival rate in patients diagnosed with pancreatic cancer. An alternate treatment for human pancreatic cancer involving targeting of Neu1 sialidase with oseltamivir phosphate (Tamiflu®) was investigated in human pancreatic cancer (PANC1) cells with acquired resistance to cisplatin and gemcitabine. Its efficacy in overcoming the intrinsic resistance of the cell to chemotherapeutics and metastasis was evaluated. Methods Microscopic imaging, immunocytochemistry, immunohistochemistry, and WST-1 cell viability assays were used to evaluate cell survival, morphologic changes, and expression levels of E-cadherin, N-cadherin, and VE-cadherin before and after treatment with oseltamivir phosphate in PANC1 cells with established resistance to cisplatin, gemcitabine, or a combination of the two agents, and in archived paraffin-embedded PANC1 tumors grown in RAGxCγ double mutant mice. Results Oseltamivir phosphate overcame the chemoresistance of PANC1 to cisplatin and gemcitabine alone or in combination in a dose-dependent manner, and disabled the cancer cell survival mechanism(s). Oseltamivir phosphate also reversed the epithelial-mesenchymal transition characteristic of the phenotypic E-cadherin to N-cadherin changes associated with resistance to drug therapy. Low-dose oseltamivir phosphate alone or in combination with gemcitabine in heterotopic xenografts of PANC1 tumors growing in RAGxCγ double mutant mice did not prevent metastatic spread to the liver and lung. Conclusion Therapeutic targeting of Neu1 sialidase with oseltamivir phosphate at the growth factor receptor level disables the intrinsic signaling platform for cancer cell survival in human pancreatic cancer with acquired chemoresistance. These findings provide evidence for oseltamivir phosphate (Tamiflu) as a potential therapeutic agent for pancreatic cancer resistant to drug therapy. PMID:24470763

  17. An immunoassay for the detection of triclosan-O-glucuronide, a primary human urinary metabolite of triclosan

    PubMed Central

    Ranganathan, Anupama; Gee, Shirley J.; Hammock, Bruce D.

    2015-01-01

    Triclosan-O-glucuronide (TCSG) is one of the primary urinary metabolites of the antibacterial compound triclosan or TCS that is found in many personal care products and consumer goods. We have developed a competitive, indirect heterologous ELISA for the detection of the target TCSG in urine. Such an ELISA for TCSG could be developed as a useful tool to measure this important biomarker of human exposure to TCS. Immunogens were prepared by conjugating TCSG to thyroglobulin, via heterobifunctional cross-linkers AEDP or 3-[(2-aminoethyl)dithio] propionic acid•hydrochloride and TFCS or N-[ε-trifluoroacetylcaproyloxy]succinimide ester. The coating antigen was prepared by the direct conjugation of TCSG to bovine serum albumin. Antibodies raised in rabbits 2619, 2621 (immunogen TCSG-AEDP-Thy) and 2623 (immunogen TCSG-TFCS-Thy) and the coating antigen were screened and characterized to determine their optimal concentrations. The optimized ELISA, developed with antibody 2621, gave an IC50 value of 2.85 ng/mL, with the linear range (IC20 – IC80) determined to be 2.6 – 24.8 ng/mL. Selectivity of the assay was assessed by measuring cross-reactivity of antibody 2621 to related congeners such as the aglycone TCS, triclosan-O-sulfate, triclocarban, a polybrominated diphenyl ether derivative and 3-phenoxybenzyl alcohol glucuronide. There was virtually no recognition by antibody 2621 to any of these cross-reactants. PMID:26255293

  18. An immunoassay for the detection of triclosan-O-glucuronide, a primary human urinary metabolite of triclosan.

    PubMed

    Ranganathan, Anupama; Gee, Shirley J; Hammock, Bruce D

    2015-09-01

    Triclosan-O-glucuronide (TCSG) is one of the primary urinary metabolites of the antibacterial compound triclosan or TCS that is found in many personal care products and consumer goods. We have developed a competitive, indirect heterologous ELISA for the detection of the target TCSG in urine. Such an ELISA for TCSG could be developed as a useful tool to measure this important biomarker of human exposure to TCS. Immunogens were prepared by conjugating TCSG to thyroglobulin, via heterobifunctional cross-linkers AEDP or 3-[(2-aminoethyl)dithio] propionic acid•hydrochloride and TFCS or N-[ε-trifluoroacetylcaproyloxy]succinimide ester. The coating antigen was prepared by the direct conjugation of TCSG to bovine serum albumin. Antibodies raised in rabbits 2619, 2621 (immunogen TCSG-AEDP-Thy), and 2623 (immunogen TCSG-TFCS-Thy), and the coating antigen were screened and characterized to determine their optimal concentrations. The optimized ELISA, developed with antibody 2621, gave an IC50 value of 2.85 ng/mL, with the linear range (IC20-IC80) determined to be 2.6-24.8 ng/mL. Selectivity of the assay was assessed by measuring cross-reactivity of antibody 2621 to related congeners such as the aglycone TCS, triclosan-O-sulfate, triclocarban, a polybrominated diphenyl ether derivative, and 3-phenoxybenzyl alcohol glucuronide. There was virtually no recognition by antibody 2621 to any of these cross-reactants. Graphical Abstract Urinary biomarker analysis of triclosan glucuronide.

  19. Detection of the marijuana metabolite 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid in oral fluid specimens and its contribution to positive results in screening assays.

    PubMed

    Moore, Christine; Ross, Wayne; Coulter, Cynthia; Adams, Laura; Rana, Sumandeep; Vincent, Michael; Soares, James

    2006-09-01

    The detection of the marijuana metabolite 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in oral fluid specimens is described, and its contribution to an immunoassay for the detection of cannabinoids is investigated. Oral fluid specimens, screened using an enzyme-linked immunosorbent immunoassay (ELISA), were carried forward to confirmation for both tetrahydrocannabinol (THC) and THC-COOH using gas chromatography-mass spectrometry (GC-MS). One hundred and fifty-three specimens were analyzed, of which 143 screened positive for cannabinoids. Ninety-five (66.4%) of these specimens were positive for both THC and THC-COOH; 14 (9.7%) were positive for THC-COOH only, and 27 (18.8%) were positive for THC only. The GC-MS assay for the detection of THC-COOH in oral fluid was linear to 160 pg/mL with a limit of quantitation of 2 pg/mL. The detection of the marijuana metabolite, THC-COOH, in 76.2% of oral fluid specimens screening positive for cannabinoids is reported. As a potential defense against passive exposure claims, proposed SAMHSA regulations may require the simultaneous collection of a urine sample when oral fluid samples are used. The detection of the metabolite, THC-COOH, is a significant alternative to this approach because its presence in oral fluid minimizes the argument for passive exposure to marijuana in drug testing cases.

  20. Use of LC-QqQ-MS for the detection of emodin metabolites in rat bile and urine.

    PubMed

    Wu, Songyan; Zhang, Yaqing; Zhang, Zunjian; Song, Rui

    2017-03-25

    Emodin is the representative form of rhubarb, which is widely used in traditional Chinese medicine for the treatment of purgative, anti-inflammatory, antioxidative and antiviral, etc. Previous reports demonstrated that emodin glucuronide was the major metabolite in plasma. Due to the extensive conjugation reactions of polyphenols, the aim of this study was to identify the metabolites of emodin in rat bile and urine. Neutral loss and precursor ion scan methods of triple-quadrupole mass spectrometer revealed 13 conjugated metabolites in rat bile and 22 metabolites in rat urine, which including 4 phase I and 18 phase II metabolites. The major metabolites in rat bio-samples were emodin glucuronoconjugates. Moreover, rhein monoglucuronide, chrysophanol monoglucuronide and rhein sulfate were proposed for the first time after oral administration of emodin. Overall, liquid chromatography hybrid triple-quadrupole mass spectrometry (LC-QqQ-MS) analysis leads to the discovery of several novel emodin metabolites in rat bile and urine and underscores that conjugated with glucuronic acid is the main metabolic pathway.

  1. Determination of methandrostenolone and its metabolites in equine plasma and urine by coupled-column liquid chromatography with ultraviolet detection and confirmation by tandem mass spectrometry.

    PubMed

    Edlund, P O; Bowers, L; Henion, J

    1989-02-24

    Monitoring steroid use requires an understanding of the metabolism in the species in question and development of sensitive methods for screening of the steroid or its metabolites in urine. Qualitative information for confirmation of methandrostenolone and identification of its metabolites was primarily obtained by coupled-column high-performance liquid chromatography-tandem mass spectrometry. The steroids and a sulphuric acid conjugate were isolated and identified by their daughter ion mass spectra in the urine of both man and the horse following administration of methandrostenolone. Spontaneous hydrolysis of methandrostenolone sulphate gave 17-epimethandrostenolone and several dehydration products. This reaction had a half-life of 16 min in equine urine at 27 degrees C. Mono- and dihydroxylated metabolites were also identified. Several screening methods were evaluated for detection and confirmation of methandrostenolone use including thin-layer chromatography and high-performance liquid chromatography. Coupled-column liquid chromatography was used for automated clean-up of analytes difficult to isolate by manual methods. The recovery of methandrostenolone was 101 +/- 3.3% (mean +/- S.D.) at 6.5 ng/ml and both methandrostenolone and 17-epimethandrostenolone were quantified in urine by ultraviolet detection up to six days after a 250-mg intramuscular dose to a horse. The utility of on-line tandem mass spectrometry for confirmation of suspected metabolites is also shown.

  2. Echo-Planar Imaging Based J-Resolved Spectroscopic Imaging for Improved Metabolite Detection in Prostate Cancer

    DTIC Science & Technology

    2012-10-01

    phantom containing several metabolites which have been reported in prostate tissues, and to optimize the EP-JJRESI sequence and other acquisition...parameters using the phantom (Months 6-12). Accomplished during September 29, 2011-October 28 2012: The sequence was tested using a prostate phantom ...containing 10 different metabolites at physiological concentrations (pH set to 7.2). A 500 ml prostate phantom was prepared containing the following

  3. Determination of loratadine and its active metabolite in human plasma by high-performance liquid chromatography with mass spectrometry detection.

    PubMed

    Vlase, Laurian; Imre, Silvia; Muntean, Dana; Leucuta, Sorin E

    2007-07-27

    A new sensitive and selective liquid chromatography coupled with mass spectrometry (LC/MS/MS) method for quantification of loratadine (LOR) and its active metabolite descarboethoxyloratadine (DSL) in human plasma was validated. After addition of the internal standard, metoclopramide, the human plasma samples (0.3 ml) were precipitated using acetonitrile (0.75 ml) and the centrifuged supernatants were partially evaporated under nitrogen at 37 degrees C at approximately 0.3 ml volume. The LOR, DSL and internal standard were separated on a reversed phase column (Zorbax SB-C18, 100 mmx3.0 mm i.d., 3.5 microm) under isocratic conditions using a mobile phase of an 8:92(v/v) mixture of acetonitrile and 0.4% (v/v) formic acid in water. The flow rate was 1 ml/min and the column temperature 45 degrees C. The detection of LOR, DSL and internal standard was in MRM mode using an ion trap mass spectrometer with electrospray positive ionisation. The ion transitions were monitored as follows: 383-->337 for LOR, 311-->(259+294+282) for DSL and 300-->226.8 for internal standard. Calibration curves were generated over the range of 0.52-52.3 ng/ml for both LOR and DSL with values for coefficient of determination greater than 0.994 by using a weighted (1/y) quadratic regression. The lower limits of quantification were established at 0.52 ng/ml LOR and DSL, respectively, with an accuracy and precision less than 20%. Both analytes demonstrated good short-term, long-term, post-preparative and freeze-thaw stability. Besides its simplicity, the sample treatment allows obtaining a very good recovery of both analytes, around 100%. The validated LC/MS/MS method has been applied to a pharmacokinetic study of loratadine tablets on healthy volunteers.

  4. Enantioselective determination of metoprolol and its metabolites in human urine high-performance liquid chromatography with fluorescence detection (HPLC-FLD) and tandem mass spectrometry (MS/MS).

    PubMed

    Baranowska, Irena; Adolf, Weronika; Magiera, Sylwia

    2015-11-01

    A sensitive, stereoselective assay using solid phase extraction and high-performance liquid chromatography (HPLC) with fluorescence detection (FLD) was developed and validated for the analysis of enantiomers of metoprolol and its metabolites (α-hydroxymetoprolol, O-desmethylmetoprolol). Chiral separation was achieved using a CHIRALCEL OD-RH column, packed with cellulose tris-(3,5-dimethylphenyl-carbamate) stationary phase, employing a mobile phase composed by a mixture of 0.2% diethylamine in water and acetonitrile in gradient elution mode. Linear calibration curves were obtained over the range of 0.025-2.0μg/mL (R(2)>0.994) in urine for both enantiomers of metoprolol and its metabolites with quantitation limit of 0.025μg/mL. Intra and inter-day precision and accuracy were below 15% for both metoprolol and metabolites enantiomers. The recovery of enantiomer of metoprolol and its metabolite was greater than 68.0%, utilizing a SPE procedure. The method was tested with urine quality control samples and human urine fractions after administration of 50mg rac-metoprolol.

  5. Electrochemical detection of DNA damage induced by acrylamide and its metabolite at the graphene-ionic liquid-Nafion modified pyrolytic graphite electrode.

    PubMed

    Qiu, Yanyan; Qu, Xiangjin; Dong, Jing; Ai, Shiyun; Han, Ruixia

    2011-06-15

    A new electrochemical biosensor for directly detecting DNA damage induced by acrylamide (AA) and its metabolite was presented in this work. The graphene-ionic liquid-Nafion modified pyrolytic graphite electrode (PGE) was prepared, and then horseradish peroxidase (HRP) and natural double-stranded DNA were alternately assembled on the modified electrode by the layer-by-layer method. The PGE/graphene-ionic liquid-Nafion and the construction of the (HRP/DNA)(n) film were characterized by electrochemical impedance spectroscopy. With the guanine signal in DNA as an indicator, the damage of DNA was detected by differential pulse voltammetry after PGE/graphene-ionic liquid-Nafion/(HRP/DNA)(n) was incubated in AA solution or AA+H(2)O(2) solution at 37°C. This method provides a new model to mimic and directly detect DNA damage induced by chemical pollutants and their metabolites in vitro. The results indicated that, in the presence of H(2)O(2), HRP was activated and catalyzed the transformation of AA to glycidamide, which could form DNA adducts and induce more serious damage of DNA than AA. In order to further verify these results, UV-vis spectrophotometry was also used to investigate DNA damage induced by AA and its metabolites in solution and the similar results were obtained.

  6. 'Dilute-and-shoot' RSLC-MS-MS method for fast detection of nerve and vesicant chemical warfare agent metabolites in urine.

    PubMed

    Rodin, Igor; Braun, Arcady; Stavrianidi, Andrey; Baygildiev, Timur; Shpigun, Oleg; Oreshkin, Dmitry; Rybalchenko, Igor

    2015-01-01

    A sensitive screening method based on fast liquid chromatography tandem mass-spectrometry (RSLC-MS-MS) has shown the feasibility of separation and detection of low concentration β-lyase metabolites of sulfur mustard and of nerve agent phosphonic acids in urine. The analysis of these compounds is of interest because they are specific metabolites of the chemical warfare agents (CWAs), sulfur mustard (HD), sarin (GB), soman (GD), VX and Russian VX (RVX). The 'dilute-and-shoot' RSLC-MS-MS method provides a sensitive and direct approach for determining CWA exposure in non-extracted non-derivatized samples from urine. Chromatographic separation of the metabolites was achieved using a reverse phase column with gradient mobile phases consisting of 0.5% formic acid in water and acetonitrile. Identification and quantification of species were achieved using electrospray ionization-tandem mass-spectrometry monitoring two precursor-to-product ion transitions for each compound. The method demonstrates linearity over at least two orders of magnitude and had detection limits of 0.5 ng/mL in urine.

  7. Evaluation of a simple in-house test to presumptively differentiate Mycobacterium tuberculosis complex from nontuberculous mycobacteria by detection of p-nitrobenzoic acid metabolites.

    PubMed

    Wang, Guirong; Yu, Xia; Liang, Qian; Chen, Suting; Wilson, Stuart; Huang, Hairong

    2013-01-01

    The timely differentiation of Mycobacterium tuberculosis complex (MTC) and non-tubercular mycobacterium (NTM) species is urgently needed in patient care since the routine laboratory method is time consuming and cumbersome. An easy and cheap method which can successfully distinguish MTC from NTM was established and evaluated. 38 mycobacterial type and reference strains and 65 clinical isolates representing 10 species of mycobacterium were included in this study. Metabolites of p-nitrobenzoic acid (PNB) reduction were identified using liquid chromatography and tandem mass spectrometry (LC/MS/MS). A spectrophotometric method was developed to detect these metabolites, which was evaluated on a number of MTC and NTM species. All of the tested NTM species and strains reduced PNB to p-aminobenzoic acid (PABA), while none of the MTC strains showed a similar activity. Spectrophotometric detection of PABA had 100% sensitivity and specificity for MTC and NTM differentiation among the type strains and the clinical isolates tested. PABA was identified as one of the metabolites of PNB reduction. All the tested NTM species metabolized PNB to PABA whereas the MTC members lacked this activity. A simple, specific and cost-effective method based on PABA production was established in order to discriminate MTC from NTM from cultured organisms.

  8. A derivatization-enhanced detection strategy in mass spectrometry: analysis of 4-hydroxybenzoates and their metabolites after keratinocytes are exposed to UV radiation

    PubMed Central

    Lee, Yi-Hsuan; Lin, Ying-Chi; Feng, Chia-Hsien; Tseng, Wei-Lung; Lu, Chi-Yu

    2017-01-01

    4-Hydroxybenzoate is a phenolic derivative of alkyl benzoates and is a widely used preservative in cosmetic and pharmaceutical products. The presence of 4-hydroxybenzoates in the human body may result from the use of pharmaceutical and personal care products. These compounds are also known to exhibit estrogenic and genotoxic activities. The potential adverse effects of these compounds include endocrine disruption, oxidative and DNA damage, contact dermatitis, and allergic reactions. This study used two mass spectrometry methods that are applicable when using a derivatization-enhanced detection strategy (DEDS) to screen 4-hydroxybenzoates and their metabolites. Chemical derivatization was used to enhance the detection of these compounds. To evaluate the metabolic process triggered by UV radiation, human keratinocyte HaCaT cells treated with these 4-hydroxybenzoates were further exposed to UVA, UVB and UVC radiation. Metabolites transformed by human keratinocytes in the chemical derivatization procedure were identified by a nano ultra-performance liquid chromatographic system (nanoUPLC) coupled with LTQ Orbitrap. The experiments confirmed the feasibility of this method for identifying 4-hydroxybenzoate metabolites and for high-throughput screening of 4-hydroxybenzoate in commercial products (50 samples) by the DEDS. PMID:28057923

  9. High temperature liquid chromatography hyphenated with ESI-MS and ICP-MS detection for the structural characterization and quantification of halogen containing drug metabolites.

    PubMed

    de Vlieger, Jon S B; Giezen, Mark J N; Falck, David; Tump, Cornelis; van Heuveln, Fred; Giera, Martin; Kool, Jeroen; Lingeman, Henk; Wieling, Jaap; Honing, Maarten; Irth, Hubertus; Niessen, Wilfried M A

    2011-07-18

    In this paper we describe the hyphenation of high temperature liquid chromatography with ICP-MS and ESI-MS for the characterization of halogen containing drug metabolites. The use of temperature gradients up to 200°C enabled the separation of metabolites with low organic modifier content. This specific property allowed the use of detection methods that suffer from (significant) changes in analyte response factors as a function of the organic modifier content such as ICP-MS. Metabolites of two kinase inhibitors (SB-203580-Iodo and MAPK inhibitor VIII) produced by bacterial cytochrome P450 BM3 mutants and human liver microsomes were identified based on high resolution MS(n) data. Quantification was done using their normalized and elemental specific response in the ICP-MS. The importance of these kinds of quantification strategies is stressed by the observation that the difference of the position of one oxygen atom in a structure can greatly affect its response in ESI-MS and UV detection.

  10. Analysis of lidocaine and its major metabolite, monoethylglycinexylidide, in elk velvet antler by liquid chromatography with UV detection and confirmation by electrospray ionization tandem mass spectrometry.

    PubMed

    Bagonluri, Mukasa T; Woodbury, Murray R; Reid, R Stephen; Boison, Joe O

    2005-04-06

    A sensitive liquid chromatographic (LC) method with UV detection was developed for the determination of residues of lidocaine (LID) and its major metabolite, monoethylglycinexylidide (MEGX), in elk velvet antler. The drugs were extracted from alkaline velvet antler homogenates, cleaned up on a C(18) solid-phase extraction cartridge, and separated on an Inertsil ODS-3 (3.0 x 250 mm, 5 microm) column using an isocratic mobile phase made up of 0.05 M phosphate buffer (pH 4.0)/acetonitrile (88:12, v/v) at a flow rate of 1.0 mL/min. The limits of quantification for LID and its major metabolite, MEGX, were 10 and 20 ng/g, respectively. The method was validated and used to measure the concentration of residues of LID and MEGX in elk velvet antlers harvested after either LID anesthesia or application of a drug-free control method (electro-anesthesia, EA). No LID or MEGX residues were detected in any of the antlers harvested after EA application. No MEGX residues were detected in any of the velvet antlers harvested after LID application, but residues of LID ranging in concentration from 68 to 4300 ng/g were detected in the three sections of the velvet antlers harvested after LID administration. LC-tandem mass spectrometry was used to confirm the presence of lidocaine detected in the velvet antlers.

  11. Toxicological detection of the designer drug 3,4-methylenedioxyethylamphetamine (MDE, "Eve") and its metabolites in urine by gas chromatography-mass spectrometry and fluorescence polarization immunoassay.

    PubMed

    Ensslin, H K; Kovar, K A; Maurer, H H

    1996-08-30

    Studies are presented on the toxicological detection of the designer drug methylenedioxyethylamphetamine [MDE, rac-N-ethyl-(3,4-methylenedioxyphenyl)-propane-2-amine] in urine after a single oral dose of 140 mg of MDE by GC-MS and fluorescence polarization immunoassay (FPIA). After acid hydrolysis, extraction and acetylation MDE and its metabolites could be detected by mass chromatography with the selected ions m/z 72, 86, 114, 150, 162 and 164, followed by identification of the peaks underlying full mass spectra by computer library search. The following metabolites could be detected: unchanged MDE and 3,4-dihydroxyethylamphetamine (DHE) for 33-62 h, 3,4-methylenedioxyamphetamine (MDA) for 32-36 h and 4-hydroxy-3-methoxyethylamphetamine (HME) for 7-8 days. 3,4-Dihydroxyamphetamine (DHA), 4-hydroxy-3-methoxyamphetamine (HMA), piperonyl acetone, 3,4-dihydroxyphenyl acetone and 4-hydroxy-3-methoxyphenyl acetone could only be detected in trace amounts within the first few hours. The Abbott TD x FPIA assay amphetamine/metamphetamine II gave positive results in urine for 33-62 h. Therefore, positive immunoassay results could be confirmed by the GC-MS procedure which also allowed the differentiation of MDE and its homologues 3,4-methylenedioxymethamphetamine (MDMA) and MDA as well as other amphetamine derivatives interfering with the TD x assay. Furthermore, this GC-MS procedure allowed the simultaneous detection of most of the toxicologically relevant drugs.

  12. Combined derivatization and high-performance liquid chromatography with fluorescence and ultraviolet detection for simultaneous analysis of octreotide and gabexate mesylate metabolite in human pancreatic juice samples.

    PubMed

    Carlucci, Giuseppe; Selvaggi, Federico; Sulpizio, Sara; Bassi, Claudio; Carlucci, Maura; Cotellese, Roberto; Ferrone, Vincenzo; Innocenti, Paolo; Locatelli, Marcello

    2015-06-01

    A simple and sensitive method based on the combination of derivatization and high-performance liquid chromatography with ultraviolet and fluorimetric detection was developed for the simultaneous determination of octreotide and gabexate mesylate metabolite in human pancreatic juice samples. Parameters of the derivatization procedure affecting extraction efficiency were optimized. The developed method was validated according to the International Conference on Harmonization guidelines. The calibration curves were linear over a range of 0.1-15 µg/mL for octreotide and 0.20-15 µg/mL for gabexate mesylate metabolite. Derivatized products of octreotide and gabexate mesylate metabolite were separated on a Luna C18 column (4.6 × 250 mm; 5 µm particle size) using a gradient with a run time of 36 min, without further purification. The limits of detection were 0.025 and 0.05, respectively, for octreotide and gabexate mesylate metabolite. This paper reports the validation of a quantitative high performance liquid chromatography-photodiode array-fluorescence (HPLC-PDA-FL) method for the simultaneous analysis of octreotide and gabexate mesylate metabolite in pancreatic juice by protein precipitation using zinc sulfate-methanol-acetonitrile containing the derivatizing reagent, 4-fluoro-7-nitro-[2,1,3]-benzoxadiazole (NBD-F). Derivatized products of octreotide and gabexate mesylate metabolite were separated on a Luna C18 column (4.6 × 250 mm; 5 µm particle size) using a gradient with a run time of 36 min, without further purification. The method was validated over the concentration ranges 0.1-15 and 0.2-15 µg/mL for octreotide and gabexate mesylate metabolite, respectively, in human pancreatic juice. Biphalin and methyl-p-hydroxybenzoate were used as the internal standards. This method was successfully utilized to support clinical studies in humans. The results from assay validations show that the method is selective, sensitive and robust. The limit

  13. Screening procedure for detection of diuretics and uricosurics and/or their metabolites in human urine using gas chromatography-mass spectrometry after extractive methylation.

    PubMed

    Beyer, Jochen; Bierl, Anabelle; Peters, Frank T; Maurer, Hans H

    2005-08-01

    A gas chromatography-mass spectrometry (GC-MS)-based screening procedure was developed for the detection of diuretics, uricosurics, and/or their metabolites in human urine after extractive methylation. Phase-transfer catalyst remaining in the organic phase was removed by solid-phase extraction on a diol phase. The compounds were separated by GC and identified by MS in the full-scan mode. The possible presence of the following drugs and/or their metabolites could be indicated using mass chromatography with the given ions: m/z 267, 352, 353, 355, 386, and 392 for thiazide diuretics bemetizide, bendroflumethiazide, butizide, chlorothiazide, cyclopenthiazide, cyclothiazide, hydrochlorothiazide, metolazone, polythiazide, and for canrenoic acid and spironolactone; m/z 77, 81, 181, 261, 270, 295, 406, and 438 for loop diuretics bumetanide, ethacrynic acid, furosemide, piretanide, torasemide, as well as the uricosurics benzbromarone, probenecid, and sulfinpyrazone; m/z 84, 85, 111, 112, 135, 161, 249, 253, 289, and 363 for the other diuretics acetazolamide, carzenide, chlorthalidone, clopamide, diclofenamide, etozoline, indapamide, mefruside, tienilic acid, and xipamide. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with reference spectra. This method allowed the detection of the abovementioned drugs and/or their metabolites in human urine samples, except torasemide. The limits of detection ranged from 0.001 to 5 mg/L in the full-scan mode. Recoveries of selected diuretics and uricosurics, representing the different chemical classes, ranged from 46% to 99% with coefficients of variation of less than 21%. After ingestion of the lowest therapeutic doses, furosemide was detectable in urine samples for 67 hours, hydrochlorothiazide for 48 hours, and spironolactone for 52 hours (via its target analyte canrenone). The procedure described here is part of a systematic toxicological analysis

  14. Simultaneous determination of aromatic acid metabolites of styrene and styrene-oxide in rat urine by gas chromatography-flame ionization detection.

    PubMed

    Cosnier, Frédéric; Nunge, Hervé; Cossec, Benoît; Gaté, Laurent

    2012-06-01

    A convenient and reliable gas chromatographic method was developed for the simultaneous determination of six aromatic acid metabolites of styrene and styrene-oxide in rat urine; i.e., benzoic (BA), phenylacetic (PAA), mandelic (MA), phenylglyoxylic (PGA), hippuric (HA) and phenylaceturic (PAUA) acids. The method involves a one-pot esterification-extraction procedure, performed directly on urine without prior treatment. Analyses were performed on a RTX-1701 capillary column and the recovered isopropyl esters derivatives were detected by flame ionization detection. The analytical method was validated for selectivity, linearity, detection and quantification limits, recovery and intra-day and inter-day precisions. Calibration curves showed linearity in the range of 8-800 mg/L, except for HA and PAUA (40-800 mg/L). Limits of detection were between 0.2 (PPA) and 7.0 (PAUA) mg/L. The intra-day precisions determined at three concentrations levels were less than 5% for BA, PAA, MA and PGA and 9% for HA and PAUA, respectively. The corresponding mean inter-day precisions for these two groups were 8 and 16%, respectively. The method was successfully applied to quantitatively analyze styrene, styrene-oxide, ethylbenzene and toluene metabolites in urine samples from rats exposed by inhalation to these compounds at levels close to the occupational threshold limit values. Provided that this method can be transposed to human urine, it could have applications as part of biological monitoring for workers exposed to styrene or related compounds.

  15. Determination of zuclopenthixol and its main N-dealkylated metabolite in biological fluids using high-performance liquid chromatography with post-column photochemical derivatization and fluorescence detection.

    PubMed

    Hansen, B B; Hansen, S H

    1994-08-19

    A highly sensitive high-performance liquid chromatographic (HPLC) method for the assay of cis-(Z)-clopenthixol (zuclopenthixol) in urine and plasma has been developed. Following solid-phase extraction, the samples are chromatographed using reversed-phase ion-pairing HPLC. After separation, the solutes, having a thioxanthene structure, are transformed on-line into thioxanthones in a photochemical reactor. The thioxanthones are highly fluorescent compounds, and therefore, low detection limits are obtained when using fluorescence detection. Detection limits for zuclopenthixol and its N-dealkylated metabolite, in plasma as well as in urine, using fluorescence detection with excitation at 260 nm and emission at 435 nm, were found to be 0.05 ng/ml and 0.2 ng/ml, respectively. The chromatographic system separates the cis-(Z)- and trans-(E)-isomers of clopenthixol from its main dealkylated metabolite. Furthermore, the chromatographic system is very suitable for study of the photochemical reaction, since the chloro-thioxanthone and thioxanthone are well separated from the isomers of clopenthixol.

  16. Effects of Water Models on Binding Affinity: Evidence from All-Atom Simulation of Binding of Tamiflu to A/H5N1 Neuraminidase

    PubMed Central

    Nguyen, Trang Truc; Viet, Man Hoang

    2014-01-01

    The influence of water models SPC, SPC/E, TIP3P, and TIP4P on ligand binding affinity is examined by calculating the binding free energy ΔGbind of oseltamivir carboxylate (Tamiflu) to the wild type of glycoprotein neuraminidase from the pandemic A/H5N1 virus. ΔGbind is estimated by the Molecular Mechanic-Poisson Boltzmann Surface Area method and all-atom simulations with different combinations of these aqueous models and four force fields AMBER99SB, CHARMM27, GROMOS96 43a1, and OPLS-AA/L. It is shown that there is no correlation between the binding free energy and the water density in the binding pocket in CHARMM. However, for three remaining force fields ΔGbind decays with increase of water density. SPC/E provides the lowest binding free energy for any force field, while the water effect is the most pronounced in CHARMM. In agreement with the popular GROMACS recommendation, the binding score obtained by combinations of AMBER-TIP3P, OPLS-TIP4P, and GROMOS-SPC is the most relevant to the experiments. For wild-type neuraminidase we have found that SPC is more suitable for CHARMM than TIP3P recommended by GROMACS for studying ligand binding. However, our study for three of its mutants reveals that TIP3P is presumably the best choice for CHARMM. PMID:24672329

  17. Effects of water models on binding affinity: evidence from all-atom simulation of binding of tamiflu to A/H5N1 neuraminidase.

    PubMed

    Nguyen, Trang Truc; Viet, Man Hoang; Li, Mai Suan

    2014-01-01

    The influence of water models SPC, SPC/E, TIP3P, and TIP4P on ligand binding affinity is examined by calculating the binding free energy ΔG(bind) of oseltamivir carboxylate (Tamiflu) to the wild type of glycoprotein neuraminidase from the pandemic A/H5N1 virus. ΔG(bind) is estimated by the Molecular Mechanic-Poisson Boltzmann Surface Area method and all-atom simulations with different combinations of these aqueous models and four force fields AMBER99SB, CHARMM27, GROMOS96 43a1, and OPLS-AA/L. It is shown that there is no correlation between the binding free energy and the water density in the binding pocket in CHARMM. However, for three remaining force fields ΔG(bind) decays with increase of water density. SPC/E provides the lowest binding free energy for any force field, while the water effect is the most pronounced in CHARMM. In agreement with the popular GROMACS recommendation, the binding score obtained by combinations of AMBER-TIP3P, OPLS-TIP4P, and GROMOS-SPC is the most relevant to the experiments. For wild-type neuraminidase we have found that SPC is more suitable for CHARMM than TIP3P recommended by GROMACS for studying ligand binding. However, our study for three of its mutants reveals that TIP3P is presumably the best choice for CHARMM.

  18. Lysophosphatidylcholine and amide as metabolites for detecting alzheimer disease using ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry-based metabonomics.

    PubMed

    Cui, Yu; Liu, Xiuqin; Wang, Maoqing; Liu, Liyan; Sun, Xiaohong; Ma, Lan; Xie, Wei; Wang, Chao; Tang, Sisi; Wang, Decai; Wu, Qunhong

    2014-10-01

    Alzheimer disease (AD) can be diagnosed by clinical and neuropsychologic tests and at autopsy, but there are no simple effective diagnostic methods for detecting biomarkers in patients at early stages of cognitive impairment. Early metabolic alterations that may facilitate AD diagnosis have not been thoroughly explored. We applied a nontargeted metabonomic approach using ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry to analyze serum and urine samples from 46 patients with AD and 36 healthy controls. Metabolite profiles were processed using multivariate analysis to identify potential metabolites, which were further confirmed using tandem mass spectrometry. Ultrahigh-performance liquid chromatography mass spectrometry methods were additionally used to quantify potentially important biomarkers. Independent samples were then selected to validate the identified biomarkers. There was a clear separation between healthy controls and AD patients; AD patient samples had disordered amino acid and phospholipid metabolism and dysregulated palmitic amide. Receiver operator characteristic curve and quantification suggested that palmitic amide, lysophosphatidylcholine (LysoPC, 18:0), LysoPC(18:2), L-glutamine, and 5-L-glutamylglycine were the optimal metabolites. In addition, areas under the curve from the palmitic amide, LysoPC(18:2), and 5-L-glutamylglycine in the validation study were 0.714, 0.996, and 0.734, respectively. These data elucidate the metabolic alterations associated with AD and suggest new biomarkers for AD diagnosis, thereby permitting early intervention designed to prevent disease progression.

  19. Determination of metoprolol and its two metabolites in human plasma and urine by high performance liquid chromatography with fluorescence detection and its application in pharmacokinetics.

    PubMed

    Xu, Tao; Bao, Shihui; Geng, Peiwu; Luo, Jun; Yu, Lei; Pan, Peipei; Chen, Yi; Hu, Guoxin

    2013-10-15

    A simple, specific and sensitive HPLC method has been developed for the determination of metoprolol and its two metabolites in human plasma and urine. Separation of metoprolol, α-hydroxymetoprolol, O-desmethylmetoprolol and esmolol (internal standard) was achieved on an Agilent XDB-C18 column (150mm×4.6mm, 5μm) using fluorescence detection with Ex 216nm and Em 312nm. The mobile phase consisted of ACN-H2O-0.1%TFA. The analysis was performed in less than 16min with a flow rate of 0.8mL/min. The assay was linear over the concentration range of 5-600ng/mL and 2.5-300ng/mL for metoprolol and its metabolites, respectively. The LOQ were 5.0 and 2.5ng/mL for plasma and urine, respectively. Good precision and accuracy for metoprolol and its two metabolites were obtained. The extraction recoveries were found to be more than 86.91% both in plasma and urine. At the same time, the method was successfully applied to nine healthy volunteers who had been given an oral tablet of 100mg metoprolol.

  20. Detection of 5α-androst-2-en-17-one and variants: Identification of main urinary metabolites in human urine samples by GC-MS and NMR.

    PubMed

    Ayotte, Christiane; Sylvestre, Alexandre; Charlebois, Alain; Poirier, Donald

    2016-11-01

    Two steroids were identified in a supplement named D-2 following the detection of unknown compounds during the routine testing of an athlete's sample. The main glucuroconjugated metabolites were isolated from this urine by high performance liquid chromatography (HPLC) following enzymatic hydrolysis and identified by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) analyses as being 2α-hydroxy-5α-androst-3-en-17-one (M1) and 2β,3α-dihydroxy-5α-androstan-17-one (M2). A third metabolite, 3α,4β-dihydroxy-5α-androstan-17-one (M3) was also detected, however in lower amounts. The precursor steroids, 5α-androst-2-en-17-one (1) and 5α-androst-3-en-17-one (2) were present in the first D-2 products offered on the Internet. Later, the corresponding 17-hydroxyl compounds were offered as such or as esters (acetate, cypionate) in different relative ratios. Both M2 and M3 were synthesized from the trans-diaxial hydrolysis of the corresponding 2α,3α- and 3α,4α-epoxides (3). These were excreted in the hours following the controlled administration of the commercial product called D-2 R to a male volunteer and were also produced from the incubation of 1 and 2 with S9 liver fractions. Some preparations contain predominantly the alkene in C-2 and, therefore, an efficient detection method must include both primary metabolites M1 and M2. The latter was found equally in the fractions extracted following the enzymatic hydrolysis with β-glucuronidase and the chemical solvolysis, which may ease its identification. Copyright © 2016 John Wiley & Sons, Ltd.

  1. Differences in metabolite-detecting, adrenergic, and immune gene expression following moderate exercise in chronic fatigue syndrome, multiple sclerosis and healthy controls

    PubMed Central

    White, Andrea T.; Light, Alan R.; Hughen, Ronald W.; VanHaitsma, Timothy A.; Light, Kathleen C.

    2011-01-01

    Objective Chronic fatigue syndrome (CFS) and multiple sclerosis (MS) are characterized by debilitating fatigue, yet evaluation of this symptom is subjective. We examined metabolite-detecting, adrenergic, and immune gene expression (mRNA) in patients with CFS (n=22) vs. MS (n=20) vs. healthy controls (n=23) and determined their relationship to fatigue and pain before and after exercise. Methods Blood samples and fatigue and pain ratings were obtained at baseline and 0.5, 8, 24, and 48 hours following sustained moderate exercise. Leukocyte mRNA of 4 metabolite-detecting receptors (ASIC3, P2X4, P2X5, TRPV1), 4 adrenergic (α-2a, β-1, β-2 receptors, COMT) and 5 immune markers (CD14, TLR4, IL-6, IL-10, LTa) was examined using quantitative PCR. Results CFS patients had greater post-exercise increases in fatigue and pain (10–29 pts above baseline, p<.001) and greater mRNA increases in P2X4, TRPV1, CD14 and all adrenergic receptors than controls (1.3 ± .14 to 3.4 ± .90 fold increase above baseline, p=.04 – .005). CFS patients with co-morbid fibromyalgia (n=18) also showed greater increases in ASIC3 and P2X5 (p<.05). MS patients had greater post-exercise increases than controls in β-1 and β-2 adrenergic receptor expression (1.4 ± .27 and 1.3 ± .06 fold increase, respectively, p=.02 and <.001) and greater decreases in TLR4 (p=.02). In MS, IL-10 and TLR4 decreases correlated with higher fatigue scores. Conclusion Post-exercise mRNA increases in metabolite-detecting receptors were unique to CFS patients while both MS and CFS showed abnormal increases in adrenergic receptors. Among MS patients, greater fatigue was correlated with blunted immune marker expression. PMID:22210239

  2. Comparison of the Microgenics CEDIA heroin metabolite (6-AM) and the Roche Abuscreen ONLINE opiate immunoassays for the detection of heroin use in forensic urine samples.

    PubMed

    Holler, Justin M; Bosy, Thomas Z; Klette, Kevin L; Wiegand, Russel; Jemionek, John; Jacobs, Aaron

    2004-09-01

    Current Department of Defense (DoD) and Department of Health and Human Services (HHS) procedures for the detection of heroin abuse by testing urine utilize an initial opiate (codeine/morphine) immunoassay (IA) screen followed by gas chromatography-mass spectrometry (GC-MS) confirmation of 6-acetylmorphine (6-AM), if the morphine concentration is above established cutoff. An alternative to the current opiates screen for heroin abuse is the direct IA for the metabolite of heroin, 6-acetylmorphine. In this regard, the performance of the Microgenics CEDIA heroin metabolite (6-AM) screening reagent was assessed. This evaluation was conducted on the P module of a Hitachi Modular automated IA analyzer calibrated using 6-AM at 10 ng/mL. Reproducibility, linearity, accuracy, sensitivity, and interferences associated with use of the 6-AM IA reagent were evaluated. The IA reagent precision (percent coefficient of variation (%CV)) around each of seven standards was less than 0.63%, with a linearity (r(2)) value of 0.9951. A total of 37,713 active duty service members' urine samples were analyzed simultaneously using the CEDIA heroin metabolite (6-AM) reagent and the Roche Abuscreen ONLINE opiate reagent to evaluate both the prevalence rate of 6-AM in the demographic group and the sensitivity and specificity of the reagents for the detection of heroin use. Of the 37,713 samples tested using the CEDIA heroin metabolite (6-AM) reagent, three samples screened positive at the DoD and HHS cutoff of 10 ng/mL. One of the three samples confirmed positive for 6-AM by GC-MS above the cutoff of 10 ng/mL, the two remaining samples confirmed negative for 6-AM at a GC-MS limit of detection (LOD) of 2.1 ng/mL. In contrast, the Roche Abuscreen ONLINE opiate IA produced 74 opiate-positive results for codeine/morphine, with 6 of the 74 specimens confirming positive for morphine above the DoD cutoff concentration of 4000 ng/mL (8% DoD morphine confirmation rate), only one of the 74 opiate

  3. Multi-residue enantiomeric analysis of human and veterinary pharmaceuticals and their metabolites in environmental samples by chiral liquid chromatography coupled with tandem mass spectrometry detection.

    PubMed

    Camacho-Muñoz, Dolores; Kasprzyk-Hordern, Barbara

    2015-12-01

    Enantiomeric profiling of chiral pharmacologically active compounds (PACs) in the environment has hardly been investigated. This manuscript describes, for the first time, a multi-residue enantioselective method for the analysis of human and veterinary chiral PACs and their main metabolites from different therapeutic groups in complex environmental samples such as wastewater and river water. Several analytes targeted in this paper have not been analysed in the environment at enantiomeric level before. These are aminorex, carboxyibuprofen, carprofen, cephalexin, 3-N-dechloroethylifosfamide, 10,11-dihydro-10-hydroxycarbamazepine, dihydroketoprofen, fenoprofen, fexofenadine, flurbiprofen, 2-hydroxyibuprofen, ifosfamide, indoprofen, mandelic acid, 2-phenylpropionic acid, praziquantel and tetramisole. The method is based on chiral liquid chromatography utilising a chiral α1-acid glycoprotein column and tandem mass spectrometry detection. Excellent chromatographic separation of enantiomers (Rs≥1.0) was achieved for chloramphenicol, fexofenadine, ifosfamide, naproxen, tetramisole, ibuprofen and their metabolites: aminorex and dihydroketoprofen (three of four enantiomers), and partial separation (Rs = 0.7-1.0) was achieved for ketoprofen, praziquantel and the following metabolites: 3-N-dechloroethylifosfamide and 10,11-dihydro-10-hydroxycarbamazepine. The overall performance of the method was satisfactory for most of the compounds targeted. Method detection limits were at low nanogram per litre for surface water and effluent wastewater. Method intra-day precision was on average under 20% and sample pre-concentration using solid phase extraction yielded recoveries >70% for most of the analytes. This novel, selective and sensitive method has been applied for the quantification of chiral PACs in surface water and effluent wastewater providing excellent enantioresolution of multicomponent mixtures in complex environmental samples. It will help with better understanding

  4. Simultaneous determination of a novel calcium entry blocker, monatepil maleate, and its metabolites in rat plasma by means of solid-phase extraction and reversed-phase liquid chromatography with electrochemical detection.

    PubMed

    Kurono, M; Suzuki, K; Yoshida, K; Naruto, S

    1997-02-21

    A reversed-phase LC method with electrochemical detection is described for the simultaneous determination of monatepil maleate (AJ-2615, AJ), a novel calcium entry blocker, and its three S-oxidized metabolites in plasma. These compounds were extracted from plasma by solid-phase extraction and injected onto an ODS column. The determination limit in plasma (0.5 ml) was 10 ng/ml for AJ and 5 ng/ml for the three metabolites. The method was applied to the determination of AJ and the metabolites in rat plasma samples.

  5. Metabolite identification of a radiotracer by electrochemistry coupled to liquid chromatography with mass spectrometric and radioactivity detection.

    PubMed

    Baumann, Anne; Faust, Andreas; Law, Marylin P; Kuhlmann, Michael T; Kopka, Klaus; Schäfers, Michael; Karst, Uwe

    2011-07-01

    Radioligands, which specifically bind to a receptor or enzyme (target), enable molecular imaging of the target expression by positron emission tomography (PET). One very promising PET tracer is (S)-1-(4-(2-[(18)F]-fluoroethoxy)benzyl)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (isatin), a caspase-3 inhibitor, which has been developed at the University Hospital of Münster to image cell death (apoptosis). The translation of this novel tracer from preclinical evaluation to clinical examinations requires biodistribution studies, which characterize the pharmakodynamics and metabolic fate of the compound. This information is used to further optimize the radioligands and to interpret radioactive signals from tissues upon injection of the radioligand in vivo with respect to their specificity. The analysis of the metabolism of radioligands is hampered by the low amount of the compound being typically injected (nano/picomolar amount per injection). In the present study, electrochemistry (EC) is applied to elucidate the oxidative metabolism pathway of the radiotracer. Previous studies have demonstrated that EC can be utilized as a complementary tool to conventional in vitro approaches in drug metabolism studies. Thereby, potential oxidative metabolites of the isatin are determined by EC coupled to electrospray ionization mass spectrometry (EC/ESI-MS). Moreover, using EC/liquid chromatography (LC) and ESI-ion trap MS(n), structural elucidation of the oxidation products is performed. Comparatively to EC, in vitro metabolism studies with rat liver microsomes are conducted. Finally, the developed LC/ESI-MS method is applied to determine metabolites in body fluids and cell extracts from in vivo studies with the nonradioactive ((19)F) and radioactive isatin ((18)F). On the basis of the electrochemically generated oxidation products of the radioligand, the major radioactive metabolite occurring in vivo was successfully identified.

  6. Detection of metabolites by frequency-pulsed electron capture gas-liquid chromatography in serum and cerebrospinal fluid of a patient with Nocardia infection.

    PubMed

    Brooks, J B; Kasin, J V; Fast, D M; Daneshvar, M I

    1987-02-01

    Serum (SR) and cerebrospinal fluid (CSF) from a patient suspected of having tuberculous meningitis were submitted to our laboratory for analysis by frequency-pulsed electron capture gas-liquid chromatography (FPEC GLC). The samples were tested for the presence of carboxylic acids, alcohols, hydroxy acids, and amines by methods described previously (C. C. Alley, J. B. Brooks, and D. S. Kellogg, Jr., J. Clin. Microbiol. 9:97-102, 1977; J. B. Brooks, C. C. Alley, and J. A. Liddle, Anal. Chem. 46:1930-1934, 1974; J. B. Brooks, D. S. Kellogg, Jr., M. E. Shepherd, and C. C. Alley, J. Clin. Microbiol. 11:45-51, 1980; J. B. Brooks, D. S. Kellogg, Jr., M. E. Shepherd, and C. C. Alley, J. Clin. Microbiol. 11:52-58, 1980). The results were different from previous FPEC GLC profiles of SR and CSF from patients with known tuberculous meningitis. Both the SR and CSF contained several unidentified compounds that were not previously detected in tuberculous meningitis or any of our other studies of body fluids. Nocardia brasiliensis was later isolated from the patient. Detection of these metabolites by FPEC GLC could prove to be useful for rapid diagnosis of Nocardia disease, and their identification will provide a better understanding of metabolites produced by Nocardia sp. in vivo.

  7. Simultaneous analysis of acetaminophen, p-aminophenol and aspirin metabolites by hydrophilic interaction and strong anion exchange capillary liquid chromatography coupled to amperometric detection.

    PubMed

    Zheng, Minmin; Wu, Yimin; Lu, Lanxiang; Ding, Kang; Tang, Fengxiang; Lin, Zian; Wu, Xiaoping

    2011-08-01

    A simple and sensitive method has been developed for the simultaneous determination of polar nonsteroidal pharmaceuticals and metabolites, including acetaminophen, p-aminophenol and several aspirin metabolites (salicylic acid, gentisic acid, salicyluric acid and 2,3-dihydroxybenzoic acid), by capillary liquid chromatography with amperometric detection. Using a capillary monolithic column with mixed mode stationary phases and a mobile phase composed of acetonitrile and Tris buffer, rapid separation of six polar analytes was achieved within 8 min, and a hydrophilic interaction and strong anion exchange separation mechanism were exhibited. Method detection limits of six analytes ranged from 10 to 50 ng/mL. In terms of precision, the intra- and interday relative standard deviation values in all analytes never exceeded 3.1% for migration time and 8.9% for peak areas, respectively. This method provided a simple, rapid and cost-effective approach for the analysis of polar pharmaceuticals. The applicability of the method in pharmacokinetics was verified by spiking human serum samples with the compounds and analyzing the recoveries.

  8. Solid phase extraction with silicon dioxide microsphere adsorbents in combination with gas chromatography-electron capture detection for the determination of DDT and its metabolites in water samples.

    PubMed

    Zhou, Qingxiang; Wu, Wei; Xie, Guohong

    2013-01-01

    The goal of the present study was to investigate the feasibility of silicon dioxide (SiO(2)) microspheres without special modification to enrich dichlorodiphenyltrichloroethane (DDT) and its main metabolites, p,p'-dichlorodiphenyl-2,2-dichloroethylene (p,p'-DDD) and p,p'-dichlorodiphenyldichloroethylene (DDE) in combination with gas chromatography-electron-capture detection. The experimental results indicated that an excellent linear relationship between the recoveries and the concentrations of DDT and its main metabolites was obtained in the range of 0.2-30 ng mL(-1) and the correlation coefficients were in the range of 99.96-99.99%. The detection limits based on the ratio of signal to the baseline noise (S/N = 3) were 2.2, 2.9, 3.8 and 4.1 ng L(-1) for p,p'-DDD, p,p'-DDT, o,p'-DDT, and p,p'-DDE, respectively. The precisions of the proposed method were all below 10% (n = 6). Four real water samples were utilized for validation of the proposed method, and satisfactory spiked recoveries in the range of 72.4-112.9% were achieved. These results demonstrated that the developed method was a simple, sensitive, and robust analytical method for the monitoring of pollutants in the environment.

  9. Determination of herbicides and its metabolite in soil and water samples by capillary electrophoresis-laser induced fluorescence detection using microwave-assisted derivatization.

    PubMed

    Cao, Liwei; Deng, Tao; Liang, Siliu; Tan, Xiaofang; Meng, Jianxin

    2014-01-01

    Methods were developed to determine glufosinate (GLUF), glyphosate (GLYP) and its metabolite, aminomethylphosphonic acid (AMPA) by capillary electrophoresis-laser induced fluorescence detection using 5-(4,6-dichlorotriazinylamino) fluorescein (DTAF) and fluorescein isothiocyanate (FITC) as the derivatizing reagents. To accelerate the labeling speed, a microwave-assisted derivatization method was adopted. The derivatizing reaction time was reduced to 180 and 150 s for DTAF and FITC, whose reaction time for conventional labeling was 50 min and 5 h, respectively. The optimum separation conditions for derivatives were as follows: a back ground electrolyte (BGE) of 30 mmol L(-1) sodium tetraborate containing 15 mmol L(-1) brij-35, hydrodynamic injection 15 s and a 10 kV separation voltage. Under these conditions, the LODs (S/N = 3) for DTAF derivatives were 0.32, 0.19 and 0.15 nmol L(-1) for GLUF, GLYP, and AMPA, respectively. The LODs (S/N = 3) for FITC derivatives were 2.60, 3.88 and 2.42 nmol L(-1) for GLUF, GLYP, and AMPA, respectively. The applicability of the developed method was demonstrated by the detection of the above herbicides and metabolite in water and soil samples.

  10. Detection of a novel neurotoxic metabolite of Parkinson's disease-related neurotoxin, 1-benzyl-1,2,3,4- tetrahydroisoquinoline.

    PubMed

    Kotake, Yaichiro; Sekiya, Yoko; Okuda, Katsuhiro; Ohta, Shigeru

    2014-01-01

    Naturally occurring low-molecular weight compounds with a chemical structure like that of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, such as 1-benzyl-1,2,3,4-tetrahydroisoquinoline(1BnTIQ), are candidates for the endogenous neurotoxins that cause Parkinson's disease (PD). 1BnTIQ is an endogenous amine in human CSF and increases in the CSF of patients with PD. It inhibits complex Iand elicits PD-like behavioral abnormalities in monkey and mouse. In this study, we searched metabolites of 1BnTIQ by rat liver S9 using liquid chromatography-tandem mass spectrometry, and identified a dehydrated metabolite, 1-benzyl-3,4-dihydroisoquinoline (1BnDIQ). 1BnDIQ was identified by corresponding mass spectra and precursor ion scans in authentic and complete enzyme samples. Multiple reaction monitoring analysis showed microsome-dependent 1BnDIQ production. We previously reported that 1BnDIQ is more toxic than 1BnTIQ in cytotoxicity study in SH-SY5Y neuroblastoma cells. In addition, 1BnTIQ is reported to pass through the blood-brain barrier of the rat brain, and 1BnDIQ is supposed to be more lipophilic than 1BnTIQ. 1BnDIQ may easily reach the brain, and it might contribute to PD-related neurotoxicity.

  11. New synthesis and characterization of (+)-lysergic acid diethylamide (LSD) derivatives and the development of a microparticle-based immunoassay for the detection of LSD and its metabolites.

    PubMed

    Li, Z; Goc-Szkutnicka, K; McNally, A J; Pilcher, I; Polakowski, S; Vitone, S; Wu, R S; Salamone, S J

    1997-01-01

    In this paper are reported the synthesis and characterization of three LSD derivatives. On the basis of several analytical characterization studies, the most stable derivative has been selected and a procedure to covalently link the derivative to polystyrene microparticles through a carrier protein has been developed. In addition, two new LSD immunogens have been synthesized and characterized, and from these immunogens antibodies that recognize not only LSD but also several major LSD metabolites have been generated. Using the selected derivative and antibody, a homogeneous microparticle-based immunoassay has been developed for the detection of LSD in human urine with the required sensitivity and specificity for an effective screening assay. The performance of this LSD OnLine assay has been evaluated using the criteria of precision, cross-reactivity, correlation to the Abuscreen LSD RIA and GC/MS/MS, assay specificity, and limit of detection.

  12. Detection of metabolite changes in response to a varying visual stimulation paradigm using short-TE (1) H MRS at 7 T.

    PubMed

    Mekle, Ralf; Kühn, Simone; Pfeiffer, Harald; Aydin, Semiha; Schubert, Florian; Ittermann, Bernd

    2017-02-01

    The two-fold benefit of (1) H magnetic resonance spectroscopy (MRS) at high B0 fields - enhanced sensitivity and increased spectral dispersion - has been used previously to study dynamic changes in metabolite concentrations in the human brain in response to visual stimulation. In these studies, a strong visual on/off stimulus was combined with MRS data acquisition in a voxel location in the occipital cortex determined by an initial functional magnetic resonance imaging experiment. However, 1) to exclude the possibility of systemic effects (heartbeat, blood flow, etc.), which tend to be different for on/off conditions, a modified stimulation condition not affecting the target voxel needs to be employed, and 2) to assess important neurotransmitters of low concentration, in particular γ-aminobutyric acid (GABA), it may be advantageous to analyze steady-state, rather than dynamic, conditions. Thus, the aim of this study was to use short-TE (1) H MRS methodology at 7 T to detect differences in steady-state metabolite levels in response to a varying stimulation paradigm in the human visual cortex. The two different stimulation conditions were termed voxel and control activation. Localized MR spectra were acquired using the SPECIAL (spin-echo full-intensity acquired localized) sequence. Data were analyzed using LCModel. Fifteen individual metabolites were reliably quantified. On comparison of steady-state concentrations for voxel versus control activation, a decrease in GABA of 0.05 mmol/L (5%) and an increase in lactate of 0.04 mmol/L (7%) were found to be the only significant effects. The observed reduction in GABA can be interpreted as reduced neuronal inhibition during voxel activation, whereas the increase in lactate hints at an intensification of anaerobic glycolysis. Differences from previous studies, notably the absence of any changes in glutamate, are attributed to the modified experimental conditions. This study demonstrates that the use of advanced (1) H

  13. In vitro estrogen receptor binding of PCBs: measured activity and detection of hydroxylated metabolites in a recombinant yeast assay.

    PubMed

    Layton, Alice C; Sanseverino, John; Gregory, Betsy W; Easter, James P; Sayler, Gary S; Schultz, T Wayne

    2002-05-01

    The estrogenic activities of 17beta-estradiol, biphenyl, chlorinated biphenyls, and Aroclor mixtures 1221, 1242, and 1248 were measured with a modified recombinant yeast estrogen assay (i.e., a Saccharomyces cerevisiae-based lac-Z (beta-galactosidase) reporter assay). Modifications of the assay included the use of glass vials instead of plastic microtiter plates and the addition of the medium and yeast before the test substrate. 14C-labeled compounds were used to follow improvements in the assay procedures. 14C-17beta-estradiol recovery from plastic microtiter plates and glass vials using the standard or the modified procedure was approximately 89%. However, 14C-4-CB (4-chlorobiphenyl) recovery was considerably less, ranging from 3% in plastic microtiter plates using the standard procedure to 26% in vials using the modified procedure. These results suggest that the toxicity of strongly hydrophobic chemicals may be underestimated. Using the modified yeast estrogen assay, full agonist activity was observed for 4-CB, 2,4,6-CB, and 2,5-CB while each of the Aroclor mixtures were only partial agonists. The equivalent EC50 values in ppm were in environmentally relevant concentrations for biphenyl (19 ppm), 4-CB (4.5 ppm), 2,5-CB (21 ppm), 2,4,6-CB (0.8 ppm), Aroclor 1221 (2.9 ppm), Aroclor 1242 (0.65 ppm), and Aroclor 1248 (2.3 ppm). Estrogen receptor binding for the individual PCB congeners was 25- to 650-fold less than the reported estrogen binding for the corresponding hydroxylated PCB metabolite. Gas chromatographic/mass spectrometric analysis of yeast extracts indicated that S. cerevisiae hydroxylated the individual PCB congeners in the ppb range. With the exception of biphenyl, the concentration of hydroxylated metabolites obtained from incubation of S. cerevisiae with PCB congeners was consistent with the concentration necessary to elicit a positive estrogen receptor-binding response. This work provides evidence that S. cerevisiae are capable of metabolic

  14. Analytical Methodologies for Detection of Gamma-Valerolactone, Delta-Valerolactone, Acephate and Azinphos Methyl and Their Associated Metabolites in Complex Biological Matrices

    SciTech Connect

    Zink, E.; Clark, R.; Grant, K.; Campbell, J.; Hoppe, E.

    2005-01-01

    Non-invasive biomonitoring for chemicals of interest in law enforcement and similar monitoring of pesticides, together with their metabolites, can not only save money but can lead to faster medical attention for individuals exposed to these chemicals. This study describes methods developed for the analysis of gamma-valerolactone (GVL), delta-valerolactone (DVL), acephate, and azinphos methyl in saliva and serum. Liquid chromatography/mass spectrometry (LC/MS) operated in the negative and positive ion mode and gas chromatography/mass spectrometry (GC/MS) were used to analyze GVL and DVL. Although both analytical techniques worked well, lower detection limits were obtained with GC/MS. The lactones and their corresponding sodium salts were spiked into both saliva and serum. The lactones were isolated from saliva or serum using newly developed extraction techniques and then subsequently analyzed using GC/MS. The sodium salts of the lactones are nonvolatile and require derivatization prior to analysis by this method. N-methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA) was ultimately selected as the reagent for derivatization because the acidic conditions required for reactions with diazomethane caused the salts to undergo intramolecular cyclization to the corresponding lactones. In vitro studies were conducted using rat liver microsomes to determine other metabolites associated with these compounds. Azinphos methyl and acephate are classified as organophosphate pesticides, and are known to be cholinesterase inhibitors in humans and insects, causing neurotoxicity. For this reason they have both exposure and environmental impact implications. These compounds were spiked into serum and saliva and prepared for analysis by GC/MS. Continuation of this research would include analysis by GC/MS under positive ion mode to determine the parent ions of the unknown metabolites. Further research is planned through an in vivo analysis of the lactones and pesticides. These

  15. Analytical Methodologies for Detection of Gamma-valerolactone, Delta-valerolactone, Acephate, and Azinphos Methyl and their Associated Metabolites in Complex Biological Matrices

    SciTech Connect

    Zink, Erika M.; Clark, Ryan J.; Grant, Karen E.; Campbell, James A.; Hoppe, Eric W.

    2005-01-01

    Non-invasive biomonitoring for chemicals of interest in law enforcement and similar monitoring of pesticides together with their metabolites can not only save money but can lead to faster medical attention for individuals exposed to these chemicals. This study describes methods developed for the analysis of gamma-valerolactone (GVL), delta-valerolactone (DVL), acephate, and azinphos methyl in saliva and serum. Liquid chromatography/mass spectrometry (LC/MS) operated in the negative ion mode and in the positive ion mode and gas chromatography/mass spectrometry (GC/MS) were used to analyze GVL and DVL. Although both analytical techniques worked well, lower detection limits were obtained with GC/MS. The lactones and their corresponding sodium salts were spiked into both saliva and serum. The lactones were isolated from saliva or serum using newly developed extraction techniques and then subsequently analyzed using GC/MS. The sodium salts of the lactones are nonvolatile and require derivatization prior to analysis by this method. N-methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA) was ultimately selected as the reagent for derivatization because the acidic conditions required for reactions with diazomethane caused the salts to undergo intramolecular cyclization to the corresponding lactones. In vitro studies were conducted using rat liver microsomes to determine other metabolites associated with these compounds. Azinphos methyl and acephate are classified as organophosphate pesticides, and are known to be cholinesterase inhibitors in humans and insects, causing neurotoxicity. For this reason they have both exposure and environmental impact implications. These compounds were spiked into serum and saliva and prepared for analysis by GC/MS. Continuation of this research would include analysis by GC/MS under positive ion mode to determine the parent ions of the unknown metabolites. Further research is planned through an in vivo analysis of the lactones and

  16. Quantification of drugs in plasma without primary reference standards by liquid chromatography-chemiluminescence nitrogen detection: application to tramadol metabolite ratios.

    PubMed

    Ojanperä, Suvi; Rasanen, Ilpo; Sistonen, Johanna; Pelander, Anna; Vuori, Erkki; Ojanperä, Ilkka

    2007-08-01

    Lack of availability of reference standards for drug metabolites, newly released drugs, and illicit drugs hinders the analysis of these substances in biologic samples. To counter this problem, an approach is presented here for quantitative drug analysis in plasma without primary reference standards by liquid chromatography-chemiluminescence nitrogen detection (LC-CLND). To demonstrate the feasibility of the method, metabolic ratios of the opioid drug tramadol were determined in the setting of a pharmacogenetic study. Four volunteers were given a single 100-mg oral dose of tramadol, and a blood sample was collected from each subject 1 hour later. Tramadol, O-desmethyltramadol, and nortramadol were determined in plasma by LC-CLND without reference standards and by a gas chromatography-mass spectrometry reference method. In contrast to previous CLND studies lacking an extraction step, a liquid-liquid extraction system was created for 5-mL plasma samples using n-butyl chloride-isopropyl alcohol (98 + 2) at pH 10. Extraction recovery estimation was based on model compounds chosen according to their similar physicochemical characteristics (retention time, pKa, logD). Instrument calibration was performed with a single secondary standard (caffeine) using the equimolar response of the detector to nitrogen. The mean differences between the results of the LC-CLND and gas chromatography-mass spectrometry methods for tramadol, O-desmethyltramadol, and nortramadol were 8%, 32%, and 19%, respectively. The sensitivity of LC-CLND was sufficient for therapeutic concentrations of tramadol and metabolites. A good correlation was obtained between genotype, expressed by the number of functional genes, and the plasma metabolite ratios. This experiment suggests that a recovery-corrected LC-CLND analysis produces sufficiently accurate results to be useful in a clinical context, particularly in instances in which reference standards are not readily accessible.

  17. Metabolite profiling of tomato (Lycopersicon esculentum) using 1H NMR spectroscopy as a tool to detect potential unintended effects following a genetic modification.

    PubMed

    Le Gall, Gwénaëlle; Colquhoun, Ian J; Davis, Adrienne L; Collins, Geoff J; Verhoeyen, Martine E

    2003-04-23

    The maize transcription factors LC and C1 were simultaneously overexpressed in tomato with the aim of producing lines with increased amounts of flavonols. The metabolite composition of these genetically modified tomatoes has been compared with that of azygous (nonmodified) controls grown side-by-side under the same conditions. It has been possible to observe metabolic changes in both types at different stages of maturity. (1)H NMR spectra showed that the levels of glutamic acid, fructose, and some nucleosides and nucleotides gradually increase from the immature to the ripe stage, whereas some amino acids such as valine and gamma-aminobutyric acid were present in higher amounts in unripe tomatoes. Apart from the significantly increased content of six main flavonoid glycosides (mainly kaempferol-3-O-rutinoside, with additional increases in kaempferol-3,7-di-O-glucoside (1), kaempferol-3-O-rutinoside-7-O-glucoside (2), kaempferol-3-O-glucoside, a dihydrokaempferol-O-hexoside (3), and naringenin-7-O-glucoside), the levels of at least 15 other metabolites were found to be different between the two types of red tomato. Among them were citric acid, sucrose, phenylalanine, and trigonelline. However, although statistically significant, these changes in mean values were relatively minor (less than 3-fold) and within the natural variation that would be observed in a field-grown crop. Nevertheless, this study clearly showed that NMR combined with chemometrics and univariate statistics can successfully trace even small differences in metabolite levels between plants and therefore represents a powerful tool to detect potential unintended effects in genetically modified crops.

  18. Simultaneous determination of OSI-774 and its major metabolite OSI-420 in human plasma by using HPLC with UV detection.

    PubMed

    Zhang, Wenjiang; Siu, Lillian L; Moore, Malcolm J; Chen, Eric X

    2005-01-05

    A new method was developed and validated for quantitating OSI-774 and its metabolite OSI-420 in human plasma. Sample preparation involved initial extraction with methyl t-butyl ether followed by back extraction with HCl and re-extraction with methyl t-butyl ether. This extraction process resulted in significant improvement in the specificity, reproducibility and sensitivity. The analytes were separated on a Water Symmetry C18 analytical column and the mobile phase consisted of acetonitrile-0.05 M potassium phosphate buffer (42:58, v/v) (pH 4.8), and monitored at a wavelength 345 nm. Values of between- and within-day precision and accuracy for both OSI-774 and OSI-420 were <20%. This method was successfully applied to study steady-state pharmacokinetics of OSI-774 and OSI-420 in a phase II clinical trial.

  19. Identification of metabolites of Si-Ni-San, a traditional Chinese medicine formula, in rat plasma and urine using liquid chromatography/diode array detection/triple-quadrupole spectrometry.

    PubMed

    Yan, Zhixiang; Chen, Ying; Li, Tianxue; Zhang, Jie; Yang, Xinghao

    2012-02-15

    Si-Ni-San (SNS) is a widely used traditional Chinese medicine formula (TCMF) in treating various diseases. However, the in vivo integrated metabolism of its multiple components remains unknown. In this paper, a liquid chromatography coupled with diode array detection and triple-quadrupole spectrometry (LC-DAD-MS/MS) method was developed for detection and identification of SNS metabolites in rat plasma and urine at a normal clinical dosage. Accurate structural elucidation was performed using MS/MS, UV data and n-octanol/water partition coefficient. Based on the proposed strategy, 36 absorbed compounds and 29 metabolites in plasma and 33 metabolites in urine were detected by a highly sensitive MRM method. Our results indicated that phase II reactions (e.g., methylation, glucuronidation and sulfation) were the main metabolic pathways of gallic acid and flavanones, while phase I reactions (e.g., hydroxylation) were the major metabolic reaction for triterpenoid saponins. The metabolite profile analysis of SNS provided a comprehensive understanding of the in vivo metabolic fates of constituents in SNS. Moreover, the results in this work demonstrated the present strategy based on the combination of chromatographic, spectrophotometric, mass-spectrometric, and software prediction to detect and identify metabolites was effective and reliable. And such a strategy may also be extended to investigate the metabolism of other TCMF.

  20. Detection of fenspiride and identification of in vivo metabolites in horse body fluids by capillary gas chromatography-mass spectrometry: administration, biotransformation and urinary excretion after a single oral dose.

    PubMed

    Dumasia, M C; Houghton, E; Hyde, W; Greulich, D; Nelson, T; Peterson, Jackie

    2002-02-05

    Studies related to the in vivo biotransforrmation and urinary excretion of fenspiride hydrochloride in the horse are described. After oral administration, the drug is metabolised by both phase I functionalisation and phase II conjugation pathways. Following enzymatic deconjugation, fenspiride and its phase I metabolites were isolated from post-administration biofluids using bonded co-polymeric mixed mode solid-phase extraction cartridges to isolate the basic compounds. Following trimethylsilylation (TMS), the parent drug and metabolites were identified by capillary gas chromatography-mass spectrometry (GC-MS). Fenspiride (A) and seven metabolites (B-->G) arising from oxidation on both the aromatic and heterocyclic substructures were detected in urine. The positive ion electron ionisation mass spectra of the TMS derivatives of fenspiride and its metabolites provided useful information on its metabolism. Positive ion methane chemical ionisation-GC-MS of the derivatives provided both derivatised molecular mass and structural information. Unchanged fenspiride can be detected in post-administration plasma and urine samples for up to 24 h. Maximum urinary levels of 100-200 ng ml(-1) were observed between 3 and 5 h after administration. After enzymatic deconjugation, the major phenolic metabolite (G) can be detected in urine for up to 72 h. This metabolite is the analyte of choice in the GC-MS screening of post-race equine urine samples for detection of fenspiride use. However, a distinct difference was observed in the urinary excretion of this metabolite between the thoroughbred horses used in UK study and the quarterbred and standardbred horses used for the USA administrations.

  1. Gas chromatography-combustion-isotope ratio mass spectrometry analysis of 19-norsteroids: application to the detection of a nandrolone metabolite in urine.

    PubMed

    Mathurin, J C; Herrou, V; Bourgogne, E; Pascaud, L; de Ceaurriz, J

    2001-08-15

    Determination of whether the major metabolite of nandrolone in urine, 19-norandrosterone (19-NA), is exogenous or endogenous in origin is one of the most exciting challenges for antidoping laboratories. Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) can be used to differentiate these two origins by carbon isotopic ratio analysis. A complete method for purification of 19-NA in urine has been established. Acetylated ketosteroids, and in particular 19-NA, are isolated from the urine matrix before analysis after hydrolysis and purification of urine by reversed-phase and normal solid-phase extraction. The limit of detection for 19-NA was about 60 ng with recoveries of 54-60%. Evidence of exogenous administration of 19-NA may be established from isotope ratio determination from the 13C/12C ratios of several synthetic 19-norsteroids compared to those obtained for endogenous steroids.

  2. Use of Fluorinated Compounds To Detect Aromatic Metabolites from m-Cresol in a Methanogenic Consortium: Evidence for a Demethylation Reaction

    PubMed Central

    Londry, Kathleen L.; Fedorak, Phillip M.

    1993-01-01

    Anaerobic sewage sludge was used to enrich a methanogenic m-cresol-degrading consortium. 6-Fluoro-3-methylphenol was synthesized and added to subcultures of the consortium with m-cresol. This caused the accumulation of 4-hydroxy-2-methylbenzoic acid. In a separate experiment, the addition of 3-fluorobenzoic acid caused the transient accumulation of 4-hydroxybenzoic acid. Inhibition with bromoethanesulfonic acid caused the accumulation of benzoic acid. Thus, the proposed degradation pathway was m-cresol → 4-hydroxy-2-methylbenzoic acid → 4-hydroxybenzoic acid → benzoic acid. The m-cresol-degrading consortium was able to convert exogenous 4-hydroxybenzoic acid and benzoic acid to methane. In addition, for each metabolite of m-cresol identified, the corresponding fluorinated metabolite was detected, giving the following sequence: 6-fluoro-3-methylphenol → 5-fluoro-4-hydroxy-2-methylbenzoic acid → 3-fluoro-4-hydroxybenzoic acid → 3-fluorobenzoic acid. The second step in each of these pathways is a novel demethylation which was rate limiting. This demethylation reaction would likely facilitate the transformation of the methyl group to methane, which is consistent with the results of a previous study that showed that the methyl carbon of m-[methyl-14C]cresol was recovered predominantly as [14C]methane (D. J. Roberts, P. M. Fedorak, and S. E. Hrudey, Can. J. Microbiol. 33:335-338, 1987). The final aromatic compound in the proposed route for m-cresol metabolism was benzoic acid, and its detection in these cultures merges the pathway for the methanogenic degradation of m-cresol with those for the anaerobic metabolism of many phenols. PMID:16348996

  3. Development and validation of a fast monoclonal based disequilibrium enzyme-linked immunosorbent assay for the detection of triphenylmethane dyes and their metabolites in fish.

    PubMed

    Oplatowska, Michalina; Connolly, Lisa; Stevenson, Paul; Stead, Sara; Elliott, Christopher T

    2011-07-18

    Malachite Green (MG), Crystal Violet (CV) and Brilliant Green (BG) are antibacterial, antifungal and antiparasitic agents that have been used for treatment and prevention of diseases in fish. These dyes are metabolized into reduced leuco forms (LMG, LCV, LBG) that can be present in fish muscles for a long period. Due to the carcinogenic properties they are banned for use in fish for human consumption in many countries including the European Union and the United States. HPLC and LC-MS techniques are generally used for the detection of these compounds and their metabolites in fish. This study presents the development of a fast enzyme-linked immunosorbent assay (ELISA) method as an alternative for screening purposes. A first monoclonal cell line producing antibodies to MG was generated using a hybridoma technique. The antibody had good cross-reactivates with related chromatic forms of triphenylmethane dyes such as CV, BG, Methyl Green, Methyl Violet and Victoria Blue R. The monoclonal antibody (mAb) was used to develop a fast (20 min) disequilibrium ELISA screening method for the detection of triphenylmethanes in fish. By introducing an oxidation step with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) during sample extraction the assay was also used to detect the presence of the reduced metabolites of triphenylmethanes. The detection capability of the assay was 1 ng g(-1) for MG, LMG, CV, LCV and BG which was below the minimum required performance limit (MRPL) for the detection method of total MG (sum of MG and LMG) set by the Commission Decision 2004/25/EC (2 ng g(-1)). The mean recoveries for fish samples spiked at 0.5 MRPL and MRPL levels with MG and LMG were between 74.9 and 117.0% and inter- and intra-assay coefficients of variation between 4.7 and 25.7%. The validated method allows the analysis of a batch of 20 samples in two to three hours. Additionally, this procedure is substantially faster than other ELISA methods developed for MG/LMG thus far. The stable

  4. Secondary metabolite components of kiwifruit.

    PubMed

    McGhie, Tony K

    2013-01-01

    Both green and gold kiwifruit contain high concentrations of vitamin C, and much of the "health story" of kiwifruit involves this vitamin. Kiwifruit also contain other compounds that are bioactive and beneficial to health. In this chapter, the secondary metabolite composition of kiwifruit is presented. Although there are limited compositional data for kiwifruit published in the scientific literature, the concentrations of 42 compounds have been documented. Included are compounds that are often associated with "healthfulness," such as the vitamins (A, C, E, and K), carotenoids (lutein and β-carotene), folate, and antioxidant phenolic compounds. Metabolite discovery is advancing rapidly with the introduction of "metabolomic" studies where the goal is to identify and measure the complete metabolite composition of a sample. In a metabolomic experiment using liquid chromatography and high-resolution mass spectrometry, it was possible to measure more than 500 metabolites in kiwifruit extracts. The large number of detectable metabolites present suggests that there is an abundance of kiwifruit metabolites still to be discovered. Such studies will provide a more complete understanding of the metabolite composition of kiwifruit that will lead to new and improved hypotheses as to the function and effects of kiwifruit metabolites, including their relevance to human health.

  5. Analysis of blood and urine samples for hydroxychloroquine and three major metabolites by high-performance liquid chromatography with fluorescence detection.

    PubMed

    Williams, S B; Patchen, L C; Churchill, F C

    1988-12-09

    A high-performance liquid chromatographic (HPLC) method using fluorescence detection is described for the quantification of hydroxychloroquine (HCQ) and three of its metabolites in blood and urine samples. The method is selective, permitting quantification of analytes without interferences from chloroquine or quinine in the sample. Detection limits for HCQ, desethylhydroxychloroquine, desethylchloroquine, and bisdesethylchloroquine are 10, 30, 5, and 5 ppb, respectively, for a 100-microliters blood or urine sample. The internally standardized method requires only one extraction step and utilizes normal-phase HPLC conditions including an amine modifier in the mobile phase. These conditions facilitate fluorescence detection, selective separation, and acceptable peak shapes. A mobile phase of 0.5% n-butylamine in methanol-hexane-methyl tert. butyl ether (1:1:1) is used in the analysis. Analysis of blood and urine samples from two healthy volunteers given 400 mg of Plaquenil (310 mg of HCQ base) weekly for four weeks provided data on HCQ metabolism for the two persons during the recommended chemoprophylactic regimen for malaria.

  6. High-performance liquid chromatographic method with amperometric detection employing boron-doped diamond electrode for the determination of sildenafil, vardenafil and their main metabolites in plasma.

    PubMed

    Bartošová, Zdenka; Jirovský, David; Horna, Aleš

    2011-11-04

    A simple, fast and sensitive HPLC method with electrochemical detection employing boron-doped diamond electrode (BDD) for the determination of sildenafil (Viagra™), vardenafil (Levitra™) and their main metabolites, N-desmethyl sildenafil and N-desethyl vardenafil in human plasma is presented. The assay involved drug extraction by tert-butyl methyl ether and isocratic reversed-phase liquid chromatography with amperometric detection. Complete separation of all analytes was achieved within 12 min. The mobile phase consisted of 20mM sodium dihydrogen phosphate with 40 mM sodium perchlorate/acetonitrile (70:30, v/v), pH 3.5. The electrode working potential was +1520 mV (vs. Pd/H(2)). Calibration curves were linear over the concentration range of 10-400 ng mL(-1). Phloretin was used as an internal standard. The limits of detection (LOD) and quantification (LOQ) for the studied analytes were within the range of 2-4 ng mL(-1) and 7.0-13.4 ng mL(-1), respectively. The developed method was applied to human plasma samples spiked with analytes at therapeutic concentrations. The study confirms the method's suitability for both pharmacokinetic studies and therapeutic monitoring.

  7. The detection of piroxicam, tenoxicam and their metabolites in equine urine by electrospray ionisation ion trap mass spectrometry.

    PubMed

    McKinney, Andrew R; Suann, Craig J; Stenhouse, Allen M

    2004-01-01

    An investigation has been conducted into the metabolism and urinary excretion of orally administered piroxicam and tenoxicam in the horse. The major component detected in urine after the administration of piroxicam was 5'-hydroxypiroxicam, which was detectable up to 24 h post-administration. Unchanged piroxicam was present only as a minor component. In contrast, unchanged tenoxicam was the major component observed after the administration of tenoxicam, being detectable for 72 h post-administration, while 5'-hydroxytenoxicam was a minor component. Phase II beta-glucuronide conjugation in each case was found to be negligible. The ion trap mass spectral characteristics of piroxicam, tenoxicam, 5'-hydroxypiroxicam and 5'-hydroxytenoxicam under electrospray ionisation conditions were examined in some detail.

  8. Simultaneous metabolite fingerprinting of hydrophilic and lipophilic compounds in Echinacea pallida by high-performance liquid chromatography with diode array and electrospray ionization-mass spectrometry detection.

    PubMed

    Pellati, Federica; Orlandini, Giulia; Benvenuti, Stefania

    2012-06-15

    In this study, a detailed phytochemical characterization of Echinacea pallida (Nutt.) Nutt. root extracts and dietary supplements was carried out for the first time by developing advanced chromatographic techniques, based on HPLC with diode array (DAD) and electrospray ionization-mass spectrometry (ESI-MS) detection (with ion trap and triple quadrupole mass analyzers), for the simultaneous analysis of hydrophilic and lipophilic secondary metabolites. The HPLC analyses were carried out on an Ascentis C(18) column (250 mm × 4.6 mm I.D., 5 μm), with a mobile phase composed by H(2)O and ACN both containing 0.1% formic acid, under gradient elution. The UV spectra, in combination with MS and MS/MS data, allowed the identification of fourteen compounds, including caffeic acid derivatives, polyacetylenes and polyenes, in the analyzed samples. MS and MS/MS data were discussed in detail and the typical fragmentation patterns of each class of secondary metabolites were identified. For the first time, a hydroperoxide intermediate was characterized as an oxidation product of one of E. pallida monocarbonylic acetylenes, providing a confirmation of the mechanism that leads to the generation of hydroxylated derivatives. The HPLC method was fully validated in agreement with ICH guidelines and then applied to real samples. The quantitative analysis indicated that there was a great variability in the amount of the active compounds in the dietary supplements containing E. pallida root extracts: the content of total caffeic acid derivatives ranged from 2.31 to 11.45 mg/g and the amount of total polyacetylenes and polyenes from 6.38 to 30.54 mg/g. In the analyzed samples, the most abundant caffeic acid derivative was found to be echinacoside. Regarding polyacetylenes and polyenes, the most representative compounds were found to be tetradec-(8Z)-ene-11,13-diyn-2-one, pentedeca-(8Z,11Z)-dien-2-one and pentadec-(8Z)-en-2-one. The developed method can be considered suitable for metabolite

  9. Time-course measurements of caffeine and its metabolites extracted from fingertips after coffee intake: a preliminary study for the detection of drugs from fingerprints.

    PubMed

    Kuwayama, Kenji; Tsujikawa, Kenji; Miyaguchi, Hajime; Kanamori, Tatsuyuki; Iwata, Yuko T; Inoue, Hiroyuki

    2013-05-01

    The aim of this study was to determine whether an ingested drug and its metabolites could be detected in the subject's fingerprints. Caffeine (CF) was chosen as the model drug. Three healthy subjects were asked to consume a cup of coffee (ca. 100 mL) containing 80 micro micro mg CF as the total dose, which is the normal amount in one cup of coffee. After washing hands with water to remove external contaminants, each subject pressed the index fingertip to a collecting matrix just before consuming the test cup of coffee, and then again pressed the index fingertip to the collecting matrix after 1, 3, 5, and 7 h. The time curve of the amounts of CF and its metabolites-theobromine (TB), paraxanthine (PX), and theophylline (TP)-in fingerprints and blood was determined using liquid chromatography/tandem mass spectrometry (LC/MS). A filter paper wetted with water (50 μL) was an efficient collecting matrix for extracting the analytes from the fingertip. With optimized sample preparation and LC/MS conditions, the total operating time, from taking the fingerprints to obtaining the analytical result, was approximately 10 min. The lower limits of quantification for CF, TB, PX, and TP were 0.5, 5, 0.5, and 5 ng/fingerprint, respectively. The amount of CF or PX determined in fingerprints obtained over 7 h after coffee intake was significantly greater than the amount determined in fingerprints taken before drinking coffee. Fingerprints were a more efficient source for drug testing than other biological samples, such as blood and sweat, because the procedures for sampling and extracting the drugs were simpler and took less time. The method could be used to prove drug intake in criminal investigations.

  10. Volatile Metabolites

    PubMed Central

    Rowan, Daryl D.

    2011-01-01

    Volatile organic compounds (volatiles) comprise a chemically diverse class of low molecular weight organic compounds having an appreciable vapor pressure under ambient conditions. Volatiles produced by plants attract pollinators and seed dispersers, and provide defense against pests and pathogens. For insects, volatiles may act as pheromones directing social behavior or as cues for finding hosts or prey. For humans, volatiles are important as flavorants and as possible disease biomarkers. The marine environment is also a major source of halogenated and sulfur-containing volatiles which participate in the global cycling of these elements. While volatile analysis commonly measures a rather restricted set of analytes, the diverse and extreme physical properties of volatiles provide unique analytical challenges. Volatiles constitute only a small proportion of the total number of metabolites produced by living organisms, however, because of their roles as signaling molecules (semiochemicals) both within and between organisms, accurately measuring and determining the roles of these compounds is crucial to an integrated understanding of living systems. This review summarizes recent developments in volatile research from a metabolomics perspective with a focus on the role of recent technical innovation in developing new areas of volatile research and expanding the range of ecological interactions which may be mediated by volatile organic metabolites. PMID:24957243

  11. Development of a new E. coli strain to detect oxidative mutation and its application to the fungicide o-phenylphenol and its metabolites.

    PubMed

    Tani, Shusuke; Yonezawa, Yusuke; Morisawa, Shinsuke; Nishioka, Hajime

    2007-04-02

    Oxidative mutation is mainly induced by reactive oxygen species (ROS), such as the superoxide anion radical (O(2)(-)) and hydrogen peroxide (H(2)O(2)). However, in Escherichia coli (E. coli), ROS are eliminated by enzymes such as superoxide dismutase and catalase, which are coded by sodAB and katEG genes. In this study, to detect mutagens that induce oxidative mutation, a mutant (WP2katEGsodAB) with katEG and sodAB deleted was constructed by gene manipulation of E. coli WP2. H(2)O(2) and menadione sodium bisulfite generated mutation in WP2katEGsodAB but not in WP2. o-Phenylphenol (OPP) and its metabolites (phenylhydroquinone (PHQ) and phenyl-1,4-benzoquinone (PBQ)), which had been shown to be negative in the Ames test but reported to be carcinogenic, induced mutation in WP2katEGsodAB but not in WP2. These results suggest that the new assay may be useful for the detection of oxidative mutagens.

  12. [The detection and identification of alpha-pyrrolidino-valerophenone (α-PVP) and its metabolites in the objects of the forensic chemical examination].

    PubMed

    Moskaleva, E V; Eroshchenko, N N; Kiryushin, A N; Kardonsky, D A; Eganov, A A

    2017-01-01

    The combined application of the IK 200609 chemical toxicological analyzer and the diagnostic biosensor (reagent) for the determination of synthetic cations (with due regard for compliance with the instruction for analysis) made it possible to detect during 15 minutes the presence of cations in the urine samples at the concentration of 376.64 ng/ml. This result was further confirmed by HPLC-Ms/MS and GH-MS. The use of the analyzer allowed the screening of the urine samples to be performed within several minutes at the preliminary stage of the study. A simplified method of tissue sample preparation with the use of the kits for extraction and solid-phase purification is proposed together with a variant of blood sample preparation with the use of filtration. The proposed approach can be employed for the rapid detection and identification of alpha-pyrrolidino-valerophenone (α-PVP) and its metabolites in urine, blood and tissues of various organs for the purpose of practical toxicological investigations in the framework of forensic chemical expertise.

  13. Comparison of sulfo-conjugated and gluco-conjugated urinary metabolites for detection of methenolone misuse in doping control by LC-HRMS, GC-MS and GC-HRMS.

    PubMed

    Fragkaki, A G; Angelis, Y S; Kiousi, P; Georgakopoulos, C G; Lyris, E

    2015-05-01

    Methenolone (17β-hydroxy-1-methyl-5α-androst-1-en-3-one) misuse in doping control is commonly detected by monitoring the parent molecule and its metabolite (1-methylene-5α-androstan-3α-ol-17-one) excreted conjugated with glucuronic acid using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) for the parent molecule, after hydrolysis with β-glucuronidase. The aim of the present study was the evaluation of the sulfate fraction of methenolone metabolism by LC-high resolution (HR)MS and the estimation of the long-term detectability of its sulfate metabolites analyzed by liquid chromatography tandem mass spectrometry (LC-HRMSMS) compared with the current practice for the detection of methenolone misuse used by the anti-doping laboratories. Methenolone was administered to two healthy male volunteers, and urine samples were collected up to 12 and 26 days, respectively. Ethyl acetate extraction at weak alkaline pH was performed and then the sulfate conjugates were analyzed by LC-HRMS using electrospray ionization in negative mode searching for [M-H](-) ions corresponding to potential sulfate structures (comprising structure alterations such as hydroxylations, oxidations, reductions and combinations of them). Eight sulfate metabolites were finally detected, but four of them were considered important as the most abundant and long term detectable. LC clean up followed by solvolysis and GC/MS analysis of trimethylsilylated (TMS) derivatives reveal that the sulfate analogs of methenolone as well as of 1-methylene-5α-androstan-3α-ol-17-one, 3z-hydroxy-1β-methyl-5α-androstan-17-one and 16β-hydroxy-1-methyl-5α-androst-1-ene-3,17-dione were the major metabolites in the sulfate fraction. The results of the present study also document for the first time the methenolone sulfate as well as the 3z-hydroxy-1β-methyl-5α-androstan-17-one sulfate as metabolites of methenolone in human urine. The time window for the detectability

  14. Detection of cysteine- and lysine-based protein adductions by reactive metabolites of 2,5-dimethylfuran.

    PubMed

    Wang, Kai; Li, Weiwei; Chen, Jiaming; Peng, Ying; Zheng, Jiang

    2015-10-08

    Many furan-containing compounds are known to be toxic and/or carcinogenic. Metabolic activation of toxic furans to cis-enediones (cis-enedials or γ-ketoenals) is generally considered as the initial step towards the processes of their toxicities. Sequential modification of key proteins by the electrophilic reactive intermediates is suggested to be an important mechanism of the toxic actions. In the present study, we developed a novel and simple analytical platform to detect protein modification resulting from metabolic activation of model compound 2,5-dimethylfuran (DMF). 4-Bromobenzylamine and 4-bromobenzylmercaptan were employed to trap protein adductions at cysteine and lysine residues, respectively. The resulting protein samples were proteolytically digested by chymotrypsin and Pronase E, followed by LC-MS/MS analysis. Modifications of cysteine and lysine residues of proteins were observed in microsomal incubations and animals after exposure to DMF. In conclusion, the approach established has been proven highly selective and reliable. This advance allows us not only to detect the protein adductions but also to define the structural identities of amino acid residues modified. This technique provides a unique platform to assess protein modifications arising from metabolic activation of potentially harmful furan-containing compounds. Hepatic protein adductions were found to be proportional to the hepatotoxicity of DMF.

  15. Methods of analysis by the U.S. Geological Survey Organic Geochemistry Research Group; determination of chloroacetanilide herbicide metabolites in water using high-performance liquid chromatography-diode array detection and high-performance liquid chromatography/mass spectrometry

    USGS Publications Warehouse

    Zimmerman, L.R.; Hostetler, K.A.; Thurman, E.M.

    2000-01-01

    Analytical methods using high-performance liquid chromatography-diode array detection (HPLC-DAD) and high-performance liquid chromatography/mass spectrometry (HPLC/MS) were developed for the analysis of the following chloroacetanilide herbicide metabolites in water: acetochlor ethanesulfonic acid (ESA), acetochlor oxanilic acid (OXA), alachlor ESA, alachlor OXA, metolachlor ESA, and metolachlor OXA. Good precision and accuracy were demonstrated for both the HPLC-DAD and HPLC/MS methods in reagent water, surface water, and ground water. The mean HPLC-DAD recoveries of the chloroacetanilide herbicide metabolites from water samples spiked at 0.25, 0.50, and 2.0 mg/L (micrograms per liter) ranged from 84 to 112 percent, with relative standard deviations of 18 percent or less. The mean HPLC/MS recoveries of the metabolites from water samples spiked at 0.05, 0.20, and 2.0 mg/L ranged from 81 to 125 percent, with relative standard deviations of 20 percent or less. The limit of quantitation (LOQ) for all metabolites using the HPLC-DAD method was 0.20 mg/L, whereas the LOQ using the HPLC/MS method was 0.05 mg/L. These metabolite-determination methods are valuable for acquiring information about water quality and the fate and transport of the parent chloroacetanilide herbicides in water.

  16. Quantitative determination of total methamphetamine and active metabolites in rat tissue by liquid chromatography with tandem mass spectrometric detection.

    PubMed

    Hendrickson, Howard; Laurenzana, Elizabeth; Owens, S Michael

    2006-11-22

    High-throughput liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) methodology for the determination of methamphetamine (METH), amphetamine (AMP), 4-hydroxymethamphetamine (4-OH-METH), and 4-hydroxyamphetamine (4-OH-AMP) was developed and validated using simple trichloroacetic acid sample treatment. The method was validated in rat serum, brain, and testis. Lower limits-of-quantitation (LOQ) for METH and AMP were 1 ng x mL(-1) using positive ion electrospray tandem mass spectrometry (MS/MS). The accuracy of the method was within 25% of the actual values over a wide range of analyte concentrations. The within-assay precision was better than 12% (coefficient of variation). The method was linear over a wide dynamic range (0.3-1000 ng x mL(-1)). Quantitation was possible in all 3 matrices using only serum standards because of minimal matrix-associated ion effects or the use of an internal standard. Finally, the LC-MS/MS method was used to determine serum, brain, and testis METH and AMP concentrations during a subcutaneous infusion (5.6 mg kg(-1) day(-1)) of METH in rats. Concentrations of 4-OH-AMP and 4-OH-METH were below the LOQ in experimental samples. The bias introduced by using serum calibrators for the determination of METH and AMP concentrations in testis and brain was less than 8% and insignificant relative to the interanimal variability.

  17. Evaluation of the potential of surface enhancement Raman spectroscopy for detection of tricyclic psychotropic drugs. Case studies on imipramine and its metabolite.

    PubMed

    Jaworska, Aleksandra; Wietecha-Posłuszny, Renata; Woźniakiewicz, Michał; Kościelniak, Paweł; Malek, Kamilla

    2011-11-21

    The potential use of surface Raman enhanced spectroscopy (SERS) for confirmatory identification and the semi-quantitative analysis of selected tricyclic antidepressants (TCAs) is examined utilizing a conventional silver colloid. Raman and SERS spectra of aqueous solutions of imipramine (Imi) and its metabolite, desipramine (Des), were recorded as the function of concentration using NIR excitation. A good linear correlation is observed for the dependence of the SERS signal at 684 cm(-1) (R(2) = 0.9997) on Imi concentration over the range of 0.75-7.5 μM. The limit of detection of imipramine in the silver colloidal solution is 0.98 μM. SERS spectra of Imi and Des were also recorded for blood plasma samples without prior purification as well as after the use of standard solid phase extraction. All spectra show the characteristic spectral profile of the molecules and moreover, stronger signal enhancement is observed for Imi in the "raw" samples as opposed to Imi extracted from a biological matrix.

  18. The enantioselective determination of chlorpheniramine and its major metabolites in human plasma using chiral chromatography on a beta-cyclodextrin chiral stationary phase and mass spectrometric detection.

    PubMed

    Fried, Karen M; Young, Andrea E; Usdin Yasuda, Sally; Wainer, Irving W

    2002-01-15

    A sensitive enantioselective high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of plasma concentrations of (-)(R)- and (+)(S)-chlorpheniramine (CP) and their metabolites, desmethyl-chlorpheniramine (DCP), didesmethyl-chorpheniramine (DDCP) and chlorpheniramine N-oxide (CPNO). Enantioselective separations were achieved on a beta-cyclodextrin chiral stationary phase (CYCLOBOND I 2000) with a mobile phase consisting of diethylamine acetate (0.25%, pH 4.4):methanol:acetonitrile [85:7.5:7.5, (v/v/v)]and a flow-rate of 0.5 ml/min. For CP, the enantioselectivity (alpha) of the separation was 1.12 with a resolution factor (R(s)) of 1.17. The method was validated for CP by using mass spectroscopy detection (MSD). Concentrations of each enantiomer could be measured down to 125 pg/ml from a 1-ml plasma sample. Extracted calibration curves were linear from 0.13 to 50.00 ng/ml for each enantiomer. The method was applied to samples from two clinical studies.

  19. Method development and validation for simultaneous determination of lumefantrine and its major metabolite, desbutyl lumefantrine in human plasma using RP-HPLC/UV detection.

    PubMed

    Khuda, Fazli; Iqbal, Zafar; Shah, Yasar; Ahmmad, Lateef; Nasir, Fazli; Khan, Amir Zada; Amanullah; Shahbaz, Naila

    2014-01-01

    A simple, specific, precise and rapid RP-HPLC-UV method was developed for simultaneous determination of lumefantrine and its metabolite desbutyl lumefantrine in human plasma. Experimental parameters were optimized and the method was validated according to standard guidelines. The method showed adequate separation for lumefantrine and desbutyl lumefantrine and best resolution was achieved with Supelco Discovery HS C18 RP (150mm×4.6mm, 5μm) column using acetonitrile and 0.05% trifluroacetic acid (70:30, v/v) as a mobile phase pumped at a flow rate of 1.0ml/min and wavelength of 335nm. The method was linear over the concentration range of 10-12,000ng/ml. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for lumefantrine were 10.0 and 18.0ng/ml, while for desbutyl lumefantrine were 7.5 and 15.0ng/ml, respectively. The proposed method was efficiently applied for determination of lumefantrine and desbutyl lumefantrine concentrations in plasma samples for pharmacokinetic studies.

  20. Detection of Pancreatic Cancer-induced Cachexia using a Fluorescent Myoblast Reporter System and Analysis of Metabolite Abundance

    PubMed Central

    Winnard, Paul T.; Bharti, Santosh; Penet, Marie-France; Marik, Radharani; Mironchik, Yelena; Wildes, Flonne; Maitra, Anirban; Bhujwalla, Zaver M.

    2016-01-01

    The dire effects of cancer-induced cachexia undermine treatment and contribute to decreased survival rates. Therapeutic options for this syndrome are limited, and therefore efforts to identify signs of precachexia in cancer patients are necessary for early intervention. The applications of molecular and functional imaging that would enable a whole-body “holistic” approach to this problem may lead to new insights and advances for diagnosis and treatment of this syndrome. Here we have developed a myoblast optical reporter system with the purpose of identifying early cachectic events. We generated a myoblast cell line expressing a dual tdTomato:GFP construct that was grafted onto the muscle of mice bearing human pancreatic cancer xenografts to provide noninvasive live imaging of events associated with cancer-induced cachexia (i.e., weight loss). Real time optical imaging detected a strong tdTomato fluorescent signal from skeletal muscle grafts in mice with weight loses of only 1.2 to 2.7% and tumor burdens of only ~79 to ~170 mm3. Weight loss in cachectic animals was also associated with a depletion of lipid, cholesterol, valine, and alanine levels, which may provide informative biomarkers of cachexia. Taken together, our findings demonstrate the utility of a reporter system that is capable of tracking tumor-induced weight loss, an early marker of cachexia. Future studies incorporating resected tissue from human pancreatic ductal adenocarcinoma (PDAC) into a reporter-carrying mouse may be able to provide a risk assessment of cachexia with possible implications for therapeutic development. PMID:26719527

  1. Identification and determination of phase II nabumetone metabolites by high-performance liquid chromatography with photodiode array and mass spectrometric detection.

    PubMed

    Nobilis, M; Holcapek, M; Kolárová, L; Kopecký, J; Kunes, M; Svoboda, Z; Kvetina, J

    2004-03-26

    Chromatographic analyses play an important role in the identification and determination of phase I and phase II drug metabolites. While the chemical standards of phase I metabolites are usually available from commercial sources or by various synthetic, degradation or isolation methods, the phase II drug metabolites have usually more complicated structures, their standards are in general inaccessible and their identification and determination require a comprehensive analytical approach involving the use of xenobiochemical methods and the employment of hyphenated analytical techniques. In this work, various high-performance liquid chromatography (HPLC) methods were employed in the evaluation of xenobiochemical experiments leading to the identification and determination of phase II nabumetone metabolites. Optimal conditions for the quantitative enzymatic deconjugation of phase II metabolites were found for the samples of minipig bile, small intestine contents and urine. Comparative HPLC analyses of the samples of above-mentioned biomatrices and of the same biomatrices after their enzymatic treatment using beta-glucuronidase and arylsulfatase afforded the qualitative and quantitative information about phase II nabumetone metabolites. Hereby, three principal phase II nabumetone metabolites (ether glucuronides) were discovered in minipig's body fluids and their structures were confirmed using liquid chromatography (LC)-electrospray ionization mass spectrometric (MS) analyses.

  2. Impact of halides on the simultaneous separation of aromatic amines and their acidic metabolites by capillary electrophoresis with laser-induced native fluorescence detection under acidic conditions.

    PubMed

    Hsieh, Ming-Mu; Chang, Huan-Tsung

    2006-01-13

    This paper describes a simple, sensitive, efficient, and rapid method for simultaneous analysis of biologically active amines and acids by capillary electrophoresis in conjunction with laser-induced native fluorescence detection (CE-LINF) using a diode pumped solid state nanolaser at 266 nm. In order to optimize resolution of the amines that were prepared in 10.0 mM formate-Tris (FT) solutions, 10.0 mM FT solutions with and without containing halides were used to fill the capillary and reservoirs, respectively. The electrophoretic mobilities of tryptamine (TA) and serotonin (5-HT) at pH 4.0 decrease with the increase in halide concentration (0-10.0 mM). Taken together with a great effect of iodide than other halides, we suggest that the formation of ion pairs is a main contributor for altering the migration of the amines. In order to simultaneously analyze the amines and their metabolites (acids) at low pH, a high bulk EOF is required. The analysis of 10 anlytes including amines and acids was completed within 12 min by CE-LINF using a capillary treated with 0.5M NaOH and then filled with 10.0 mM FT solutions (pH 4.0) containing 10.0 mM KCl prior to analysis. The limits of detection for TA and 5-hydroxyindole-3-acetic acid (5-HIAA) are 0.12 and 6.0 nM, respectively. The present method has been further validated by analyzing urine samples, with an RSD less than 3.1% (migration times) and 3.9% (concentration).

  3. [To the question of the optimization of methods for detection of vinyl chloride and 1,2-dichloroethane, and their metabolites in biological fluids in workers involved in production of polyvinyl chloride].

    PubMed

    Zhurba, O M; Alekseenko, A N

    2014-01-01

    There is considered the improvement of methodological approaches to the gas chromatographic methods- of the detection of vinyl chloride and 1,2-dichloroethane and their metabolites--chloroethanol and monochloroacetic acid in biological fluids. There were evaluated such metrological characteristics of methods, as repeatability, interlaboratoty precision, relevance and accuracy. The value of relative expanded uncertainty does not exceed 30%. There are reported optimal regimes of gas chromatographic analysis, conditions for sample preparation. The results of the contents ofthese chemical compounds and their metabolites in biological fluids from persons working in contact with chlorinated hydrocarbons are presented These techniques can be used for the detection ofthe fact of exposure to toxic substances, assessment of the level of exposure and biomonitoring.

  4. Automated analysis of oxidative metabolites

    NASA Technical Reports Server (NTRS)

    Furner, R. L. (Inventor)

    1974-01-01

    An automated system for the study of drug metabolism is described. The system monitors the oxidative metabolites of aromatic amines and of compounds which produce formaldehyde on oxidative dealkylation. It includes color developing compositions suitable for detecting hyroxylated aromatic amines and formaldehyde.

  5. High-performance liquid chromatography-tandem mass spectrometry method for simultaneous detection of ochratoxin A and relative metabolites in Aspergillus species and dried vine fruits.

    PubMed

    Zhang, Xiaoxu; Li, Jingming; Cheng, Zhan; Zhou, Ziying; Ma, Liyan

    2016-08-01

    A simple, sensitive and reliable quantification and identification method was developed for simultaneous analysis of ochratoxin A (OTA) and its related metabolites ochratoxin alpha (OTα), ochratoxin B (OTB) and mellein. The method was assessed by spiking analytes into blank culture media and dried vine fruits. Performance was tested in terms of accuracy, selectivity and repeatability. The method involves an ultrasonic extraction step for culture samples using methanol aqueous solution (7:3, v/v); the mycotoxin is quantified by high-performance liquid chromatography coupled with electrospray ionisation and triple quadrupole mass spectrometry (LC-ESI-MS/MS). The recoveries were 74.5-108.8%, with relative standard deviations (RSDs) of 0.4-8.4% for fungal culture. The limits of detection (LODs) were in the range of 0.03-0.87 μg l(-)(1), and the limits of quantification (LOQs) ranged from 0.07 to 2.90 μg l(-)(1). In addition, the extraction solutions and clean-up columns were optimised specifically for dried vine fruit samples to improve the performance of the method. Methanol-1% sodium bicarbonate extraction solution (6:4, v/v) was determined to be the most efficient. Solid-phase extraction (SPE) was performed as a clean-up step prior to HPLC-MS/MS analysis to reduce matrix effects. Recoveries ranged from 80.1% to 110.8%. RSDs ranged from 0.1% to 3.6%. LODs and LOQs ranged from 0.06 to 0.40 μg kg(-)(1) and from 0.19 to 1.20 μg kg(-)(1), respectively. The analytical method was established and used to identify and quantify OTA and related compounds from Aspergillus carbonarius and Aspergillus ochraceus in cultures and dried vine fruits. The results showed that A. carbonarius produced OTα, OTB and OTA, whereas A. ochraceus produced OTB, OTA and mellein after 7 days of cultivation. Of 30 commercial samples analysed, 10 were contaminated with ochratoxins; OTB, OTα and mellein were also detected in different samples.

  6. Metabolite profiling of licorice (Glycyrrhiza glabra) from different locations using comprehensive two-dimensional liquid chromatography coupled to diode array and tandem mass spectrometry detection.

    PubMed

    Montero, Lidia; Ibáñez, Elena; Russo, Mariateresa; di Sanzo, Rosa; Rastrelli, Luca; Piccinelli, Anna Lisa; Celano, Rita; Cifuentes, Alejandro; Herrero, Miguel

    2016-03-24

    Profiling of the main metabolites from several licorice (Glycyrrhiza glabra) samples collected at different locations is carried out in this work by using comprehensive two-dimensional liquid chromatography (LC × LC) coupled to diode array (DAD) and mass spectrometry (MS) detectors. The optimized method was based on the application of a HILIC-based separation in the first dimension combined with fast RP-based second dimension separation. This set-up was shown to possess powerful separation capabilities allowing separating as much as 89 different metabolites in a single sample. Identification and grouping of metabolites according to their chemical class were achieved using the DAD, MS and MS/MS data. Triterpene saponins were the most abundant metabolites followed by glycosylated flavanones and chalcones, whereas glycyrrhizic acid, as expected, was confirmed as the main component in all the studied samples. LC × LC-DAD-MS/MS was able to resolve these complex licorice samples providing with specific metabolite profiles to the different licorice samples depending on their geographical origin. Namely, from 19 to 50 specific compounds were exclusively determined in the 2D-chromatograms from the different licorice samples depending on their geographical origin, which can be used as a typical pattern that could potentially be related to their geographical location and authentication.

  7. A novel integrated method for large-scale detection, identification, and quantification of widely targeted metabolites: application in the study of rice metabolomics.

    PubMed

    Chen, Wei; Gong, Liang; Guo, Zilong; Wang, Wensheng; Zhang, Hongyan; Liu, Xianqing; Yu, Sibin; Xiong, Lizhong; Luo, Jie

    2013-11-01

    Liquid chromatography-mass spectrometry (LC-MS)-based metabolomics has been facilitated by the construction of MS(2) spectral tag (MS2T) library from the total scan ESI MS/MS data, and the development of widely targeted metabolomics method using MS/MS data gathered from authentic standards. In this report, a novel strategy called stepwise multiple ion monitoring-enhanced product ions (stepwise MIM-EPI) was developed to construct the MS2T library, in which stepwise MIM was used as survey scans to trigger the acquisition of EPI. A total number of 698 (almost) non-redundant metabolites with MS(2) spectra were obtained, of which 135 metabolites were identified/annotated. Integrating the data gathered from our MS2T library and other available multiple reaction monitoring (MRM) information, a widely targeted metabolomics method was developed to quantify 277 metabolites, including some phytohormones. Evaluation of the dehydration responses and natural variations of these metabolites in rice leaf not only suggested the coordinated regulation of abscisic acid (ABA) with metabolites such as serotonin derivative(s), polyamine conjugates under drought stress, but also revealed some C-glycosylated flavones as the potential markers for the discrimination of indica and japonica rice subspecies. The new MS2T library construction and widely targeted metabolomics strategy could be used as a tool for rice functional genomics.

  8. Determination of vitamins D2, D3, K1 and K3 and some hydroxy metabolites of vitamin D3 in plasma using a continuous clean-up-preconcentration procedure coupled on-line with liquid chromatography-UV detection.

    PubMed

    Ortiz Boyer, F; Fernández Romero, J M; Luque de Castro, M D; Quesada, J M

    1999-03-01

    A semi-automatic procedure for the continuous clean-up and concentration of several fat-soluble vitamins prior to their separation by HPLC and UV detection is reported. The procedure is based on the use of a minicolumn packed with aminopropylsilica as sorbent located prior to the chromatographic detection system. The overall process was developed and applied to the main liposoluble vitamins (A, D2, D3, E, K1, K3) and several hydroxy metabolites of vitamin D3 [25-(OH)-D3,24,25-(OH)2-D3 and 1,25-(OH)2-D3]. All the analytes were monitored at a compromise wavelength of 270 nm. Calibration graphs were constructed between 0.01 and 100 ng ml-1 for vitamin D2 and D3 and their hydroxy metabolites, between 0.1 and 100 ng ml-1 for vitamin A, K1 and K3 and between 1 and 100 ng ml-1 for vitamin E, with excellent regression coefficients (> or = 0.9901) in all cases. The precision was established at two concentration levels with acceptable RSDs in all instances (between 3.6 and 8.7%). The method was appropriate for the determination of vitamin D2, D3, K1 and K3 and the 24,25-dihydroxy and 25-hydroxy metabolites of vitamin D3 in human plasma. The method was applied to plasma samples spiked with the target analytes and the recoveries ranged between 78 and 109%.

  9. Validation of a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair using LC-MS/MS and its application to human and rat hair.

    PubMed

    Kim, Jihyun; Lee, Sooyeun; In, Sanghwan; Choi, Hwakyung; Chung, Heesun

    2011-04-15

    Benzodiazepines and zolpidem are controlled in many countries due to their inherent adverse effects of a high degree of tolerance and dependence. Recently, as some of these drugs have become distributed illegally and available through media such as the Internet, their abuse is becoming a serious social problem. Hair is a useful specimen to prove chronic drug use. In the present study, a simultaneous analytical method for the detection of 27 benzodiazepines and metabolites and zolpidem in hair was established and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The drugs and their metabolites in hair were extracted using methanol, filtered and injected on the LC-MS/MS. The following validation parameters of the method were satisfactory: selectivity, linearity, matrix effect, recovery, process efficiency, intra- and inter-assay precision and accuracy and processed sample stability. The limit of detection (LOD) and the limit of quantification (LOQ) were the total drug detected from the sample. The LODs ranged from 0.005 ng (zolpidem) to 0.5 ng (bromazepam and chlordiazepoxide) and the LOQs were 0.25 ng in every analyte except for bromazepam and chlordiazepoxide, for which they were 0.5 ng. The developed method was successfully applied to five legal cases involving use of benzodiazepines and zolpidem and to an animal study on drug incorporation into hair. Diazepam and its three metabolites, as well as lorazepam, were detected in hair from both the multiple- and single-dose administration groups of lean Zucker rats. The concentration of diazepam was higher than those of its metabolites in both dark grey and white hair from the multiple-dose administration groups, with the mean concentration ranges from 0.16 to 0.51 ng/mg and from 0.10 to 0.24 ng/mg, respectively. The mean concentration ranges of lorazepam were from 0.05 to 0.37 ng/mg in dark grey hair and from 0.11 to 0.45 ng/mg in white hair from the multiple-dose administration groups. Hair

  10. Characterization of proflavine metabolites in rainbow trout.

    PubMed

    Yu, Z; Hayton, W L; Chan, K K

    1997-04-01

    Proflavine (3,6-diaminoacridine) has potential for use as an antiinfective in fish, and its metabolism by rainbow trout was therefore studied. Fourteen hours after intraarterial bolus administration of 10 mg/kg of proflavine, three metabolites were found in liver and bile, and one metabolite was found in plasma using reversed-phase HPLC with UV detection at 262 nm. Treatment with hydrochloric acid converted the three metabolites to proflavine, which suggested that the metabolites were proflavine conjugates. Treatment with beta-glucuronidase and saccharic acid 1,4-lactone, a specific beta-glucuronidase inhibitor, revealed that two metabolites were proflavine glucuronides. For determination of UV-VIS absorption and mass spectra, HPLC-purified metabolites were isolated from liver. Data from these experiments suggested that the proflavine metabolites were 3-N-glucuronosyl proflavine (PG), 3-N-glucuronosyl,6-N-acetyl proflavine (APG), and 3-N-acetylproflavine (AP). The identities of the metabolites were verified by chemical synthesis. When synthetic PG and AP were compared with the two metabolites isolated from trout, they had the same molecular weight as determined by matrix-assisted, laser desorption ionization, time-of-flight MS. In addition, they coeluted on HPLC under different mobile phase conditions. Finally, the in vitro incubation with liver subcellular preparations confirmed this characterization and provided the evidence that APG can be formed by glucuronidation of AP or acetylation of PG.

  11. Secondary metabolite profiles of leaves of healthy and huanglongbing-infected orange (Citrus sinensis L.) seedlings measured by HPLC-fluorescence detection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Leaves of greenhouse-grown ‘Hamlin’ and ‘Valencia’ orange (Citrus sinensis L.) seedlings were analyzed by high performance liquid chromatography in a study of the progression of changes in secondary metabolite profiles resulting from infection by Candidatus Liberibacter asiaticus (CLas), the Huanglo...

  12. Detection of normal aging effects on human brain metabolite concentrations and microstructure with whole brain MR spectroscopic imaging and quantitative MR imaging

    PubMed Central

    Eylers, Vanessa V.; Maudsley, Andrew A.; Bronzlik, Paul; Dellani, Paulo R.; Lanfermann, Heinrich; Ding, Xiao-Qi

    2015-01-01

    Background and purpose Whole brain 1H-MR spectroscopic imaging (wbMRSI) was used in combination with quantitative MRI (qMRI) to study the effects of normal aging on healthy human brain metabolites and microstructure. Materials and Methods Sixty healthy volunteers aged 21 to 70 years were studied. Brain maps of the metabolites NAA, Cr, and Cho, and the tissue irreversible and reversible transverse relaxation times, T2 and T2′, were derived from the datasets. The relative metabolite concentrations [NAA], [tCr] and [Cho] as well as the values of relaxation times were measured with ROIs placed within frontal and parietal WM, centrum semiovale (CSO), splenium of the corpus callosum (SCC), hand motor area (HK), occipital GM, putamen, thalamus, pons ventral/dorsal (BSv/BSd), cerebellar white matter (CbWM) and posterior lobe (CbGM). Linear regression analysis and Pearson’s correlation tests were used to analyze the data. Results Aging resulted in decreased [NAA] in occipital GM, putamen, SCC, and BSv, and decreased [tCr] in BSd and putamen. [Cho] did not change significantly in selected brain regions. T2 increased in CbWM and decreased in SCC with aging, while the T2′ decreased in the occipital GM, HK, putamen, and increased in the SCC. Correlations were found between [NAA] and T2′ in occipital GM and putamen and between [tCr] and T2′ in the putamen. Conclusion The effects of normal aging on brain metabolites and microstructure are regional dependent. Correlations between both processes are evident in the gray matter. The obtained data could be used as references for future studies on patients. PMID:26564440

  13. Novel and sensitive reversed-phase high-pressure liquid chromatography method with electrochemical detection for the simultaneous and fast determination of eight biogenic amines and metabolites in human brain tissue.

    PubMed

    Van Dam, Debby; Vermeiren, Yannick; Aerts, Tony; De Deyn, Peter Paul

    2014-08-01

    A fast and simple RP-HPLC method with electrochemical detection (ECD) and ion pair chromatography was developed, optimized and validated in order to simultaneously determine eight different biogenic amines and metabolites in post-mortem human brain tissue in a single-run analytical approach. The compounds of interest are the indolamine serotonin (5-hydroxytryptamine, 5-HT), the catecholamines dopamine (DA) and (nor)epinephrine ((N)E), as well as their respective metabolites, i.e. 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), 5-hydroxy-3-indoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG). A two-level fractional factorial experimental design was applied to study the effect of five experimental factors (i.e. the ion-pair counter concentration, the level of organic modifier, the pH of the mobile phase, the temperature of the column, and the voltage setting of the detector) on the chromatographic behaviour. The cross effect between the five quantitative factors and the capacity and separation factors of the analytes were then analysed using a Standard Least Squares model. The optimized method was fully validated according to the requirements of SFSTP (Société Française des Sciences et Techniques Pharmaceutiques). Our human brain tissue sample preparation procedure is straightforward and relatively short, which allows samples to be loaded onto the HPLC system within approximately 4h. Additionally, a high sample throughput was achieved after optimization due to a total runtime of maximally 40min per sample. The conditions and settings of the HPLC system were found to be accurate with high intra and inter-assay repeatability, recovery and accuracy rates. The robust analytical method results in very low detection limits and good separation for all of the eight biogenic amines and metabolites in this complex mixture of biological analytes.

  14. Quantitative analysis of the principle soy isoflavones genistein, daidzein and glycitein, and their primary conjugated metabolites in human plasma and urine using reversed-phase high-performance liquid chromatography with ultraviolet detection.

    PubMed

    Thomas, B F; Zeisel, S H; Busby, M G; Hill, J M; Mitchell, R A; Scheffler, N M; Brown, S S; Bloeden, L T; Dix, K J; Jeffcoat, A R

    2001-09-05

    Soy isoflavones are becoming of increasing interest as nutritional agents which can be used to combat osteoporosis and hyperlipidemia, and are also being considered as potential cancer chemopreventive compounds. However, prior to their formulation and distribution as therapeutic agents, thorough pharmacokinetic and toxicological assessment needs to be completed in men and women in a variety of health conditions in order to ensure their therapeutic efficacy and safety. At this time, studies of purified soy isoflavones are possible, and are being designed to fully evaluate the pharmacological utility of these preparations. In support of these studies, quantitative analysis of soy isoflavones in biological fluids can be accomplished with a wide variety of methods and analytical instrumentation. However, the relatively ubiquitous presence of high-performance liquid chromatography with ultraviolet detection (HPLC-UV) in most analytical laboratories, the relative ease of its operation, and the lesser expense of this instrumentation as compared to more sophisticated techniques such as liquid chromatography-mass spectrometry, offers some distinct advantages for its use in pharmacokinetic studies. In this manuscript, the development and validation of an HPLC-UV method for the quantitation of the principal soy isoflavones, genistein, daidzein, and glycitein, and their primary metabolites, in human plasma and urine is described. This analytical approach allows for pharmacologically relevant concentrations of the analytes and their principle metabolites to be detected, and has been validated in close agreement with the US Food and Drug Administration's guidelines for the validation of methods to be used in support of pharmacokinetic studies.

  15. Using multi-walled carbon nanotubes as solid phase extraction adsorbents to determine dichlorodiphenyltrichloroethane and its metabolites at trace level in water samples by high performance liquid chromatography with UV detection.

    PubMed

    Zhou, Qingxiang; Xiao, Junping; Wang, Weidong

    2006-09-01

    Carbon nanotubes (CNTs) are a kind of new carbon-based nano-materials which have drawn great attention in many application fields. The potential of multi-walled carbon nanotubes (MWNTs) as solid-phase extraction (SPE) adsorbents for the preconcentration of environmental pollutants has been investigated in recent years. The goal of this work was to investigate the feasibility of MWNTs used as SPE adsorbents to enrich dichlorodiphenyltrichloroethane (DDT) and its metabolites including 1,1-dichloro-2,2-bis-(4'-chlorophenyl)ethane (DDD) and 1,1-dichloro-2,2-bis-(4'-chlorophenyl)ethane (DDE) at trace level which are typical persistent organic pollutants in environment. Parameters that maybe influence the extraction efficiency such as the eluent volume, sample flow rate, sample pH and the sample volume were optimized in detail. The experimental results showed the excellent linear relationship between peak area and the concentration of DDT and its metabolites over the range of 0.2-60 microg L(-1), and the precisions (RSD) were 2.3-2.5% under the optimal conditions. The detection limits of proposed method could reach 4-13 ng L(-1) based on the ratio of chromatographic signal to base line noise (S/N = 3). Satisfied results were achieved when the proposed method was applied to determine the four target compounds in realworld water samples with spiked recoveries over the range of 89.7-115.5%. All these facts indicated that MWCNTs as SPE packing materials coupled to HPLC was an excellent alternative for the routine analysis of DDT and its metabolites at trace level in environment.

  16. Interest of molecularly imprinted polymers in the fight against doping. Extraction of tamoxifen and its main metabolite from urine followed by high-performance liquid chromatography with UV detection.

    PubMed

    Claude, Bérengère; Morin, Philippe; Bayoudh, Sami; de Ceaurriz, Jacques

    2008-07-04

    A molecular imprinted polymer (MIP) has been synthesized in order to specifically extract tamoxifen, a nonsteroidal antiestrogen, and its metabolites from urine by solid-phase extraction (SPE) before HPLC-UV analysis. Clomiphene, a chlorinated tamoxifen analogue, was selected as template for MIP synthesis. Polymerisation was achieved by thermal polymerisation of methacrylic acid (MAA) as functional monomer, ethylene glycol dimethacrylate (EDMA) as cross-linking agent and acetonitrile as porogen. The efficient elimination of the urinary matrix has been obtained by MIP-SPE but the elution recovery of tamoxifen was initially too low ( approximately 14%). This problem has been overcome following two ways. At first, a preliminary HLB-SPE of the urine has enabled to discard endogenous salts and to percolate an organic sample through the MIP cartridge. Extraction recoveries are equal to 56 and 74% for tamoxifen and 4-hydroxytamoxifen, respectively. Then, a second MIP has been prepared with styrene and MAA as functional co-monomers. Strong pi-pi interactions occurring between phenyl groups of styrene and tamoxifen promote rebinding of the analyte by the specific sites. The enhanced hydrophobic character of the imprinted polymer has enabled the direct percolation of urine through MIP-SPE and the easy elimination of endogenous salts from urine with only one aqueous washing step. HPLC-UV analysis has confirmed high extraction recoveries (85%) for tamoxifen and its metabolite with an enrichment factor of 8. This analytical protocol can selectively detect the presence of tamoxifen metabolites in urines and be useful as a proof of doping in competitive sports.

  17. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) in toxicological analysis. Studies on the detection of clobenzorex and its metabolites within a systematic toxicological analysis procedure by GC-MS and by immunoassay and studies on the detection of alpha- and beta-amanitin in urine by atmospheric pressure ionization electrospray LC-MS.

    PubMed

    Maurer, H H; Kraemer, T; Ledvinka, O; Schmitt, C J; Weber, A A

    1997-02-07

    GC-MS is the method of choice for toxicological analysis of toxicants volatile in GC while non-volatile and/or thermally labile toxicants need LC-MS for their determination. Studies are presented on the toxicological detection of the amphetamine-like anorectic clobenzorex in urine by GC-MS after acid hydrolysis, extraction and acetylation and by fluorescence polarization immunoassay (FPIA, TDx (meth)amphetamine II). After ingestion of 60 mg of clobenzorex, the parent compound and/or its metabolites could be detected by GC-MS for up to 84 h or by FPIA for up to 60 h. Since clobenzorex shows no cross-reactivity with the used immunoassay, the N-dealkylated metabolite amphetamine is responsible for the positive TDx results. The intake of clobenzorex instead of amphetamine can be differentiated by GC-MS detection of hydroxyclobenzorex which is detectable for at least as long as amphetamine. In addition, the described GC-MS procedure allows the simultaneous detection of most of the toxicologically relevant drugs. Furthermore, studies are described on the atmospheric pressure ionization electrospray LC-MS detection of alpha- and beta-amanitin, toxic peptides of amanita mushrooms, in urine after solid-phase extraction on RP-18 columns. Using the single ion monitoring mode with the ions m/z 919 and 920 the amanitins could be detected down to 10 ng/ml of urine which allows us to diagnose intoxications with amanita mushrooms.

  18. Quantification of the N-desmethyl metabolite of rosuvastatin in human plasma by automated SPE followed by HPLC with tandem MS detection.

    PubMed

    Hull, Caroline K; Martin, Paul D; Warwick, Michael J; Thomas, Elizabeth

    2004-05-28

    A selective, accurate and precise assay was developed for the quantification in human plasma of the N-desmethyl metabolite of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor rosuvastatin. The assay-employing automated SPE followed by HPLC with positive ion electrospray tandem MS (HPLC-MS/MS)-was validated. The standard curve range for N-desmethyl rosuvastatin in human plasma was 0.5-30 ng/ml with 0.5 ng/ml being the limit of quantification. Plasma samples were mixed 1:1 with sodium acetate buffer (pH 4.0; 0.1M) soon after separation from red blood cells. N-Desmethyl rosuvastatin was stable in plasma:buffer at room temperature for 24h and at -70 degrees C for 12 months. The assay was applied successfully to the quantification of N-desmethyl rosuvastatin in human plasma following administration of rosuvastatin.

  19. Blood styrene and urinary metabolites in styrene polymerisation.

    PubMed Central

    Wolff, M S; Lorimer, W V; Lilis, R; Selikoff, I J

    1978-01-01

    The results of the analysis of blood and urine samples for styrene and its metabolites in 491 workers in a styrene polymerisation plant in the United States are reported. The levels of exposure to styrene were estimated to be less than 10 ppm, but nevertheless styrene and metabolites were detectable in more than 50% of workers in polymerisation jobs, within 4 h of exposure. Workers involved in the manufacture and purification of styrene from ethyl benzene also had detectable blood styrene and urinary metabolites in 83% of recently exposed subjects. The relationship between styrene in blood and in subcutaneous fat and urinary metabolites as pharmacokinetic variables is discussed. PMID:737139

  20. Oxidative damage to hyaluronate and glucose in synovial fluid during exercise of the inflamed rheumatoid joint. Detection of abnormal low-molecular-mass metabolites by proton-n.m.r. spectroscopy.

    PubMed

    Grootveld, M; Henderson, E B; Farrell, A; Blake, D R; Parkes, H G; Haycock, P

    1991-01-15

    Proton Hahn spin-echo n.m.r. spectroscopy was employed to detect abnormal metabolites present in rheumatoid synovial fluid that are derived from the deleterious generation of reactive oxygen radical species during exercise of the inflamed rheumatoid joint. A resonance attributable to a low-molecular-mass N-acetylglucosamine-containing oligosaccharide formed by the oxygen-radical-mediated depolymerization of synovial-fluid hyaluronate was clearly demonstrable when subjects with inflammatory joint disease were exercised. Moreover, formate, which may be derived from the attack of OH.radical on synovial-fluid carbohydrates, was also readily detectable in these samples. gamma-Radiolysis of rheumatoid synovial fluid samples and aqueous solutions of hyaluronate also gave rise to the production of the low-molecular-mass hyaluronate-derived oligosaccharide species and markedly elevated concentrations of (non-protein-bound) formate in the biological fluids. As expected, corresponding spectra of gamma-irradiated blood serum samples obtained from normal volunteers did not contain the signal attributable to the low-molecular-mass oligosaccharide species, but the formate resonance (barely detectable in non-irradiated normal serum samples) became clearly visible. Additionally, a curious increase in the effective concentration of non-protein-bound low-molecular-mass metabolites such as acetate, citrate, lactate and glutamine was observed after gamma-radiolysis of all biological fluids studied. The hyaluronate-derived low-molecular-mass oligosaccharide species and formate are suggested as novel markers of reactive oxygen radical activity in the inflamed rheumatoid joint during exercise-induced hypoxic/reperfusion injury.

  1. The positive effects of high-frequency right dorsolateral prefrontal cortex repetitive transcranial magnetic stimulation on memory, correlated with increases in brain metabolites detected by proton magnetic resonance spectroscopy in recently detoxified alcohol-dependent patients

    PubMed Central

    Qiao, Jun; Jin, Guixing; Lei, Licun; Wang, Lan; Du, Yaqiang; Wang, Xueyi

    2016-01-01

    Objective To explore the effect of right dorsolateral prefrontal cortex (DLPFC) repetitive transcranial magnetic stimulation (rTMS) on memory, and its correlation with levels of hippocampal brain metabolites detected by proton magnetic resonance spectroscopy (1H-MRS) in recently detoxified alcohol-dependent patients. Materials and methods In this randomized, double-blind sham-controlled trial, alcohol-dependent patients were enrolled and randomized into two groups: the experimental group (rTMS, 10 Hz, on right DLPFC, 20 sessions) and the control group (sham stimulation). Memory function was assessed using Hopkins Verbal Learning Test-Revised (HVLT-R) and Brief Visuospatial Memory Test-Revised (BVMT-R) before and after treatment. 1H-MRS was used to detect the levels of N-acetyl aspartic acid (NAA), choline (Cho), and creatine (Cr) in bilateral hippocampi before and after treatment. Results Thirty-eight patients (18 in the experimental group and 20 in the control group) were included in the analyses. The experimental group showed significantly greater changes in HVLT-R, BVMT-R, NAA/Cr, and Cho/Cr after rTMS from baseline than the control group. The percentage change in BVMT-R and HVLT-R correlated with the percentage change in NAA/Cr and Cho/Cr in the right brain. Conclusion High-frequency right DLPFC rTMS was associated with improvement in memory dysfunction, which is correlated with levels of hippocampal brain metabolites detected by 1H-MRS in recently detoxified alcohol-dependent patients. PMID:27695332

  2. Screening procedure for detection of non-steroidal anti-inflammatory drugs and their metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons by gas chromatography-mass spectrometry after extractive methylation.

    PubMed

    Maurer, H H; Tauvel, F X; Kraemer, T

    2001-01-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used as analgesic and anti-rheumatic drugs, and they are often misused. A gas chromatographic-mass spectrometric (GC-MS) screening procedure was developed for their detection in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons after extractive methylation. The compounds were separated by capillary GC and identified by computerized MS in the full-scan mode. Using mass chromatography with the ions m/z 119, 135, 139, 152, 165, 229, 244, 266, 272, and 326, the possible presence of NSAIDs and their metabolites could be indicated. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with the reference spectra recorded during this study. This method allowed the detection of therapeutic concentrations of acemetacin, acetaminophen (paracetamol), acetylsalicylic acid, diclofenac, diflunisal, etodolac, fenbufen, fenoprofen, flufenamic acid, flurbiprofen, ibuprofen, indometacin, kebuzone, ketoprofen, lonazolac, meclofenamic acid, mefenamic acid, mofebutazone, naproxen, niflumic acid, phenylbutazone, suxibuzone, tiaprofenic acid, tolfenamic acid, and tolmetin in urine samples. The overall recoveries of the different NSAIDs ranged between 50 and 80% with coefficients of variation of less than 15% (n = 5), and the limits of detection of the different NSAIDs were between 10 and 50 ng/mL (S/N = 3) in the full-scan mode. Extractive methylation has proved to be a versatile method for STA of various acidic drugs, poisons, and their metabolites in urine. It has also successfully been used for plasma analysis.

  3. Simultaneous detection of sulfoxaflor and its metabolites, X11719474 and X11721061, in lettuce using a modified QuEChERS extraction method and liquid chromatography-tandem mass spectrometry.

    PubMed

    Kim, Sung-Woo; Rahman, Md Musfiqur; Abd El-Aty, A M; Kabir, Md Humayun; Na, Tae Woong; Choi, Jeong-Heui; Shin, Ho-Chul; Shim, Jae-Han

    2016-11-05

    An analytical method has been developed to quantify the residual levels of sulfoxaflor and its metabolites (X11719474 and X11721061) in/on cultivated lettuce grown under greenhouse conditions. Samples were extracted and purified using a quick, easy, cheap, effective, rugged, and safe 'QuEChERS' method (original version) following systematic method optimization and were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Good linearity with coefficient of determination ≥0.9930 was obtained and the limits of detection (LOD) and quantification (LOQ) were in the ranges of 0.003-0.006 and 0.01-0.02 mg/kg, respectively. The recovery rates of both the parent compound and its metabolites (fortified at 10 and 50× the LOQ) estimated from six replicates ranged between 81.9 and 115.5% with a relative standard deviation <12%. The validated method was applied to field-incurred samples (collected over 7 days) sprayed once or twice with a water-dispersible granule formulation. Notably, a substantial reduction in rate was observed after 3 days and the half-life was short, only 1.5 days. The developed method is simple and versatile and can be used for various leafy vegetables.

  4. 17-allyamino-17-demethoxygeldanamycin treatment results in a magnetic resonance spectroscopy-detectable elevation in choline-containing metabolites associated with increased expression of choline transporter SLC44A1 and phospholipase A2

    PubMed Central

    2010-01-01

    Introduction 17-allyamino-17-demethoxygeldanamycin (17-AAG), a small molecule inhibitor of Hsp90, is currently in clinical trials in breast cancer. However, 17-AAG treatment often results in inhibition of tumor growth rather than shrinkage, making detection of response a challenge. Magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) are noninvasive imaging methods than can be used to monitor metabolic biomarkers of drug-target modulation. This study set out to examine the MRS-detectable metabolic consequences of Hsp90 inhibition in a breast cancer model. Methods MCF-7 breast cancer cells were investigated, and MRS studies were performed both on live cells and on cell extracts. 31P and 1H MRS were used to determine total cellular metabolite concentrations and 13C MRS was used to probe the metabolism of [1,2-13C]-choline. To explain the MRS metabolic findings, microarray and RT-PCR were used to analyze gene expression, and in vitro activity assays were performed to determine changes in enzymatic activity following 17-AAG treatment. Results Treatment of MCF-7 cells with 17-AAG for 48 hours caused a significant increase in intracellular levels of choline (to 266 ± 18% of control, P = 0.05) and phosphocholine (PC; to 181 ± 10% of control, P = 0.001) associated with an increase in expression of choline transporter SLC44A1 and an elevation in the de novo synthesis of PC. We also detected an increase in intracellular levels of glycerophosphocholine (GPC; to 176 ± 38% of control, P = 0.03) associated with an increase in PLA2 expression and activity. Conclusions This study determined that in the MCF-7 breast cancer model inhibition of Hsp90 by 17-AAG results in a significant MRS-detectable increase in choline, PC and GPC, which is likely due to an increase in choline transport into the cell and phospholipase activation. 1H MRSI can be used in the clinical setting to detect levels of total choline-containing metabolite (t-Cho, composed of intracellular

  5. Enhanced metabolite generation

    DOEpatents

    Chidambaram, Devicharan [Middle Island, NY

    2012-03-27

    The present invention relates to the enhanced production of metabolites by a process whereby a carbon source is oxidized with a fermentative microbe in a compartment having a portal. An electron acceptor is added to the compartment to assist the microbe in the removal of excess electrons. The electron acceptor accepts electrons from the microbe after oxidation of the carbon source. Other transfers of electrons can take place to enhance the production of the metabolite, such as acids, biofuels or brewed beverages.

  6. Pharmacokinetics of Tyrosol Metabolites in Rats.

    PubMed

    Lee, Da-Hye; Kim, Yang-Ji; Kim, Min Jung; Ahn, Jiyun; Ha, Tae-Youl; Lee, Sang Hee; Jang, Young Jin; Jung, Chang Hwa

    2016-01-21

    Tyrosol is considered a potential antioxidant; however, little is known regarding the pharmacokinetics of its metabolites. To study the pharmacokinetics of tyrosol-derived metabolites after oral administration of a single dose of tyrosol, we attempted to identify tyrosol metabolites in rat plasma by using ultra-performance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Two tyrosol metabolites (M1 and M2) were detected in the plasma. M1 was identified as tyrosol-4-sulfate (T4S) with an [M - H](-) ion at m/z 217. While M2 showed an [M - H](-) ion at m/z 151.0, its metabolite was not identified. Pharmacokinetic analysis of T4S and M2 showed rapid uptake after oral administration of tyrosol within 1 h. The metabolites were rapidly distributed in most organs and tissues and eliminated within 4 h. The greatest T4S deposition by tissue weight was observed in the liver, followed by the kidney and spleen, while M2 was most concentrated in the kidney followed by the liver and spleen. These findings indicate that T4S and M2 were distributed mainly in tissues with an abundant blood supply and were rapidly excreted in urine.

  7. Prediction of Estrogenic Bioactivity of Environmental Chemical Metabolites.

    PubMed

    Pinto, Caroline L; Mansouri, Kamel; Judson, Richard; Browne, Patience

    2016-09-19

    The US Environmental Protection Agency's (EPA) Endocrine Disruptor Screening Program (EDSP) is using in vitro data generated from ToxCast/Tox21 high-throughput screening assays to assess the endocrine activity of environmental chemicals. Considering that in vitro assays may have limited metabolic capacity, inactive chemicals that are biotransformed into metabolites with endocrine bioactivity may be missed for further screening and testing. Therefore, there is a value in developing novel approaches to account for metabolism and endocrine activity of both parent chemicals and their associated metabolites. We used commercially available software to predict metabolites of 50 parent compounds, out of which 38 chemicals are known to have estrogenic metabolites, and 12 compounds and their metabolites are negative for estrogenic activity. Three ER QSAR models were used to determine potential estrogen bioactivity of the parent compounds and predicted metabolites, the outputs of the models were averaged, and the chemicals were then ranked based on the total estrogenicity of the parent chemical and metabolites. The metabolite prediction software correctly identified known estrogenic metabolites for 26 out of 27 parent chemicals with associated metabolite data, and 39 out of 46 estrogenic metabolites were predicted as potential biotransformation products derived from the parent chemical. The QSAR models estimated stronger estrogenic activity for the majority of the known estrogenic metabolites compared to their parent chemicals. Finally, the three models identified a similar set of parent compounds as top ranked chemicals based on the estrogenicity of putative metabolites. This proposed in silico approach is an inexpensive and rapid strategy for the detection of chemicals with estrogenic metabolites and may reduce potential false negative results from in vitro assays.

  8. Quantitative determination of regorafenib and its two major metabolites in human plasma with high-performance liquid chromatography and ultraviolet detection.

    PubMed

    Fujita, Kazuma; Miura, Masatomo; Shibata, Hiroyuki

    2016-10-01

    A simple, highly sensitive and specific high-performance liquid chromatography (HPLC) method was developed for the simultaneous quantitation of regorafenib, N-oxidemetabolite (M-2) and the desmethyl N-oxide metabolite (M-5) in human plasma. Regorafenib, M-2, M-5 and the internal standard sorafenib were separated using a mobile phase of 0.5% KH2 PO4 (pH 3.5)-acetonitrile (30:70, v/v), on a Capcell PAK MG II column at a flow rate of 0.5 mL/min and measurement at UV 260 nm. The lower limits of quantification for regorafenib, M-2 and M-5 were 10 ng/mL for each analyte. A procedure using solid-phase extraction required only a small amount of plasma (100 μL) for one analysis while providing high extraction recovery (>81% for all compounds) and good selectivity. Coefficients of variation for intra- and inter-day assays were <12.2% for regorafenib, <12.3% for M-2 and <15.1% for M-5. Accuracies for intra- and inter-day assays were <9.4% for regorafenib, <8.0% for M-2 and <12.8% for M-5 over a linear range from 10 to 10,000 ng/mL. This HPLC assay is suitable for clinical pharmacokinetic studies of regorafenib. The present HPLC method is currently in use for our observational studies of patients under treatment. Copyright © 2016 John Wiley & Sons, Ltd.

  9. Detection of reactive metabolites using isotope-labeled glutathione trapping and simultaneous neutral loss and precursor ion scanning with ultra-high-pressure liquid chromatography triple quadruple mass spectrometry.

    PubMed

    Huang, Ke; Huang, Lingyi; van Breemen, Richard B

    2015-04-07

    Metabolic activation of drugs to electrophilic species is responsible for over 60% of black box warnings and drug withdrawals from the market place in the United States. Reactive metabolite trapping using glutathione (GSH) and analysis using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) or HPLC with high resolution mass spectrometry (mass defect filtering) have enabled screening for metabolic activation to become routine during drug development. However, current MS-based approaches cannot detect all GSH conjugates present in complex mixtures, especially those present in extracts of botanical dietary supplements. To overcome these limitations, a fast triple quadrupole mass spectrometer-based approach was developed that can detect positively and negatively charged GSH conjugates in a single analysis without the need for advanced knowledge of the elemental compositions of potential conjugates and while avoiding false positives. This approach utilized UHPLC instead of HPLC to shorten separation time and enhance sensitivity, incorporated stable-isotope labeled GSH to avoid false positives, and used fast polarity switching electrospray MS/MS to detect GSH conjugates that form positive and/or negative ions. The general new method was then used to test the licorice dietary supplement Glycyrrhiza glabra, which was found to form multiple GSH conjugates upon metabolic activation. Among the GSH conjugates found in the licorice assay were conjugates with isoliquiritigenin and glabridin, which is an irreversible inhibitor of cytochrome P450 enzymes.

  10. Use of reversed-phase high-performance liquid chromatography-diode array detection for complete separation of 2,4,6-trinitrotoluene metabolites and EPA Method 8330 explosives: influence of temperature and an ion-pair reagent.

    PubMed

    Borch, Thomas; Gerlach, Robin

    2004-01-02

    Explosives such as 2,4,6-trinitrotoluene (TNT), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) are widely distributed environmental contaminants. Complete chromatographic separation is necessary in order to accurately determine and quantify explosives and their degradation products in environmental samples and in (bio)transformation studies. The present study describes a RP-HPLC method with diode array detection using a LC-8 guard column, a Supelcosil LC-8 chromatographic column, and a gradient elution system. This gradient method is capable of baseline separating the most commonly observed explosives and TNT transformation metabolites including 2,4,6-triaminotoluene (TAT) in a single run. The TNT metabolites separated were 2-hydroxylamino-4,6-dinitrotoluene, 4-hydroxylamino-2,6-dinitrotoluene, 2,4-dihydroxylamino-6-nitrotoluene, 4,4',6,6'-tetranitro-2,2'-azoxytoluene, 2,2',6,6'-tetranitro-4,4'-azoxytoluene, 4,4',6,6'-tetranitro-2,2'-azotoluene, 2,2',6,6'-tetranitro-4,4'-azotoluene, 2-amino-4,6-dinitrotoluene, 4-amino-2, 6-dinitrotoluene, 2,6-diamino-4-nitrotoluene, 2,4-diamino-6-nitrotoluene, and TAT. The same gradient method at a different column temperature can also be used to baseline separate the explosives targeted in the Environmental Protection Agency (EPA) Method 8330 with approximately 22% reduction in total run time and 48% decrease in solvent consumption compared to previously published methods. Good separation was also obtained when all TNT metabolites and EPA Method 8330 compounds (a total of 23 compounds) were analyzed together; only 2,6-DANT and HMX co-eluted in this case. The influence of temperature (35-55 degrees C) and the use of an ion-pair reagent on the chromatographic resolution and retention were investigated. Temperature was identified as the key parameter for optimal baseline separation. Increased temperature resulted in shorter retention times and better peak resolution

  11. Organochlorine isotopic pattern-enhanced detection and quantification of triclosan and its metabolites in human serum by ultra-high-performance liquid chromatography/quadrupole time-of-flight/mass spectrometry.

    PubMed

    Wu, Jian-lin; Leung, Ka-Fai; Tong, Sui-Fan; Lam, Ching-Wan

    2012-01-30

    Organochlorines possess special isotopic patterns that obey the chlorine rule. In the case of triclosan (TCS), which contains three chlorine atoms, the isotopic patterns are composed of seven obvious peaks with the calculated masses ranging from 286.9435 to 292.9350 in negative ion mode and with specific isotopic abundance ratios of 100:13.1:97.1:12.6:31.8:4.1:3.6. In this study, mass differences between the calculated and observed m/z values for all isotopic peaks of TCS were less than 3.5 ppm in the analyses of the serum samples by ultra-high-performance liquid chromatography/quadrupole time-of-flight/mass spectrometry (UHPLC-Q-TOF/MS). Combining the characteristics described above, four metabolites were identified as sulfonated TCS, glucuronidated TCS and hydroxylated sulfonated TCS. Several novel MS techniques were applied to improve the sensitivity of quantification of TCS. The limit of detection for TCS in a 250 μL serum sample was 0.05 ng/mL, which was over twenty times lower than values obtained by the LC/triple quadrupole-MS/MS method reported in the literature. The concentration of total TCS (free and conjugated) was quantified to range from 0.15 to 217 ng/mL, whereas free TCS ranged from 0.15 to 10 ng/mL. To the best of our knowledge, this is the first report on the identification of TCS and metabolites in human serum, and it also provides the most sensitive LC/MS approach for the quantification of TCS.

  12. Simultaneous quantitative determination of the synthetic antioxidant ethoxyquin and its major metabolite in Atlantic salmon (Salmo salar, L), ethoxyquin dimer, by reversed-phase high-performance liquid chromatography with fluorescence detection.

    PubMed

    Bohne, Victoria J Berdikova; Hove, Helge; Hamre, Kristin

    2007-01-01

    A method for simultaneous quantitative determination of ethoxyquin (EQ) and its major metabolite in Atlantic salmon tissues, ethoxyquin dimer (EQ dimer), has been developed. The separation was achieved on tandem coupled phenyl-hexyl and C18 columns by 2-phase gradient elution with acetonitrile-ascorbic acid-acetic acid-diethyl amine organized in a 23.5 min sequence. Compounds were extracted with hexane from samples saponified in ethanol-NaOH and protected from air- and light-mediated oxidation by addition of saturated ethylenediaminetetraacetic acid, ascorbic acid, and pyrogallol. The identity of peaks was confirmed by spiking samples with standards verified by proton nuclear magnetic resonance spectrometry, mass spectrometry, and high-performance liquid chromatography. The detection limit (at 358/433 nm) of matrix-spiked EQ was 0.02 and 0.06 microg/L for EQ dimer, with 0.5 g sample weighed and resuspension in 0.5 mL hexane. Linearity was in the range of 0.2-175 microg/L for EQ and 0.3-5100 microg/L for EQ dimer. Two more ubiquitous compounds were identified as de-ethylated EQ and quinone imine. Totally, 14 peaks sharing spectral properties of EQ were separated in a single run, including a major peak present in all muscle samples, termed unknown metabolite of EQ (UMEQ). The concentrations of EQ, EQ dimer, and de-ethylated EQ, as well as concentrations of UMEQ (in arbitrary units), in the muscle were correlated to the amount of EQ fed to the salmon, thus indicating their possible metabolic origin. The pattern of 14 peaks in the muscle showed high specificity and could be used to discriminate between wild salmon and salmon fed EQ-supplemented feed. This method will be a useful tool for studying EQ metabolism and kinetics, and for the routine surveillance of residual levels of dietary EQ in farmed Atlantic salmon.

  13. Transportable hyperpolarized metabolites

    NASA Astrophysics Data System (ADS)

    Ji, Xiao; Bornet, Aurélien; Vuichoud, Basile; Milani, Jonas; Gajan, David; Rossini, Aaron J.; Emsley, Lyndon; Bodenhausen, Geoffrey; Jannin, Sami

    2017-01-01

    Nuclear spin hyperpolarization of 13C-labelled metabolites by dissolution dynamic nuclear polarization can enhance the NMR signals of metabolites by several orders of magnitude, which has enabled in vivo metabolic imaging by MRI. However, because of the short lifetime of the hyperpolarized magnetization (typically <1 min), the polarization process must be carried out close to the point of use. Here we introduce a concept that markedly extends hyperpolarization lifetimes and enables the transportation of hyperpolarized metabolites. The hyperpolarized sample can thus be removed from the polarizer and stored or transported for use at remote MRI or NMR sites. We show that hyperpolarization in alanine and glycine survives 16 h storage and transport, maintaining overall polarization enhancements of up to three orders of magnitude.

  14. Transportable hyperpolarized metabolites

    PubMed Central

    Ji, Xiao; Bornet, Aurélien; Vuichoud, Basile; Milani, Jonas; Gajan, David; Rossini, Aaron J.; Emsley, Lyndon; Bodenhausen, Geoffrey; Jannin, Sami

    2017-01-01

    Nuclear spin hyperpolarization of 13C-labelled metabolites by dissolution dynamic nuclear polarization can enhance the NMR signals of metabolites by several orders of magnitude, which has enabled in vivo metabolic imaging by MRI. However, because of the short lifetime of the hyperpolarized magnetization (typically <1 min), the polarization process must be carried out close to the point of use. Here we introduce a concept that markedly extends hyperpolarization lifetimes and enables the transportation of hyperpolarized metabolites. The hyperpolarized sample can thus be removed from the polarizer and stored or transported for use at remote MRI or NMR sites. We show that hyperpolarization in alanine and glycine survives 16 h storage and transport, maintaining overall polarization enhancements of up to three orders of magnitude. PMID:28072398

  15. Sensitive determination of three aconitum alkaloids and their metabolites in human plasma by matrix solid-phase dispersion with vortex-assisted dispersive liquid-liquid microextraction and HPLC with diode array detection.

    PubMed

    Wang, Xiaozhong; Li, Xuwen; Li, Lanjie; Li, Min; Liu, Ying; Wu, Qian; Li, Peng; Jin, Yongri

    2016-05-01

    A simple and sensitive method for determination of three aconitum alkaloids and their metabolites in human plasma was developed using matrix solid-phase dispersion combined with vortex-assisted dispersive liquid-liquid microextraction and high-performance liquid chromatography with diode array detection. The plasma sample was directly purified by matrix solid-phase dispersion and the eluate obtained was concentrated and further clarified by vortex-assisted dispersive liquid-liquid microextraction. Some important parameters affecting the extraction efficiency, such as type and amount of dispersing sorbent, type and volume of elution solvent, type and volume of extraction solvent, salt concentration as well as sample solution pH, were investigated in detail. Under optimal conditions, the proposed method has good repeatability and reproducibility with intraday and interday relative standard deviations lower than 5.44 and 5.75%, respectively. The recoveries of the aconitum alkaloids ranged from 73.81 to 101.82%, and the detection limits were achieved within the range of 1.6-2.1 ng/mL. The proposed method offered the advantages of good applicability, sensitivity, simplicity, and feasibility, which makes it suitable for the determination of trace amounts of aconitum alkaloids in human plasma samples.

  16. A novel chemiluminescent ELISA for detecting furaltadone metabolite, 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) in fish, egg, honey and shrimp samples.

    PubMed

    Liu, Ying-Chun; Jiang, Wei; Chen, Yong-Jun; Xiao, Yan; Shi, Jin-Lei; Qiao, Yuan-Biao; Zhang, Hua-Jing; Li, Tao; Wang, Quan

    2013-09-30

    In this study, an indirect competitive enzyme-linked immunosorbent assay with chemiluminescent (CLELISA) detection for 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) was developed. A monoclonal antibody (MAb) against AMOZ was prepared through immunizing BALB/c mice with 4-carboxybenzaldehyle derivatized AMOZ (CPAMOZ), conjugated with bovine serum albumin (BSA) as antigen. The effects of the substrates luminol, p-iodophenol and urea peroxide on the performance of the assay were studied and optimized. In addition, the specificity of the MAb, estimated as the cross-reactivity values with 4-nitrobenzaldehyde derivatized AMOZ (NPAMOZ), CPAMOZ and AMOZ, was 100%, 27.45% and 0.18%, respectively. The sensitivity of the developed CLELISA was estimated as 50% inhibitory concentration (IC50) value (0.14μg/l) with a linear working range between 0.03 and 64μg/l, and a limit of detection of 0.01μg/l. The CLELISA described in this study was 5-fold more sensitive than the indirect competitive ELISA previously developed in our laboratory. Finally, this new CLELISA was compared with a commercial kit to detect NPAMOZ in spiked fish, shrimp, honey and egg samples. The recovery values from four spiked fish, shrimp, honey and egg samples with different concentrations of NPAMOZ in CLELISA were 92.1-107.7%. Thus, the immunoassay method described here has a broad detection range and high sensitivity and is a valid and cost-effective means for high throughput monitoring of residual AMOZ levels in fish, shrimps, honey and eggs with potential applications in other animal tissues.

  17. An approach based on liquid chromatography/electrospray ionization–mass spectrometry to detect diol metabolites as biomarkers of exposure to styrene and 1,3-butadiene

    PubMed Central

    Shen, Shuijie; Zhang, Fan; Zeng, Su; Zheng, Jiang

    2012-01-01

    Styrene and 1,3-butadiene are important intermediates used extensively in the plastics industry. They are metabolized mainly through cytochrome P450-mediated oxidation to the corresponding epoxides, which are subsequently converted to diols by epoxide hydrolase or through spontaneous hydration. The resulting styrene glycol and 3-butene-1,2-diol have been suggested as biomarkers of exposure to styrene and 1,3-butadiene, respectively. Unfortunately, poor ionization of the diols within electrospray mass spectrometers becomes an obstacle to the detection of the two diols by liquid chromatography/electrospray ionization–mass spectrometry (LC/ESI–MS). We developed an LC/ESI–MS approach to analyze styrene glycol and 3-butene-1,2-diol by means of derivatization with 2-bromopyridine-5-boronic acid (BPBA), which not only dramatically increases the sensitivity of diol detection but also facilitates the identification of the diols. The analytical approach developed was simple, quick, and convincing without the need for complicated chemical derivatization. To evaluate the feasibility of BPBA as a derivatizing reagent of diols, we investigated the impact of diol configuration on the affinity of a selection of diols to BPBA using the established LC/ESI–MS approach. We found that both cis and trans diols can be derivatized by BPBA. In conclusion, BPBA may be used as a general derivatizing reagent for the detection of vicinal diols by LC/MS. PMID:19111668

  18. Determination of malachite green, crystal violet and their leuco-metabolites in fish by HPLC-VIS detection after immunoaffinity column clean-up.

    PubMed

    Xie, Jie; Peng, Tao; Chen, Dong-Dong; Zhang, Qing-Jie; Wang, Guo-Min; Wang, Xiong; Guo, Qi; Jiang, Fan; Chen, Dan; Deng, Jian

    2013-01-15

    A high performance liquid chromatography method with visible detection (HPLC-VIS) for the determination of malachite green (MG), crystal violet (CV), leucomalachite green (LMG), and leucocrystal violet (LCV) in fish has been developed after clean-up through an immunoaffinity column (IAC). Residues were simultaneously extracted from fish muscle with acetonitrile and ammonium acetate buffer. The leuco-forms, LMG and LCV, were oxidized quantitatively to the chromic CV and MG by reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. Extracts were then purified on an IAC which prepared by immobilizing the anti-MG-CV antibodies by the sol-gel method. Finally, the eluents were analyzed by HPLC-VIS. The limits of detection were 0.15, 0.1, 0.18 and 0.14ng/g for MG, CV, LMG and LCV, respectively. The average recoveries in samples fortified with MG, CV, LMG and LCV over the range 0.5-10ng/g were from 71.6% to 96.8% with RSDs of 5.1-12.3% (n=6). This novel method was confirmed by liquid chromatography-tandem mass spectrometry with electrospray interface in positive mode using multiple reaction monitoring.

  19. High fat diet leads to changes in metabolite patterns in pig plasma, fecal, and urine samples detected by a ultra-high performance liquid chromatography tandem with high resolution mass spectrometry metabolomic study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Non-targeted metabolite profiling can identify robust biological markers of dietary exposure that can lead to a better understanding of causal interactions between diet and health. In this study, pigs were used as an animal model to develop an efficient procedure to discover metabolites in biolog...

  20. Analysis of selected herbicide metabolites in surface and ground water of the United States

    USGS Publications Warehouse

    Scribner, E.A.; Thurman, E.M.; Zimmerman, L.R.

    2000-01-01

    One of the primary goals of the US Geological Survey (USGS) Laboratory in Lawrence, Kansas, is to develop analytical methods for the analysis of herbicide metabolites in surface and ground water that are vital to the study of herbicide fate and degradation pathways in the environment. Methods to measure metabolite concentrations from three major classes of herbicides - triazine, chloroacetanilide and phenyl-urea - have been developed. Methods for triazine metabolite detection cover nine compounds: six compounds are detected by gas chromatography/mass spectrometry; one is detected by high-performance liquid chromatography with diode-array detection; and eight are detected by liquid chromatography/mass spectrometry. Two metabolites of the chloroacetanilide herbicides - ethane sulfonic acid and oxanilic acid - are detected by high-performance liquid chromatography with diode-array detection and liquid chromatography/mass spectrometry. Alachlor ethane sulfonic acid also has been detected by solid-phase extraction and enzyme-linked immunosorbent assay. Six phenylurea metabolites are all detected by liquid chromatography/mass spectrometry; four of the six metabolites also are detected by gas chromatography/mass spectrometry. Additionally, surveys of herbicides and their metabolites in surface water, ground water, lakes, reservoirs, and rainfall have been conducted through the USGS laboratory in Lawrence. These surveys have been useful in determining herbicide and metabolite occurrence and temporal distribution and have shown that metabolites may be useful in evaluation of non-point-source contamination. Copyright (C) 2000 Elsevier Science B.V.

  1. Microalgal metabolites: a new perspective.

    PubMed

    Shimizu, Y

    1996-01-01

    Occurrence of secondary metabolites in microalgae (protoctista) is discussed with respect to the phylogenic or taxonomic relationships of organisms. Biosynthetic mechanisms of certain metabolites such as paralytic shellfish poisoning toxins and polyether toxins are also discussed, and genetic aspects of the secondary metabolite production as well.

  2. Differential Radial Capillary Action of Ligand Assay (DRaCALA) for High-Throughput Detection of Protein-Metabolite Interactions in Bacteria.

    PubMed

    Orr, Mona W; Lee, Vincent T

    2017-01-01

    Bacteria rely on numerous nucleotide second messengers for signal transduction such as cyclic AMP, cyclic-di-GMP, and cyclic-di-AMP. Although a number of receptors responsible for known regulated phenotypes have been established, the completeness of protein receptors in any given organism remains elusive. We have developed a method called differential radial capillary action of ligand assay (DRaCALA) that allows for an unbiased, systematic high-throughput screen for the detection of ligand binding proteins encoded by a genome. DRaCALA permits interrogation of ligand binding directly to an overexpressed protein in a cell lysate and bypasses the need of protein purification. Gateway-cloning-compatible open reading frame libraries are available for a diverse range of bacterial species and permits generation of the lysates overexpressing each open reading frame. These lysates can be assessed by DRaCALA in a 96-well format to allow rapid identification of protein-ligand interactions, including previously unknown proteins. Here, we present the protocols for generating the expression library, conducting the DRaCALA screen, data analysis, and hit validation.

  3. Multiresidue Screening Method for Detection of Benzimidazoles and their Metabolites in Liver and Muscle by High-Performance Liquid Chromatography: Method Development and Validation According to Commission Decision 2002/657/EC

    PubMed Central

    Prearo, Marino; Stella, Paola; Ostorero, Federica; Abete, Maria Cesarina

    2014-01-01

    The use of veterinary drugs may cause the presence of residues in food of animal origin if appropriate withdrawal periods are not respected. A high-performance liquid chromatography (HPLC) method has been developed for the simultaneous detection of 11 benzimidazole residues, including metabolites – albendazole, albendazole sulphoxide, albendazole sulphone, fenbendazole, fenbendazole sulphoxide (oxfendazole), fenbendazole sulphone, flubendazole, mebendazole, oxibendazole, thiabendazole, 5-hydroxythiabendazole – in bovine, ovine, equine, swine, rabbit and poultry liver and in bovine, swine and fish muscle. After extraction with a dicloromethane/acetonitrile solution (35/65 v/v) containing 5% ammonium hydroxide, the solvent was evaporated to dryness, the residue was dissolved in HCl 0.1 M, defatted with hexane, purified on a strong cation exchange solid-phase extraction cartridge and analysed in HPLC with diode array and fluorescence detectors. The method was validated as screening qualitative method evaluating, according to Commission Decision 2002/657/EC criteria, specificity, CCβ and β error at cut off level of 25 μg/kg and ruggedness. PMID:27800310

  4. Ex vivo studies for the passive transdermal delivery of low-dose naltrexone from a cream; detection of naltrexone and its active metabolite, 6β-naltrexol, using a novel LC Q-ToF MS assay.

    PubMed

    Dodou, Kalliopi; Armstrong, Andrew; Kelly, Ivan; Wilkinson, Simon; Carr, Kevin; Shattock, Paul; Whiteley, Paul

    2015-01-01

    Naltrexone (NTX) is a long-acting opiate antagonist. Low-dose naltrexone (LDN) therapy has shown promising results in the treatment of several autoimmune disorders. Our aim was to formulate NTX into a cream for the delivery of LDN and develop an analytical technique for the quantification of NTX and its active metabolite 6-β-naltrexol (NTXol) during transdermal diffusion cell permeation studies. A 1% w/w NTX cream was formulated and drug permeation was examined over 24 h using static Franz diffusion cells mounted with pig skin. A Liquid Chromatography Quadrupole-Time of Flight Mass Spectrometry (LC-MS Q-ToF) method was developed for the detection of NTX and NTXol in the receptor solution, skin membrane and residual cream on the donor chamber after completion of the diffusion studies. The cream formulation exhibited steady state release of NTX over 24 h after an initial lag time of 2.74 h. The bioconversion of NTX to NTXol in the skin membrane was 1.1%. It was concluded that the cream may be an effective formulation for the sustained transdermal delivery of LDN. The novel LC Q-ToF MS method allowed the accurate measurement of NTX and NTXol levels across the diffusion cell assemblies and the quantification of NTX metabolism in the skin.

  5. On-line coupling of automated solid-phase extraction with high-performance liquid chromatography and electrochemical detection. Quantitation of oxidizable drugs of abuse and their metabolites in plasma and urine.

    PubMed

    Krämer, E; Kovar, K A

    1999-08-20

    The concentration effect of automated on-line solid-phase extraction (SPE) in combination with HPLC and very sensitive electrochemical detection was employed for the determination of N-ethyl-4-hydroxy-3-methoxy-amphetamine (HMEA, the main metabolite of the ecstasy analogue MDE), delta 9-tetrahydrocannabinol (THC) and 11-nor-delta 9-tetrahydrocannabinol-carboxylic acid (THC-COOH) in plasma and urine in comparison to a previously published psilocin assay. For the SPE either CBA (functional group: carboxypropyl)- or CH (functional group: cyclohexyl)-sorbent was used. The following separation was carried out on a reversed-phase column (LiChroCart, Superspher 60 RP select B from Merck). Depending on the hydrodynamic voltammogram of the analyzed substance the oxidation potential varied from 920 mV up to 1.2 V. In spite of using high potentials, precision and accuracy were always within the accepted statistical requirements. The limits of quantitation were between 5 ng/ml (THC, THC-COOH in plasma) and 20 ng/ml (HMEA in plasma). Advantages of on-line SPE in comparison with off-line methods were less manual effort, evidently smaller volumes (< or = 400 microliters) of plasma or urine and almost always higher recovery rates (> 93%). The assays have been successfully proven with real biological samples and found suitable for use in routine analysis.

  6. Pressurized liquid extraction for the determination of cannabinoids and metabolites in hair: Detection of cut-off values by high performance liquid chromatography-high resolution tandem mass spectrometry.

    PubMed

    Montesano, Camilla; Simeoni, Maria Chiara; Vannutelli, Gabriele; Gregori, Adolfo; Ripani, Luigi; Sergi, Manuel; Compagnone, Dario; Curini, Roberta

    2015-08-07

    Hair analysis has become a routine procedure in most forensic laboratories since this alternative matrix presents clear advantages over classical matrices; particularly wider time window, non-invasive sampling and good stability of the analytes over time. There are, however, some major challenges for the analysis of cannabinoids in hair, mainly related to the low concentrations of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THC-COOH), that is the major metabolite. In this study a fast, accurate and sensitive method for the determination of cannabinol, cannabidiol, THC and THC-COOH in hair has been developed. The extraction of analytes from hair (50mg) is based on an automated pressurized liquid extraction (PLE) using water modified with the surfactant sodium dodecyl sulphate as eluent phase. PLE extract is then cleaned up by SPE using polymeric reversed phase cartridges Strata XL before the injection in the HPLC-HRMS/MS system. Chromatographic conditions obtained with a fused-core column allowed a good separation of the analytes in less than 4min. The whole procedure has been validated according to SWGTOX guidelines. The LLOQs obtained for THC-COOH and the other analytes were respectively 0.1 and 2pg/mg. To the best of our knowledge, this is the first LC-MS/MS based method that allows the detection of THC-COOH in hair at values lower than the cut-off.

  7. Sensitive chiral high-performance liquid chromatographic determination of anthelmintic flubendazole and its phase I metabolites in blood plasma using UV photodiode-array and fluorescence detection Application to pharmacokinetic studies in sheep.

    PubMed

    Nobilis, Milan; Vybíralová, Zuzana; Krízová, Veronika; Kubícek, Vladimír; Soukupová, Marie; Lamka, Jirí; Szotáková, Barbora; Skálová, Lenka

    2008-12-01

    stereospecificity of the enzymatic carbonyl reduction of flubendazole was also observed in vivo. (+)-Reduced flubendazole was found to be the principal metabolite in ovine blood plasma and only low concentrations of hydrolyzed flubendazole, the parent flubendazole and (-)-reduced flubendazole were detected in this biomatrix.

  8. Simultaneous determination of malachite green, gentian violet and their leuco metabolites in catfish or trout tissue by high-performance liquid chromatography with visible detection.

    PubMed

    Rushing, L G; Thompson, H C

    1997-01-24

    A sensitive analytical procedure for the determination of residues of leucomalachite green (LMG)-malachite green (MG) and leucogentian violet (LGV)-gentian violet (GV) in catfish or trout tissue is presented. Frozen (-20 degrees C) fish fillets were cut into small pieces and blended in a Waring blender. A 20-g amount of homogenized fish tissue was extracted with acetonitrile-buffer, partitioned against methylene chloride, and cleaned up on tandem neutral alumina and propylsulfonic acid cation-exchange solid-phase extraction cartridges. Samples of 100 microliters (0.8 g equiv.) were chromatographed isocratically in 10 min using an acetonitrile-buffer mobile phase on a short-chain deactivated (SCD) reversed-phase column (250 x 4.6 mm I.D.) in-line with a post-column PbO2 oxidation reactor. The PbO2 post-column reactor efficiently oxidized LMG to the chromatic MG, and LGV to the chromatic GV permitting visible detection at 588 nm for all four compounds. Linearity was demonstrated with standards over the range of 0.5-50 ng per injection. Recoveries of LMG, MG, LGV and GV from catfish tissues fortified at 10 ng/g were 75.4 +/- 3.0, 61.3 +/- 4.1, 72.6 +/- 3.7 and 87.9 +/- 2.5, respectively, while trout tissues fortified at 10 ng/g yielded recoveries of 82.6 +/- 2.3, 48.6 +/- 1.8, 72.1 +/- 2.1 and 83.8 +/- 4.6 (mean +/- S.D., n = 4), respectively.

  9. Simple and sensitive determination of atrazine and its toxic metabolites in environmental water by carboxyl modified polyacrylonitrile nanofibers mat-based solid-phase extraction coupled with liquid chromatography-diode array detection.

    PubMed

    Cao, Weixin; Yang, Biyi; Qi, Feifei; Qian, Liangliang; Li, Jian; Lu, Lingeng; Xu, Qian

    2017-02-20

    Carboxyl modified polyacrylonitrile nanofibers mat (COOH-PAN NFsM) was prepared as a novel solid-phase extraction (SPE) adsorbent for the rapid and effective extraction of atrazine (ATZ) and its toxic metabolites deisopropylatrazine (DIA) and deethylatrazine (DEA) from environmental water samples. Without any pre-treatment but only with the simple filter, water samples passed through pre-conditioned COOH-PAN NFsM, which integrated extraction, purification and concentration into one single step, and the eluent was directly analyzed by high performance liquid chromatography-diode array detection (HPLC-DAD). Under the optimized conditions, the target analytes in 10mL of water sample could be completely extracted by only 4mg of COOH-PAN NFsM, and easily eluted using 400μL of methanol, indicating a high efficiency in both adsorption and desorption. Satisfactory linearity was achieved in the range of 0.4-40.0ngmL(-1) for DIA, and 0.3-40.0ngmL(-1) for DEA and ATZ. The limit of detection (LODs) were 0.12, 0.09 and 0.09ngmL(-1) for DIA, DEA and ATZ, respectively. The recoveries ranged from 81.35 to 120.32%, with the intra-day and inter-day relative standard deviations of 4.03-9.81% even after the 6-cycle usage of NFsM. And, using just 10mL loading sample, the LOD had already satisfied the demand of surface water quality monitoring levels, revealing the good sensitivity of the proposed method.

  10. Determination of organophosphorus pesticides and metabolites in cereal-based baby foods and wheat flour by means of ultrasound-assisted extraction and hollow-fiber liquid-phase microextraction prior to gas chromatography with nitrogen phosphorus detection.

    PubMed

    González-Curbelo, Miguel Ángel; Hernández-Borges, Javier; Borges-Miquel, Teresa María; Rodríguez-Delgado, Miguel Ángel

    2013-10-25

    A new method based on hollow-fiber liquid-phase microextraction (HF-LPME) has been developed for the determination of a group of organophosphorus pesticides, including some of their metabolites, in two commercial cereal-based baby foods and one wheat flour prior to gas chromatography-nitrogen phosphorus detection. Samples were first extracted by ultrasound-assisted extraction with acetonitrile (ACN) containing 1.25% (v/v) of formic acid. After evaporation and reconstitution in Milli-Q water, the HF-LPME procedure, using 1-octanol as extraction solvent, was applied followed by a desorption step in ACN, which clearly improved the performance of the technique. The effects of sample pH, ionic strength, stirring rate, extraction temperature and time as well as the desorption procedure were investigated. Under the optimum conditions that involved the extraction of the analytes from 10 mL of the water reconstituted extract at pH 7.0 containing 5% (w/v) of NaCl for 45 min at 960 rpm, the method was validated in terms of linearity, precision and accuracy. The limits of detection (LODs) were between 0.29 and 3.20 μg/kg. The extraction of Milli-Q water, as an example of the applicability of the procedure to aqueous samples, allowed achieving LODs in the range 0.01-0.04 μg/L. Such values, together with the ones achieved for the rest of the samples, are below or equal to the maximum residue limits specified by the European Union.

  11. Comparative metabolism of codeine in man, rat, dog, guinea-pig and rabbit: identification of four new metabolites.

    PubMed

    Cone, E J; Darwin, W D; Gorodetzky, C W

    1979-05-01

    The metabolism and excretion of codeine and its metabolites in untreated urine of man, rat, dog, guinea-pig and rabbit have been examined. Metabolites were identified by gas chromatography mass spectrometry operated in the chemical ionization mode (methane). Concentrations of codeine and metabolites were measured by selected ion monitoring. Both codeine and norcodeine were detected in the urine of all species but a new metabolite, hydrocodone, was found only in the urine from man, guinea-pig and dog. Additional metabolites (presumably resulting from the metabolism of hydrocodone) were also detected in man and guinea-pig. Overall recoveries of drug and metabolites from untreated urine were low for all species.

  12. Elucidation of the metabolites of the novel psychoactive substance 4-methyl-N-ethyl-cathinone (4-MEC) in human urine and pooled liver microsomes by GC-MS and LC-HR-MS/MS techniques and of its detectability by GC-MS or LC-MS(n) standard screening approaches.

    PubMed

    Helfer, Andreas G; Turcant, Alain; Boels, David; Ferec, Séverine; Lelièvre, Bénédicte; Welter, Jessica; Meyer, Markus R; Maurer, Hans H

    2015-05-01

    4-methyl-N-ethcathinone (4-MEC), the N-ethyl homologue of mephedrone, is a novel psychoactive substance of the beta-keto amphetamine (cathinone) group. The aim of the present work was to study the phase I and phase II metabolism of 4-MEC in human urine as well as in pooled human liver microsome (pHLM) incubations. The urine samples were worked up with and without enzymatic cleavage, the pHLM incubations by simple deproteinization. The metabolites were separated and identified by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-high resolution-tandem mass spectrometry (LC-HR-MS/MS). Based on the metabolites identified in urine and/or pHLM, the following metabolic pathways could be proposed: reduction of the keto group, N-deethylation, hydroxylation of the 4-methyl group followed by further oxidation to the corresponding 4-carboxy metabolite, and combinations of these steps. Glucuronidation could only be observed for the hydroxy metabolite. These pathways were similar to those described for the N-methyl homologue mephedrone and other related drugs. In pHLM, all phase I metabolites with the exception of the N-deethyl-dihydro isomers and the 4-carboxy-dihydro metabolite could be confirmed. Glucuronides could not be formed under the applied conditions. Although the taken dose was not clear, an intake of 4-MEC should be detectable in urine by the GC-MS and LC-MS(n) standard urine screening approaches at least after overdose.

  13. NMR identification of endogenous metabolites interacting with fatted and non-fatted human serum albumin in blood plasma: Fatty acids influence the HSA-metabolite interaction

    NASA Astrophysics Data System (ADS)

    Jupin, Marc; Michiels, Paul J.; Girard, Frederic C.; Spraul, Manfred; Wijmenga, Sybren S.

    2013-03-01

    Metabolites and their concentrations are direct reporters on body biochemistry. Thanks to technical developments metabolic profiling of body fluids, such as blood plasma, by for instance NMR has in the past decade become increasingly accurate enabling successful clinical diagnostics. Human Serum Albumin (HSA) is the main plasma protein (∼60% of all plasma protein) and responsible for the transport of endogenous (e.g. fatty acids) and exogenous metabolites, which it achieves thanks to its multiple binding sites and its flexibility. HSA has been extensively studied with regard to its binding of drugs (exogenous metabolites), but only to a lesser extent with regard to its binding of endogenous (non-fatty acid) metabolites. To obtain correct NMR measured metabolic profiles of blood plasma and/or potentially extract information on HSA and fatty acids content, it is necessary to characterize these endogenous metabolite/plasma protein interactions. Here, we investigate these metabolite-HSA interactions in blood plasma and blood plasma mimics. The latter contain the roughly twenty metabolites routinely detected by NMR (also most abundant) in normal relative concentrations with fatted or non-fatted HSA added or not. First, we find that chemical shift changes are small and seen only for a few of the metabolites. In contrast, a significant number of the metabolites display reduced resonance integrals and reduced free concentrations in the presence of HSA or fatted HSA. For slow-exchange (or strong) interactions, NMR resonance integrals report the free metabolite concentration, while for fast exchange (weak binding) the chemical shift reports on the binding. Hence, these metabolites bind strongly to HSA and/or fatted HSA, but to a limited degree because for most metabolites their concentration is smaller than the HSA concentration. Most interestingly, fatty acids decrease the metabolite-HSA binding quite significantly for most of the interacting metabolites. We further find

  14. NMR identification of endogenous metabolites interacting with fatted and non-fatted human serum albumin in blood plasma: Fatty acids influence the HSA-metabolite interaction.

    PubMed

    Jupin, Marc; Michiels, Paul J; Girard, Frederic C; Spraul, Manfred; Wijmenga, Sybren S

    2013-03-01

    Metabolites and their concentrations are direct reporters on body biochemistry. Thanks to technical developments metabolic profiling of body fluids, such as blood plasma, by for instance NMR has in the past decade become increasingly accurate enabling successful clinical diagnostics. Human Serum Albumin (HSA) is the main plasma protein (∼60% of all plasma protein) and responsible for the transport of endogenous (e.g. fatty acids) and exogenous metabolites, which it achieves thanks to its multiple binding sites and its flexibility. HSA has been extensively studied with regard to its binding of drugs (exogenous metabolites), but only to a lesser extent with regard to its binding of endogenous (non-fatty acid) metabolites. To obtain correct NMR measured metabolic profiles of blood plasma and/or potentially extract information on HSA and fatty acids content, it is necessary to characterize these endogenous metabolite/plasma protein interactions. Here, we investigate these metabolite-HSA interactions in blood plasma and blood plasma mimics. The latter contain the roughly twenty metabolites routinely detected by NMR (also most abundant) in normal relative concentrations with fatted or non-fatted HSA added or not. First, we find that chemical shift changes are small and seen only for a few of the metabolites. In contrast, a significant number of the metabolites display reduced resonance integrals and reduced free concentrations in the presence of HSA or fatted HSA. For slow-exchange (or strong) interactions, NMR resonance integrals report the free metabolite concentration, while for fast exchange (weak binding) the chemical shift reports on the binding. Hence, these metabolites bind strongly to HSA and/or fatted HSA, but to a limited degree because for most metabolites their concentration is smaller than the HSA concentration. Most interestingly, fatty acids decrease the metabolite-HSA binding quite significantly for most of the interacting metabolites. We further find

  15. Quantification of the HIV-integrase inhibitor raltegravir and detection of its main metabolite in human plasma, dried blood spots and peripheral blood mononuclear cell lysate by means of high-performance liquid chromatography tandem mass spectrometry.

    PubMed

    Ter Heine, R; Hillebrand, M J X; Rosing, H; van Gorp, E C M; Mulder, J W; Beijnen, J H; Huitema, A D R

    2009-02-20

    For the quantification of the HIV-integrase inhibitor raltegravir in human plasma, dried blood spots and peripheral blood mononuclear cell (PBMC) lysate, an assay was developed and validated, using liquid chromatography coupled with tandem mass spectrometry. The assay also allowed detection, but no quantification due to absence of reference substance, of the main metabolite, raltegravir-glucuronide. Raltegravir was extracted from plasma by means of protein precipitation with a mixture of methanol and acetonitrile using only 50microL plasma. Extraction from dried blood spots was performed with a simple one-step extraction with a mixture of methanol, acetonitrile and 0.2M zincsulphate in water (1:1:2, v/v/v) and extraction from cell lysate was performed in 50% methanol in water. Chromatographic separation was performed on a reversed phase C18 column (150mmx2.0mm, particle size 5microm) with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.25mL/min. The analytical run time was 10min. The triple quadrupole mass spectrometer was operated in the positive ion-mode and multiple reaction monitoring was used for drug quantification. The method was validated over a range of 50-10,000ng/mL in plasma and dried blood spots and a range of 1-500ng/mL in PBMC lysate. Dibenzepine was used as the internal standard. The method was proven to be specific, accurate, precise and robust. Accuracies ranged from 104% to 105% in plasma, from 93% to 105% in dried blood spots and from 82% to 113% in PBMC lysate. Precision over the complete concentration range was less than 6%, 11% and 13% in plasma, dried blood spots and PBMC lysate, respectively. The method is now applied for therapeutic drug monitoring and pharmacological research in HIV-infected patients treated with raltegravir.

  16. Characterization and tissue distribution of conjugated metabolites of pyrene in the rat

    PubMed Central

    SAENGTIENCHAI, Aksorn; IKENAKA, Yoshinori; DARWISH, Wageh Sobhy; NAKAYAMA, Shouta M.M.; MIZUKAWA, Hazuki; ISHIZUKA, Mayumi

    2015-01-01

    Pyrene (PY) is a polycyclic aromatic hydrocarbon (PAH) that is often used as a biomarker for human and wildlife exposure to PAHs. As the metabolites of PAHs, similar to their parent compounds, pose public health risks, it is necessary to study their characteristics and tissue-specific distribution. The present study was performed to experimentally characterize PY metabolites and analyze the tissue-specific distribution of the conjugated metabolites after oral administration of PY to rats. PY metabolites, such as pyrenediol-disulfate (PYdiol-diS), pyrenediol-sulfate (PYdiol-S), pyrene-1-sufate (PYOS), pyrene-1-glucuronide (PYOG) and 1-hydroxypyrene (PYOH), were detected in rat urine. Although glucuronide conjugate was the predominant metabolite, the metabolite composition varied among tissues. Interestingly, the proportion of PYOH was high in the large intestine. Furthermore, PYOH was the only PY metabolite detected in feces. PMID:26028020

  17. Mutagenic azide metabolite is azidoalanine

    SciTech Connect

    Owais, W.M.; Rosichan, J.L.; Ronald, R.C.; Kleinhofs, A.; Nilan, R.A.

    1981-01-01

    Sodium axide produces high mutation rates in a number of species. Azide mutagenicity is mediated through a metabolite in barley and bacteria. Many studies showed that azide affects the L-cysteine biosynthesis pathway. Cell-free extracts of Salmonella typhimurium convert azide and O-acetylserine to the mutagenic metabolite. O-acetylserine sulfhydrylase was identified as the enzyme responsible for the metabolite biosynthesis. To confirm the conclusion that the azide metabolite is formed through the ..beta..-substitution pathway of L-cysteine, we radioactively labeled the azide metabolite using /sup 14/C-labeled precursors. Moreover, the mutagenic azide metabolite was purified and identified as azidoalanine based on mass spectroscopy and elemental analysis. 26 refs., 3 figs., 1 tab.

  18. Rapamycin regulates biochemical metabolites

    PubMed Central

    Tucci, Paola; Porta, Giovanni; Agostini, Massimiliano; Antonov, Alexey; Garabadgiu, Alexander Vasilievich; Melino, Gerry; Willis, Anne E

    2013-01-01

    The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth, and deregulation of this pathway is associated with tumorigenesis. p53, and its less investigated family member p73, have been shown to interact closely with mTOR pathways through the transcriptional regulation of different target genes. To investigate the metabolic changes that occur upon inhibition of the mTOR pathway and the role of p73 in this response primary mouse embryonic fibroblast from control and TAp73−/− were treated with the macrocyclic lactone rapamycin. Extensive gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS/MS) analysis were used to obtain a rapamycin-dependent global metabolome profile from control or TAp73−/− cells. In total 289 metabolites involved in selective pathways were identified; 39 biochemical metabolites were found to be significantly altered, many of which are known to be associated with the cellular stress response. PMID:23839040

  19. The Disposition of Oxycodone and Metabolite in Human Hair.

    PubMed

    Reisfield, Gary M; Jones, Joseph T

    2015-01-01

    The disposition of oxycodone (OC) and metabolites in hair remains poorly characterized. We present a case involving a pharmacist in an impaired professionals' monitoring program in whom hair testing yielded OC on two occasions. On both occasions, his hair was negative for the oxymorphone (OM) metabolite at the cutoff concentration of 100 pg/mg. He claimed that, absent the detection of metabolite, the OC necessarily represented external contamination. This prompted a review of the laboratory's OC-positive hair results for the quarter April-June 2014. Overall, 466 specimens contained OC, with a mean (median) concentration of 2,375 (1,060) pg/mg. Of these OC-positive specimens, only 47 (10%) contained detectable OM. When OC was present at or below the mean (median) concentration, only 2.2% (1.3%) of specimens were OM-positive. In the setting of OC administration, the detection of OM in hair is unlikely at a cutoff concentration of 100 pg/mg. More consistent demonstration of OC metabolite(s) in hair will require the validation of methods to detect OM at lower concentrations and/or methods to detect noroxycodone.

  20. Vitamin D metabolites in idiopathic infantile hypercalcaemia.

    PubMed Central

    Martin, N D; Snodgrass, G J; Cohen, R D; Porteous, C E; Coldwell, R D; Trafford, D J; Makin, H L

    1985-01-01

    Metabolites of vitamin D were measured in plasma from 83 patients with idiopathic infantile hypercalcaemia syndrome who were mentally handicapped but had normal calcium values at the time of the study. No significant difference was detected in the mean plasma concentrations of 25-hydroxyvitamin D2, 1,25-dihydroxyvitamin D, 24,25-dihydroxyvitamin D3, or 25,26-dihydroxyvitamin D3 between patients and age matched controls. The mean plasma concentration of 25-hydroxyvitamin D3 was significantly lower in patients than controls but this may be a secondary phenomenon related to less sunlight exposure. In addition, two hypercalcaemic patients with this syndrome were studied during the first year of life, and were found to have normal concentrations of vitamin D metabolites. These findings do not support a role for abnormal vitamin D metabolism in the pathogenesis of this syndrome. PMID:3879160

  1. Ganoderic Acid A Metabolites and Their Metabolic Kinetics.

    PubMed

    Cao, Fang-Rui; Feng, Li; Ye, Lin-Hu; Wang, Li-Sha; Xiao, Bing-Xin; Tao, Xue; Chang, Qi

    2017-01-01

    Ganoderic acid A (GAA), a representative active triterpenoid from Ganoderma lucidum, has been reported to exhibit antinociceptive, antioxidative, cytotoxic, hepatoprotective and anticancer activities. The present study aims (1) to identify GAA metabolites, in vivo by analyzing the bile, plasma and urine after intravenous administration to rats (20 mg/kg), and in vitro by incubating with rat liver microsomes (RLMs) and human liver microsomes (HLMs); (2) to investigate the metabolic kinetics of main GAA metabolites. Using HPLC-DAD-MS/MS techniques, a total of 37 metabolites were tentatively characterized from in vivo samples based on their fragmentation behaviors. The metabolites detected in in vitro samples were similar to those found in vivo. GAA underwent extensive phase I and II metabolism. The main metabolic soft spots of GAA were 3, 7, 11, 15, 23-carbonyl groups (or hydroxyl groups) and 12, 20, 28 (29)-carbon atoms. Ganoderic acid C2 (GAC2) and 7β,15-dihydroxy-3,11,23-trioxo-lanost-26-oic acid were two main reduction metabolites of GAA, and their kinetics followed classical hyperbolic kinetics. The specific isoenzyme responsible for the biotransformation of the two metabolites in RLMs and HLMs was CYP3A. This is the first report on the comprehensive metabolism of GAA, as well as the metabolic kinetics of its main metabolites.

  2. Ganoderic Acid A Metabolites and Their Metabolic Kinetics

    PubMed Central

    Cao, Fang-Rui; Feng, Li; Ye, Lin-Hu; Wang, Li-Sha; Xiao, Bing-Xin; Tao, Xue; Chang, Qi

    2017-01-01

    Ganoderic acid A (GAA), a representative active triterpenoid from Ganoderma lucidum, has been reported to exhibit antinociceptive, antioxidative, cytotoxic, hepatoprotective and anticancer activities. The present study aims (1) to identify GAA metabolites, in vivo by analyzing the bile, plasma and urine after intravenous administration to rats (20 mg/kg), and in vitro by incubating with rat liver microsomes (RLMs) and human liver microsomes (HLMs); (2) to investigate the metabolic kinetics of main GAA metabolites. Using HPLC-DAD-MS/MS techniques, a total of 37 metabolites were tentatively characterized from in vivo samples based on their fragmentation behaviors. The metabolites detected in in vitro samples were similar to those found in vivo. GAA underwent extensive phase I and II metabolism. The main metabolic soft spots of GAA were 3, 7, 11, 15, 23-carbonyl groups (or hydroxyl groups) and 12, 20, 28 (29)-carbon atoms. Ganoderic acid C2 (GAC2) and 7β,15-dihydroxy-3,11,23-trioxo-lanost-26-oic acid were two main reduction metabolites of GAA, and their kinetics followed classical hyperbolic kinetics. The specific isoenzyme responsible for the biotransformation of the two metabolites in RLMs and HLMs was CYP3A. This is the first report on the comprehensive metabolism of GAA, as well as the metabolic kinetics of its main metabolites. PMID:28326038

  3. Identification and Structural Characterization of Three New Metabolites of Bupropion in Humans.

    PubMed

    Sager, Jennifer E; Choiniere, John R; Chang, Justine; Stephenson-Famy, Alyssa; Nelson, Wendel L; Isoherranen, Nina

    2016-08-11

    Bupropion is a widely used antidepressant and the recommended CYP2B6 probe drug. However, current understanding of bupropion elimination pathways is limited. Bupropion has three active circulating metabolites, OH-bupropion, threohydrobupropion, and erythrohydrobupropion, but together with bupropion these metabolites and their conjugates in urine represent only 23% of the dose, and the majority of the elimination pathways of bupropion result in uncharacterized metabolites. The aim of this study was to determine the structures of the uncharacterized bupropion metabolites using human clinical samples and in vitro incubations. Three new metabolites, 4'-OH-bupropion, erythro-4'-OH-hydrobupropion, and threo-4'-OH-hydrobupropion, were detected in human liver microsome incubations and were isolated from human urine. The structures of the metabolites were confirmed via comparison of UV absorbance, NMR spectra, and mass spectral data to those of the synthesized standards. In total, these metabolites represented 24% of the drug related material excreted in urine.

  4. [Effect of antibiotics on intestinal microflora and production of metabolites].

    PubMed

    Tamm, A O; Siĭgur, U Kh; Mikel'saar, M E

    1989-06-01

    Methodical approaches to detection of relation between intestinal microflora and its metabolites are described. The microbial origin of certain compounds can be asserted by a decrease in their production after exposure to antibacterial drugs or the absence of their production in microbe-free animals. The authors consider that parallel investigation of intestinal microflora and its metabolites after exposure to various agents e.g. narrow spectrum antibiotics or specific substrates is the most accurate methodical approach to detection of their interrelations. Data on the effect of four drugs i.e. kanamycin, metronidazole, cefotaxime and bactrim on production of 10 bacterial metabolites: p-cresol, phenol, indican, acetic, propionic, butyric, isobutyric, valeric, isovaleric and caproic acids in rats are presented. Correlation between the metabolites and the intestinal microflora composition was revealed. It is concluded that detection of microorganisms responsible for production of definite metabolites requires at the maximum: (1) exposure to drugs of different spectra, (2) detection of changes in intestinal microflora by biotope++ and (3) investigation of mucosa microflora which more exactly characterizes metabolism of definite biotops.

  5. Spatio-temporal distribution and natural variation of metabolites in citrus fruits.

    PubMed

    Wang, Shouchuang; Tu, Hong; Wan, Jian; Chen, Wei; Liu, Xianqing; Luo, Jie; Xu, Juan; Zhang, Hongyan

    2016-05-15

    To study the natural variation and spatio-temporal accumulation of citrus metabolites, liquid chromatography tandem mass spectrometry (LC-MS) based metabolome analysis was performed on four fruit tissues (flavedo, albedo, segment membrane and juice sacs) and different Citrus species (lemon, pummelo and grapefruit, sweet orange and mandarin). Using a non-targeted metabolomics approach, more than 2000 metabolite signals were detected, from which more than 54 metabolites, including amino acids, flavonoids and limonoids, were identified/annotated. Differential accumulation patterns of both primary metabolites and secondary metabolites in various tissues and species were revealed by our study. Further investigation indicated that flavedo accumulates more flavonoids while juice sacs contain more amino acids. Besides this, cluster analysis based on the levels of metabolites detected in 47 individual Citrus accessions clearly grouped them into four distinct clusters: pummelos and grapefruits, lemons, sweet oranges and mandarins, while the cluster of pummelos and grapefruits lay distinctly apart from the other three species.

  6. Rethinking cycad metabolite research.

    PubMed

    Snyder, Laura R; Marler, Thomas E

    2011-01-01

    Cycads are among the most ancient of extant Spermatophytes, and are known for their numerous pharmacologically active compounds. One compound in particular, β-methylamino-L-alanine (BMAA), has been implicated as the cause of amyotrophic lateral sclerosis/Parkinson dementia complex (ALS/PDC) on Guam. Previous studies allege that BMAA is produced exclusively by cyanobacteria, and is transferred to cycads through the symbiotic relationship between these cyanobacteria and the roots of cycads. We recently published data showing that Cycas micronesica seedlings grown without endophytic cyanobacteria do in fact increase in BMAA, invalidating the foundation of the BMAA hypothesis. We use this example to suggest that the frenzy centered on BMAA and other single putative toxins has hindered progress. The long list of cycad-specific compounds may have important roles in signaling or communication, but these possibilities have been neglected during decades of attempts to force single metabolites into a supposed anti-herbivory function. We propose that an unbiased, comprehensive approach may be a more appropriate means of proceeding with cycad biochemistry research.

  7. Synthesis Of Labeled Metabolites

    DOEpatents

    Martinez, Rodolfo A.; Silks, III, Louis A.; Unkefer, Clifford J.; Atcher, Robert

    2004-03-23

    The present invention is directed to labeled compounds, for example, isotopically enriched mustard gas metabolites including: [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1-[[2-(methylsulfinyl)ethyl]sulfonyl]-2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylsulfinyl)]; and, 2,2'-sulfinylbis([1,2-.sup.13 C.sub.2 ]ethanol of the general formula ##STR1## where Q.sup.1 is selected from the group consisting of sulfide (--S--), sulfone (--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), at least one C* is .sup.13 C, X is selected from the group consisting of hydrogen and deuterium, and Z is selected from the group consisting of hydroxide (--OH), and --Q.sup.2 --R where Q.sup.2 is selected from the group consisting of sulfide (--S--), sulfone(--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), and R is selected from the group consisting of hydrogen, a C.sub.1 to C.sub.4 lower alkyl, and amino acid moieties, with the proviso that when Z is a hydroxide and Q.sup.1 is a sulfide, then at least one X is deuterium.

  8. Cytochrome c Adducts with PCB Quinoid Metabolites

    PubMed Central

    Li, Miao; Teesch, Lynn M.; Murry, Daryl J.; Pope, R. Marshal; Li, Yalan; Robertson, Larry W.; Ludewig, Gabriele

    2015-01-01

    PCBs are a group of 209 individual congeners widely used as industrial chemicals. PCBs are found as by-products in dye and paint manufacture and are legacy, ubiquitous and persistent as human and environmental contaminants. PCBs with fewer chlorine atoms may be metabolized to hydroxy- and dihydroxy- metabolites and further oxidized to quinoid metabolites both in vitro and in vivo. Specifically, quinoid metabolites may form adducts on nucleophilic sites within cells. We hypothesized that the PCB-quinones covalently bind to cytochrome c and thereby cause defects in the function of cytochrome c. In this study synthetic PCB quinones (2-(4’-chlorophenyl)-1,4-benzoquinone, 2-(3’, 5’-dichlorophenyl)-1,4-benzoquinone, 2-(3’,4’, 5’-trichlorophenyl)-1,4-benzoquinone, and 2-(4’-chlorophenyl)-3,6-dichloro-1,4-benzoquinone) were incubated with cytochrome c, and adducts were detected by LC-MS and MALDI TOF. SDS PAGE gel electrophoresis was employed to separate the adducted proteins, while trypsin digestion and LC-MS/MS were applied to identify the amino acid binding sites on cytochrome c. Conformation change of cytochrome c after binding with PCB3-para-quinone was investigated by SYBYL-X simulation and cytochrome c function was examined. We found that more than one molecule of PCB-quinone may bind to one molecule of cytochrome c. Lysine and glutamic acid were identified as the predominant binding sites. Software simulation showed conformation changes of adducted cytochrome c. Additionally, cross-linking of cytochrome c was observed on the SDS PAGE gel. Cytochrome c was found to be in the reduced form after incubation with PCB quinones. These data provide evidence that the covalent binding of PCB quinone metabolites to cytochrome c may be included among the toxic effects of PCBs. PMID:26062463

  9. The metabolite profiling of coastal coccolithophorid species Pleurochrysis carterae (Haptophyta)

    NASA Astrophysics Data System (ADS)

    Zhou, Chengxu; Luo, Jie; Ye, Yangfang; Yan, Xiaojun; Liu, Baoning; Wen, Xin

    2016-07-01

    Pleurochrysis carterae is a calcified coccolithophorid species that usually blooms in the coastal area and causes aquaculture losses. The cellular calcification, blooming and many other critical species specific eco-physiological processes are closely related to various metabolic pathways. The purpose of this study is to apply the unbiased and non-destructive method of nuclear magnetic resonance (NMR) to detect the unknown holistic metabolite of P. carterae. The results show that NMR spectroscopic method is practical in the analysis of metabolites of phytoplankton. The metabolome of P. carterae was dominated by 26 metabolites involved in a number of different primary and secondary metabolic pathways. Organic acids and their derivatives, amino acids, sugars, nucleic aides were mainly detected. The abundant metabolites are that closely related to the process of cellular osmotic adjustment, which possibly reflect the active ability of P. carterae to adapt to the versatile coastal niche. DMSP (dimethylsulphoniopropionate) was the most dominant metabolite in P. carterae, up to 2.065±0.278 mg/g lyophilized cells, followed by glutamate and lactose, the contents were 0.349±0.035 and 0.301±0.073 mg/g lyophilized cells respectively. Other metabolites that had the content ranged between 0.1-0.2 mg/g lyophilized cells were alanine, isethionate and arabinose. Amino acid (valine, phenylalanine, isoleucine, tyrosine), organic acid salts (lactate, succinate), scyllitol and uracil had content ranged from 0.01 to below 0.1 mg/g lyophilized cells. Trigonelline, fumarate and formate were detected in very low content (only thousandths of 1 mg per gram of lyophilized cells or below). Our results of the holistic metabolites of P. carterae are the basic references for the further studies when multiple problems will be addressed to this notorious blooming calcifying species.

  10. TNT metabolites in animal tissues

    SciTech Connect

    Shugart, L.R.

    1990-01-01

    The overall objectives of this project are: to provide quantitative analytical procedures for the analysis of TNT and at least eight of its metabolites in animal tissues; and to obtain representative samples of tissues from animals from designated Army sites, and to determine the presence or absence of TNT and its metabolites in these samples. The study is divided into two Phases corresponding to the stated overall objectives of the project. 5 figs., 4 tabs.

  11. Development of a new high-performance liquid chromatography method with diode array and electrospray ionization-mass spectrometry detection for the metabolite fingerprinting of bioactive compounds in Humulus lupulus L.

    PubMed

    Prencipe, Francesco Pio; Brighenti, Virginia; Rodolfi, Margherita; Mongelli, Andrea; dall'Asta, Chiara; Ganino, Tommaso; Bruni, Renato; Pellati, Federica

    2014-07-04

    The study was aimed at developing a new analytical method for the metabolite fingerprinting of bioactive compounds in Humulus lupulus L. (hop), together with a simple extraction procedure. Different extraction techniques, including maceration, heat reflux extraction (HRE), ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE), were compared in order to obtain a high yield of the target analytes. Dynamic maceration for 30min with MeOH-HCOOH (99:1, v/v) as the extraction solvent provided the best result in terms of recovery of secondary metabolites. The analysis of hop constituents, including prenylflavonoids and prenylphloroglucinols (bitter acids), was carried out by means of HPLC-UV/DAD, HPLC-ESI-MS and MS(2), using an ion trap mass analyzer. An Ascentis Express C18 column (150mm×3.0mm I.D., 2.7μm) was used for the HPLC analysis, with a mobile phase composed of 0.25% formic acid in both water and acetonitrile, under gradient elution. The method validation was performed to show compliance with ICH guidelines. The validated technique was successfully applied to the phytochemical analysis of ten commercial cultivars and twenty-three wild Italian hop genotypes, thus demonstrating to be a reliable and useful tool for the comprehensive multi-component analysis of hop secondary metabolites.

  12. Production of Secondary Metabolites in Extreme Environments: Food- and Airborne Wallemia spp. Produce Toxic Metabolites at Hypersaline Conditions

    PubMed Central

    Frisvad, Jens C.; Kocev, Dragi; Džeroski, Sašo; Gunde-Cimerman, Nina

    2016-01-01

    The food- and airborne fungal genus Wallemia comprises seven xerophilic and halophilic species: W. sebi, W. mellicola, W. canadensis, W. tropicalis, W. muriae, W. hederae and W. ichthyophaga. All listed species are adapted to low water activity and can contaminate food preserved with high amounts of salt or sugar. In relation to food safety, the effect of high salt and sugar concentrations on the production of secondary metabolites by this toxigenic fungus was investigated. The secondary metabolite profiles of 30 strains of the listed species were examined using general growth media, known to support the production of secondary metabolites, supplemented with different concentrations of NaCl, glucose and MgCl2. In more than two hundred extracts approximately one hundred different compounds were detected using high-performance liquid chromatography-diode array detection (HPLC-DAD). Although the genome data analysis of W. mellicola (previously W. sebi sensu lato) and W. ichthyophaga revealed a low number of secondary metabolites clusters, a substantial number of secondary metabolites were detected at different conditions. Machine learning analysis of the obtained dataset showed that NaCl has higher influence on the production of secondary metabolites than other tested solutes. Mass spectrometric analysis of selected extracts revealed that NaCl in the medium affects the production of some compounds with substantial biological activities (wallimidione, walleminol, walleminone, UCA 1064-A and UCA 1064-B). In particular an increase in NaCl concentration from 5% to 15% in the growth media increased the production of the toxic metabolites wallimidione, walleminol and walleminone. PMID:28036382

  13. Identification of flurochloridone metabolites in rat urine using liquid chromatography/high resolution mass spectrometry.

    PubMed

    Lu, Dasheng; Zhang, Suhui; Wang, Dongli; Feng, Chao; Liu, Shihong; Jin, Yu 'e; Xu, Qian; Lin, Yuanjie; Wu, Chunhua; Tang, Liming; She, Jianwen; Wang, Guoquan; Zhou, Zhijun

    2016-05-06

    It is of great interest to develop strategic methods to enable chemicals' metabolites to be accurately and rapidly screened and identified. To screen and identify a category of metabolites with distinct isotopic distribution, this study proposed a generic strategy using in silico metabolite prediction plus accurate-mass-based isotopic pattern recognition (AMBIPR) and library identification on the data acquired via the data dependent MS/MS scan of LC-Q Exactive Orbitrap mass spectrometry. The proposed method was evaluated by the analysis of flurochloridone (FLC) metabolites in rat urine sample collected from toxicity tests. Different from the traditional isotopic pattern recognition (IPR) approach, AMBIPR here was performed based on the potential metabolites predicted via in silico metabolite prediction tools. Thus, the AMBIPR treated FLC data was only associated with FLC metabolites, consequently not only avoiding great efforts made to remove FLC-unrelated information and reveal FLC metabolites, but also increasing the percent of positive hits. Among the FLC metabolite peaks screened using AMBIPR, 87% of them (corresponding 97 metabolites and 49 biotransformation) were successfully identified via multiple MS identification techniques packaged in an established FLC's metabolites library based on Mass Frontier. Noteworthy, 34 metabolites (89%) were identified without distinct naturally isotopic distribution. The universal strategic approach based on background subtraction (BS) and mass defect filtering (MDF) was used to evaluate the AMBIPR and no more false positive and negative metabolites were detected. Furthermore, our results revealed that AMBIPR is very effective, inherently sensitive and accurate, and is easily automated for the rapidly screening and profiling chemicals related metabolites.

  14. iMet: A Network-Based Computational Tool To Assist in the Annotation of Metabolites from Tandem Mass Spectra.

    PubMed

    Aguilar-Mogas, Antoni; Sales-Pardo, Marta; Navarro, Miriam; Guimerà, Roger; Yanes, Oscar

    2017-03-21

    Structural annotation of metabolites relies mainly on tandem mass spectrometry (MS/MS) analysis. However, approximately 90% of the known metabolites reported in metabolomic databases do not have annotated spectral data from standards. This situation has fostered the development of computational tools that predict fragmentation patterns in silico and compare these to experimental MS/MS spectra. However, because such methods require the molecular structure of the detected compound to be available for the algorithm, the identification of novel metabolites in organisms relevant for biotechnological and medical applications remains a challenge. Here, we present iMet, a computational tool that facilitates structural annotation of metabolites not described in databases. iMet uses MS/MS spectra and the exact mass of an unknown metabolite to identify metabolites in a reference database that are structurally similar to the unknown metabolite. The algorithm also suggests the chemical transformation that converts the known metabolites into the unknown one. As a proxy for the structural annotation of novel metabolites, we tested 148 metabolites following a leave-one-out cross-validation procedure or by using MS/MS spectra experimentally obtained in our laboratory. We show that for 89% of the 148 metabolites at least one of the top four matches identified by iMet enables the proper annotation of the unknown metabolites. To further validate iMet, we tested 31 metabolites proposed in the 2012-16 CASMI challenges. iMet is freely available at http://imet.seeslab.net .

  15. LC/MS/MS for identification of in vivo and in vitro metabolites of jatrorrhizine.

    PubMed

    Zhang, Yi; Wu, Wenhua; Han, Fengmei; Chen, Yong

    2008-12-01

    The in vivo and in vitro metabolism of jatrorrhizine has been investigated using a specific and sensitive LC/MS/MS method. In vivo samples including rat feces, urine and plasma collected separately after dosing healthy rats with jatrorrhizine (34 mg/kg) orally, along with in vitro samples prepared by incubating jatrorrhizine with rat intestinal flora and liver microsome, respectively, were purified using a C(18) solid-phase extraction cartridge. The purified samples were then separated with a reversed-phase C(18) column with methanol-formic acid aqueous solution (70:30, v/v, pH3.5) as mobile phase and detected by on-line MS/MS. The structural elucidation of the metabolites was performed by comparing their molecular weights and product ions with those of the parent drug. As a result, seven new metabolites were found in rat urine, 13 metabolites were detected in rat feces, 11 metabolites were detected in rat plasma, 17 metabolites were identified in intestinal flora incubation solution and nine metabolites were detected in liver microsome incubation solution. The main biotransformation reactions of jatrorrhizine were the hydroxylation reaction, the methylation reaction, the demethylation reaction and the dehydrogenation reaction of parent drug and its relative metabolites. All the results were reported for the first time, except for some of the metabolites in rat urine.

  16. Control of endophytic Frankia sporulation by Alnus nodule metabolites.

    PubMed

    Hay De-Bettignies, Anne-Emmanuelle; Boubakri, Hasna; Buonomo, Antoine; Rey, Marjolaine; Meiffren, Guillaume; Cotin-Galvan, Laetitia; Comte, Gilles; Herrera-Belaroussi, Aude

    2017-01-10

    A unique case of microbial symbiont capable of dormancy within its living host cells has been reported in actinorhizal symbioses: some Frankia strains, named Sp+, are able to sporulate inside plant cells, contrarily to Sp- strains. The presence of metabolically slowed down bacterial structures in host cells alters our understanding of symbiosis based on reciprocal benefits between both partners, and its impact on the symbiotic processes remains unknown. The present work reports a metabolomic study of Sp+ and Sp- nodules (from Alnus glutinosa), in order to highlight variabilities associated with in-planta sporulation. A total of 21 amino acids (AA), 44 sugars and organic acids (SOA), and 213 secondary metabolites (M) were detected using UV and mass spectrometric-based profiling. Little change was observed in primary metabolites, suggesting that in-planta sporulation would not strongly affect the primary functionalities of the symbiosis. One secondary metabolite (M27) was detected only in Sp+ nodules. It was identified as Gentisic acid 5-O-β-D-xylopyranoside, previously reported as involved in plant defenses against microbial pathogens. This metabolite significantly increased Frankia in-vitro sporulation, unlike another metabolite significantly more abundant in Sp- nodules (M168 = (5R)-1,7-bis-(3,4-) dihydroxyphenyl)-heptane-5-O-β-D-glucopyranoside). All these results suggest that the plant could play an important role in Frankia ability to sporulate in-planta, and allow us to discuss a possible sanction emitted by the host against less cooperative Sp+ symbionts.

  17. Nuclear Magnetic Resonance Identification of New Sulfonic Acid Metabolites of Chloroacetanilide Herbicides

    USGS Publications Warehouse

    Morton, M.D.; Walters, F.H.; Aga, D.S.; Thurman, E.M.; Larive, C.K.

    1997-01-01

    The detection of the sulfonic acid metabolites of the chloroacetanilide herbicides acetochlor, alachlor, butachlor, propachlor, and, more recently, metolachlor in surface and ground water suggests that a common mechanism for dechlorination exists via the glutathione conjugation pathway. The identification of these herbicides and their metabolites is important due to growing public awareness and concern about pesticide levels in drinking water. Although these herbicides are regulated, little is known about the fate of their metabolites in soil. The sulfonic acid metabolites were synthesized by reaction of the parent compounds with an excess of sodium sulfite. Acetochlor, alachlor, butachlor, metolachlor, and propachlor and their sulfonic acid metabolites were studied by nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. This paper provides a direct method for the preparation and characterization of these compounds that will be useful in the analysis and study of chloracetanilide herbicides and their metabolites.

  18. A Carbonyl Capture Approach for Profiling Oxidized Metabolites in Cell Extracts

    PubMed Central

    Mattingly, Stephanie J.; Xu, Tao; Nantz, Michael H.; Higashi, Richard M.; Fan, Teresa W.-M.

    2012-01-01

    Fourier-transform ion-cyclotron resonance mass spectrometry (FT-ICR-MS) detection of oxidized cellular metabolites is described using isotopologic, carbonyl-selective derivatizing agents that integrate aminooxy functionality for carbonyl capture, quaternary nitrogen for electrospray enhancement, and a hydrophobic domain for sample cleanup. These modular structural features enable rapid, sensitive analysis of complex mixtures of metabolite-derivatives by FT-ICR-MS via continuous nanoelectrospray infusion. Specifically, this approach can be used to globally assess levels of low abundance and labile aldehyde and ketone metabolites quantitatively and in high throughput manner. These metabolites are often key and unique indicators of various biochemical pathways and their perturbations. Analysis of lung adenocarcinoma A549 cells established a profile of carbonyl metabolites spanning multiple structural classes. We also demonstrate a procedure for metabolite quantification using pyruvate as a model analyte. PMID:23175637

  19. Identification of amygdalin and its major metabolites in rat urine by LC-MS/MS.

    PubMed

    Ge, B Y; Chen, H X; Han, F M; Chen, Y

    2007-10-01

    Amygdalin and its metabolites in rat urine were identified using liquid chromatography-electrospray ionization (ESI) tandem ion-trap mass spectrometry. The purified rat urine sample was separated using a reversed-phase C18 column with 10 mM sodium phosphate buffer (pH 3.1) containing 30% methanol as the mobile phase, amygdalin and its metabolites were detected by on-line mass detector in selected ion monitoring (SIM) mode. The identification of the metabolites and elucidation of their structure were performed by comparing the changes in molecular masses (DeltaM), retention times and MS(2) spectral patterns of metabolites with those of parent drug. At least seven metabolites and the parent drug were found in rat urine after i.v. injection of 100 mg/kg doses of amygdalin. Among them, six metabolites were reported for the first time.

  20. Determination of AM-2201 metabolites in urine and comparison with JWH-018 abuse.

    PubMed

    Jang, Moonhee; Yang, Wonkyung; Shin, Ilchung; Choi, Hyeyoung; Chang, Hyejin; Kim, Eunmi

    2014-03-01

    With respect to the continuous emergence of new synthetic cannabinoids on the market since 2008, evaluation of the metabolism of these compounds and the development of analytical methods for the detection of these drugs including their respective metabolites in biological fluids have become essential. Other than JWH-018 or JWH-073, AM-2201 is one of the frequently identified synthetic cannabinoids in Korea. Recently, in our laboratory, several JWH-018 metabolites have been detected in some urine samples obtained from subjects who were arrested for the possession of herbal mixtures containing only AM-2201 or from those who confessed AM-2201 abuse. In the present study, we identified major urinary metabolites of AM-2201 and several metabolites of JWH-018, i.e., N-5-hydroxylated and carboxylated metabolites from rats administered AM-2201 and found that the metabolic profile in rats was similar to those in human subjects in this study. Analytical results of the urine samples from suspects who had a considerable possibility of AM-2201 or JWH-018 intake were also compared to distinguish between AM-2201 and JWH-018 abuse. The presence of 6-indole hydroxylated metabolites of each drug and N-4-hydroxy metabolite of AM-2201 was found to contribute to the decisive differences in the metabolic patterns of the two drugs. In addition, the concentration ratio of the N-(5-hydroxypentyl) metabolite to the N-(4-hydroxypentyl) metabolite of JWH-018 may be used as a criterion to differentiate between AM-2201 and JWH-018 abuse.

  1. Determination of XLR-11 and its metabolites in hair by liquid chromatography-tandem mass spectrometry.

    PubMed

    Park, Meejung; Yeon, Seonghoon; Lee, Jaesin; In, Sangwhan

    2015-10-10

    Analysis of drugs in hair is often used as a routine method to obtain detailed information about drug ingestion. However, few studies have been conducted on disposition of synthetic cannabinoids including cyclopropylindoles (UR-144 and XLR-11) and their metabolites in hair. XLR-11 has been widely abused in South Korea recently. Identification of metabolites in hair can be an important proof of synthetic cannabinoids use because it can exclude the possibility of passive smoke exposure. In this study, we described a quantitative analytical method of XLR-11 and its metabolites (UR-144, UR-144 N-5-hydroxypentyl metabolite, UR-144 N-4-hydroxypentyl metabolite, UR-144 N-pentanoic acid metabolite and XLR-11 N-4-hydroxypentyl metabolite) in hair by liquid chromatography with ESI-MS/MS. The target analytes were extracted with methanol from washed and cut hair samples and the extracts were evaporated, filtered and analyzed by LC-MS/MS with electrospray ion source in positive-ionization mode. JWH-018-d9 and JWH-018 N-5-hydroxypentyl metabolite-d5 were used as internal standards. Chromatographic separation was completed within 15 min. No interferences were detected in 10 blank hair samples. In intra- and inter-assay precision and accuracy study, CV (%) and bias (%) were below 12. The limit of detection (LOD) was 0.1∼2 pg/mg and the limit of quantification (LOQ) was 0.2-2 pg/mg, respectively. The validation results proved that the method was selective, accurate and precise with acceptable linearity within calibration range. No significant variation was observed by different sources of matrices. This method was applied to hair samples from 14 individual suspects of XLR-11 use. In this result, XLR-11, UR-144, UR-144 N-5-hydroxypentyl metabolite and UR-144 N-pentanoic acid metabolite, XLR-11 N-4-hydroxypentyl metabolite were detected. The concentration of XLR-11 as a parent drug was much higher than other metabolites. UR-144 N-5-hydroxy metabolite and UR-144 N-pentanoic acid

  2. Mining Bacterial Genomes for Secondary Metabolite Gene Clusters.

    PubMed

    Adamek, Martina; Spohn, Marius; Stegmann, Evi; Ziemert, Nadine

    2017-01-01

    With the emergence of bacterial resistance against frequently used antibiotics, novel antibacterial compounds are urgently needed. Traditional bioactivity-guided drug discovery strategies involve laborious screening efforts and display high rediscovery rates. With the progress in next generation sequencing methods and the knowledge that the majority of antibiotics in clinical use are produced as secondary metabolites by bacteria, mining bacterial genomes for secondary metabolites with antimicrobial activity is a promising approach, which can guide a more time and cost-effective identification of novel compounds. However, what sounds easy to accomplish, comes with several challenges. To date, several tools for the prediction of secondary metabolite gene clusters are available, some of which are based on the detection of signature genes, while others are searching for specific patterns in gene content or regulation.Apart from the mere identification of gene clusters, several other factors such as determining cluster boundaries and assessing the novelty of the detected cluster are important. For this purpose, comparison of the predicted secondary metabolite genes with different cluster and compound databases is necessary. Furthermore, it is advisable to classify detected clusters into gene cluster families. So far, there is no standardized procedure for genome mining; however, different approaches to overcome all of these challenges exist and are addressed in this chapter. We give practical guidance on the workflow for secondary metabolite gene cluster identification, which includes the determination of gene cluster boundaries, addresses problems occurring with the use of draft genomes, and gives an outlook on the different methods for gene cluster classification. Based on comprehensible examples a protocol is set, which should enable the readers to mine their own genome data for interesting secondary metabolites.

  3. High-resolution mass spectrometry elucidates metabonate (false metabolite) formation from alkylamine drugs during in vitro metabolite profiling.

    PubMed

    Barbara, Joanna E; Kazmi, Faraz; Muranjan, Seema; Toren, Paul C; Parkinson, Andrew

    2012-10-01

    In vitro metabolite profiling and characterization experiments are widely employed in early drug development to support safety studies. Samples from incubations of investigational drugs with liver microsomes or hepatocytes are commonly analyzed by liquid chromatography/mass spectrometry for detection and structural elucidation of metabolites. Advanced mass spectrometers with accurate mass capabilities are becoming increasingly popular for characterization of drugs and metabolites, spurring changes in the routine workflows applied. In the present study, using a generic full-scan high-resolution data acquisition approach with a time-of-flight mass spectrometer combined with postacquisition data mining, we detected and characterized metabonates (false metabolites) in microsomal incubations of several alkylamine drugs. If a targeted approach to mass spectrometric detection (without full-scan acquisition and appropriate data mining) were employed, the metabonates may not have been detected, hence their formation underappreciated. In the absence of accurate mass data, the metabonate formation would have been incorrectly characterized because the detected metabonates manifested as direct cyanide-trapped conjugates or as cyanide-trapped metabolites formed from the parent drugs by the addition of 14 Da, the mass shift commonly associated with oxidation to yield a carbonyl. This study demonstrates that high-resolution mass spectrometry and the associated workflow is very useful for the detection and characterization of unpredicted sample components and that accurate mass data were critical to assignment of the correct metabonate structures. In addition, for drugs containing an alkylamine moiety, the results suggest that multiple negative controls and chemical trapping agents may be necessary to correctly interpret the results of in vitro experiments.

  4. Mitochondrial metabolites: undercover signalling molecules

    PubMed Central

    2017-01-01

    Mitochondria are one of most characterized metabolic hubs of the cell. Here, crucial biochemical reactions occur and most of the cellular adenosine triphosphate (ATP) is produced. In addition, mitochondria act as signalling platforms and communicate with the rest of the cell by modulating calcium fluxes, by producing free radicals, and by releasing bioactive proteins. It is emerging that mitochondrial metabolites can also act as second messengers and can elicit profound (epi)genetic changes. This review describes the many signalling functions of mitochondrial metabolites under normal and stress conditions, focusing on metabolites of the tricarboxylic acid cycle. We provide a new framework for understanding the role of mitochondrial metabolism in cellular pathophysiology. PMID:28382199

  5. Toxicological significance of dihydrodiol metabolites

    SciTech Connect

    Hsia, M.T.

    1982-01-01

    Dihydrodiols are often found as the major organic-extractable metabolites of various olefinic or aromatic xenobiotics in many biological samples. Studies on the chemistry of dihydrodiol metabolites have provided insight into the pharmacokinetic behavior and the mode of action of the parent compound. The toxicology of dihydrodiol is more complex than what can be deduced solely on the basis of diminished bioavailability of the epoxide precursor, and the increased hydrophilicity associated with the dihydrodiol moiety. Dihydrodiols can be intrinsically toxic and may even represent metabolically activated species. Some of the dihydrodiol metabolites may still retain sufficient lipophilic character to serve again as substrates for microsomal oxygenases. Because of the tremendous chemical and biological diversity that existed among the various dihydrodiols, more mechanistic studies are needed to examine the toxicological properties of these compounds. It may be premature to conclude dihydrodiol formation as purely a detoxification route for xenobioties.

  6. Pesticide Urinary Metabolite Levels of Children in Eastern North Carolina Farmworker Households

    PubMed Central

    Arcury, Thomas A.; Grzywacz, Joseph G.; Barr, Dana B.; Tapia, Janeth; Chen, Haiying; Quandt, Sara A.

    2007-01-01

    Background In this investigation we documented the pesticide urinary metabolite levels of farmworker children in North Carolina, determined the number of different metabolites detected for each child, and delineated risk factors associated with the number of metabolites. Methods Urine samples were collected from 60 Latino farmworker children 1–6 years of age (34 female, 26 male). Interviews were completed by their mothers in Spanish. We analyzed urine samples for 14 pesticide metabolites, including the organophosphate pesticides chlorpyrifos, coumaphos, diazinon, isazaphos, malathion, pirimiphos, and parathion and its methyl counterpart; a common metabolite of at least 18 pyrethroid insecticides; the repellent DEET; and the herbicides 2,4,5-trichlorphenoxyacetic acid, 2,4-dichlorophenoxyacetic acid, acetochlor, atrazine, and metolachlor. Predictors included measures of paraoccupational, residential, and environmental exposure, child characteristics, and mother characteristics. Results Thirteen metabolites were present in the urine samples. Organophosphate pesticide metabolites were detected in a substantial proportion of children, particularly metabolites of parathion/methyl parathion (90.0%; geometric mean 1.00 μg/L), chlorpyrifos/chlorpyrifos methyl (83.3%; geometric mean 1.92 μg/L), and diazinon (55.0%; geometric mean 10.56 μg/L). The number of metabolites detected ranged from 0 to 7, with a mode of 4 detected (28.3%). Boys, children living in rented housing, and children with mothers working part-time had more metabolites detected. Conclusions Children in farmworker homes experience multiple sources of pesticide exposure. Pesticides may remain in their environments for long periods. Environmental and occupational health changes are needed to address these exposures. Research is needed with more precise measures of exposure and on the health effects of concurrent exposure to multiple pesticides. PMID:17687456

  7. Alterations of metabolic genes and metabolites in cancer.

    PubMed

    Oermann, Eric K; Wu, Jing; Guan, Kun-Liang; Xiong, Yue

    2012-06-01

    Altered metabolic regulation has long been observed in human cancer and broadly used in the clinic for tumor detection. Two recent findings--the direct regulation of metabolic enzymes by frequently mutated cancer genes and frequent mutations of several metabolic enzymes themselves in cancer--have renewed interest in cancer metabolism. Supporting a causative role of altered metabolic enzymes in tumorigenesis, abnormal levels of several metabolites have been found to play a direct role in cancer development. The alteration of metabolic genes and metabolites offer not only new biomarkers for diagnosis and prognosis, but also potential new targets for cancer therapy.

  8. Cocaine and metabolites by LC-MS/MS.

    PubMed

    Snozek, Christine L H; Bjergum, Matthew W; Langman, Loralie J

    2012-01-01

    Abuse of the stimulant cocaine (COC) is a common problem in the United States and elsewhere. The drug can be used either as the powder or as the free base (crack COC), and causes feelings of alertness and euphoria; both forms of COC are powerfully addictive. The assay described here is designed to detect and quantitate parent COC, its major metabolite benzoylecgonine, and a selection of metabolites that can provide specific information about sample validity (m-hydroxybenzoylecgonine), potential toxicity (norcocaine), route of administration (anhydroecgonine methyl ester), and co-utilization with ethanol (cocaethylene).

  9. Anticancer properties of Monascus metabolites.

    PubMed

    Yang, Tao; Liu, Junwen; Luo, Feijun; Lin, Qinlu; Rosol, Thomas J; Deng, Xiyun

    2014-08-01

    This review provides up-to-date information on the anticancer properties of Monascus-fermented products. Topics covered include clinical evidence for the anticancer potential of Monascus metabolites, bioactive Monascus components with anticancer potential, mechanisms of the anticancer effects of Monascus metabolites, and existing problems as well as future perspectives. With the advancement of related fields, the development of novel anticancer Monascus food products and/or pharmaceuticals will be possible with the ultimate goal of decreasing the incidence and mortality of malignancies in humans.

  10. Microbial production of primary metabolites

    NASA Astrophysics Data System (ADS)

    Demain, Arnold L.

    1980-12-01

    Microbial production of primary metabolites contributes significantly to the quality of life. Through fermentation, microorganisms growing on inexpensive carbon sources can produce valuable products such as amino acids, nucleotides, organic acids, and vitamins which can be added to food to enhance its flavor or increase its nutritive value. The contribution of microorganisms will go well beyond the food industry with the renewed interest in solvent fermentations. Microorganisms have the potential to provide many petroleum-derived products as well as the ethanol necessary for liquid fuel. The role of primary metabolites and the microbes which produce them will certainly increase in importance.

  11. Multiresidue determination of pesticides and pesticide metabolites in soil

    SciTech Connect

    Mogadati, P.S.; Rosen, J.D.

    1995-12-31

    Methods for the multiresidue extraction, cleanup and GC/MS determination of 142 pesticides and pesticide metabolites in soil have been developed. The use of solid phase extraction cartridges makes it possible to clean up the soil sufficiently so that the equivalent of 40 mg. soil may be injected onto the GC capillary column without overloading or harming the column. Combining this clean-up method with chemical ionization ion trap detection allowed for very low limits of detection.

  12. Methodological considerations for measuring glucocorticoid metabolites in feathers

    PubMed Central

    Berk, Sara A.; McGettrick, Julie R.; Hansen, Warren K.; Breuner, Creagh W.

    2016-01-01

    In recent years, researchers have begun to use corticosteroid metabolites in feathers (fCORT) as a metric of stress physiology in birds. However, there remain substantial questions about how to measure fCORT most accurately. Notably, small samples contain artificially high amounts of fCORT per millimetre of feather (the small sample artefact). Furthermore, it appears that fCORT is correlated with circulating plasma corticosterone only when levels are artificially elevated by the use of corticosterone implants. Here, we used several approaches to address current methodological issues with the measurement of fCORT. First, we verified that the small sample artefact exists across species and feather types. Second, we attempted to correct for this effect by increasing the amount of methanol relative to the amount of feather during extraction. We consistently detected more fCORT per millimetre or per milligram of feather in small samples than in large samples even when we adjusted methanol:feather concentrations. We also used high-performance liquid chromatography to identify hormone metabolites present in feathers and measured the reactivity of these metabolites against the most commonly used antibody for measuring fCORT. We verified that our antibody is mainly identifying corticosterone (CORT) in feathers, but other metabolites have significant cross-reactivity. Lastly, we measured faecal glucocorticoid metabolites in house sparrows and correlated these measurements with corticosteroid metabolites deposited in concurrently grown feathers; we found no correlation between faecal glucocorticoid metabolites and fCORT. We suggest that researchers should be cautious in their interpretation of fCORT in wild birds and should seek alternative validation methods to examine species-specific relationships between environmental challenges and fCORT. PMID:27335650

  13. MetDIA: Targeted Metabolite Extraction of Multiplexed MS/MS Spectra Generated by Data-Independent Acquisition.

    PubMed

    Li, Hao; Cai, Yuping; Guo, Yuan; Chen, Fangfang; Zhu, Zheng-Jiang

    2016-09-06

    With recent advances in mass spectrometry, there is an increased interest in data-independent acquisition (DIA) techniques for metabolomics. With DIA technique, all metabolite ions are sequentially selected and isolated using a wide window to generate multiplexed MS/MS spectra. Therefore, DIA strategy enables a continuous and unbiased acquisition of all metabolites and increases the data dimensionality, but presents a challenge to data analysis due to the loss of the direct link between precursor ion and fragment ions. However, very few DIA data processing methods are developed for metabolomics application. Here, we developed a new DIA data analysis approach, namely, MetDIA, for targeted extraction of metabolites from multiplexed MS/MS spectra generated using DIA technique. MetDIA approach considers each metabolite in the spectral library as an analysis target. Ion chromatograms for each metabolite (both precursor ion and fragment ions) and MS(2) spectra are readily detected, extracted, and scored for metabolite identification, referred as metabolite-centric identification. A minimum metabolite-centric identification score responsible for 1% false positive rate of identification is determined as 0.8 using fully (13)C labeled biological extracts. Finally, the comparisons of our MetDIA method with data-dependent acquisition (DDA) method demonstrated that MetDIA could significantly detect more metabolites in biological samples, and is more accurate and sensitive for metabolite identifications. The MetDIA program and the metabolite spectral library is freely available on the Internet.

  14. Reactive Arrays of Colorimetric Sensors for Metabolite and Steroid Identification

    PubMed Central

    Batres, Gary; Jones, Talia; Johnke, Hannah; Wilson, Mark; Holmes, Andrea E.; Sikich, Sharmin

    2014-01-01

    The work described herein examines a rapid mix-and-measure method called DETECHIP suitable for screening of steroids and metabolites. The addition of steroids and metabolites to reactive arrays of colorimetric sensors generated characteristic color “fingerprints” that were used to identify the analyte. A color analysis tool was used to identify the analyte pool that now includes biologically relevant analytes. The mix-and-measure arrays allowed the detection of disease metabolites, orotic acid and argininosuccinic acid; and the steroids androsterone, 1,4-androstadiene, testosterone, stanozolol, and estrone. The steroid 1,4-androstadiene was also detected by this method while dissolved in synthetic urine. Some of the steroids, such as androstadiene, stanozolol, and androsterone were co-dissolved with (2-hydroxypropyl)-β-cyclodextrin in order to increase solubility in aqueous buffered solutions. The colorimetric arrays do not intend to eliminate ELISA or mass spectroscopy based screening, but to possibly provide an alternative analytical detection method for steroids and metabolites. PMID:25019034

  15. Pesticides in ground water: Do atrazine metabolites matter?

    USGS Publications Warehouse

    Liu, S.; Yen, S.T.; Kolpin, D.W.

    1996-01-01

    Atrazine and atrazine-residue (atrazine + two metabolites - deethylatrazine and deisopropylatrazine) concentrations were examined to determine if consideration of these atrazine metabolites substantially adds to our understanding of the distribution of this pesticide in groundwater of the midcontinental United States. The mean of atrazine.residue concentrations was 53 percent greater than that of atrazine alone for those observations above the detection limit (> 0.05 μg/l). Furthermore, a censored regression analysis using atrazine-residue concentrations revealed significant factors not identified when only atrazine concentrations were used. Thus, knowledge of concentrations of these atrazine metabolites is required to obtain a true estimation of risk of using these aquifers as sources for drinking water, and such knowledge also provides information that ultimately may be important for future management policies designed to reduce atrazine concentrations in ground water.

  16. Mass defect filter technique and its applications to drug metabolite identification by high-resolution mass spectrometry.

    PubMed

    Zhang, Haiying; Zhang, Donglu; Ray, Kenneth; Zhu, Mingshe

    2009-07-01

    Identification of drug metabolites by liquid chromatography/mass spectrometry (LC/MS) involves metabolite detection in biological matrixes and structural characterization based on product ion spectra. Traditionally, metabolite detection is accomplished primarily on the basis of predicted molecular masses or fragmentation patterns of metabolites using triple-quadrupole and ion trap mass spectrometers. Recently, a novel mass defect filter (MDF) technique has been developed, which enables high-resolution mass spectrometers to be utilized for detecting both predicted and unexpected drug metabolites based on narrow, well-defined mass defect ranges for these metabolites. This is a new approach that is completely different from, but complementary to, traditional molecular mass- or MS/MS fragmentation-based LC/MS approaches. This article reviews the mass defect patterns of various classes of drug metabolites and the basic principles of the MDF approach. Examples are given on the applications of the MDF technique to the detection of stable and chemically reactive metabolites in vitro and in vivo. Advantages, limitations, and future applications are also discussed on MDF and its combinations with other data mining techniques for the detection and identification of drug metabolites.

  17. Nontargeted Modification-Specific Metabolomics Investigation of Glycosylated Secondary Metabolites in Tea (Camellia sinensis L.) Based on Liquid Chromatography-High-Resolution Mass Spectrometry.

    PubMed

    Dai, Weidong; Tan, Junfeng; Lu, Meiling; Xie, Dongchao; Li, Pengliang; Lv, Haipeng; Zhu, Yin; Guo, Li; Zhang, Yue; Peng, Qunhua; Lin, Zhi

    2016-09-07

    Glycosylation on small molecular metabolites modulates a series of biological events in plants. However, a large number of glycosides have not been discovered and investigated using -omics approaches. Here, a general strategy named "nontargeted modification-specific metabolomics" was applied to map the glycosylation of metabolites. The key aspect of this method is to adopt in-source collision-induced dissociation to dissociate the glycosylated metabolite, causing a characteristic neutral loss pattern, which acts as an indicator for the glycosylation identification. In an exemplary application in green teas, 120 glucosylated/galactosylated, 38 rhamnosylated, 21 rutinosylated, and 23 primeverosylated metabolites were detected simultaneously. Among them, 61 glycosylated metabolites were putatively identified according to current tea metabolite databases. Thanks to the annotations of glycosyl moieties in advance, the method aids metabolite identifications. An additional 40 novel glycosylated metabolites were tentatively elucidated. This work provides a feasible strategy to discover and identify novel glycosylated metabolites in plants.

  18. CHARACTERIZATION OF METABOLITES IN SMALL FISH BIOFLUIDS AND TISSUES BY NMR SPECTROSCOPY

    EPA Science Inventory

    Nuclear magnetic resonance (NMR) spectroscopy has been utilized for assessing ecotoxicity in small fish models by means of metabolomics. Two fundamental challenges of NMR-based metabolomics are the detection limit and characterization of metabolites (or NMR resonance assignments...

  19. Quantification of 22 phthalate metabolites in human urine.

    PubMed

    Silva, Manori J; Samandar, Ella; Preau, James L; Reidy, John A; Needham, Larry L; Calafat, Antonia M

    2007-12-01

    Phthalates are ubiquitous industrial chemicals with high potential for human exposure. Validated analytical methods to measure trace concentrations of phthalate metabolites in humans are essential for assessing exposure to phthalates. Previously, we developed a sensitive and accurate automated analytical method for measuring up to 16 phthalate metabolites in human urine by using on-line solid phase extraction coupled with isotope dilution-high performance liquid chromatography (HPLC)-electrospray ionization-tandem mass spectrometry. To include the measurement of seven additional analytes, including oxidative metabolites of diisononyl and diisodecyl phthalates, two chemicals used extensively in numerous consumer products, we used a novel nontraditional HPLC solvent gradient program. With this approach, we achieved adequate resolution and sensitivity for all 22 analytes with limits of detection in the low ng/mL range, without increasing the analytical run time. The method also has high accuracy with automatic recovery correction, high precision, and excellent sample throughput with minimal matrix effects. Although it is possible to measure these 22 phthalate metabolites with adequate precision and accuracy at sub-parts-per-billion levels, additional information, including toxicokinetic data, is needed to demonstrate the usefulness of these phthalate metabolites for exposure assessment purposes.

  20. Natural products: Hunting microbial metabolites

    NASA Astrophysics Data System (ADS)

    Schmidt, Eric W.

    2015-05-01

    Symbiotic bacteria synthesize many specialized small molecules; however, establishing the role these chemicals play in human health and disease has been difficult. Now, the chemical structure and mechanism of the Escherichia coli product colibactin provides insight into the link between this secondary metabolite and colorectal cancer.

  1. Primary expectations of secondary metabolites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant secondary metabolites (e.g., phenolics) are important for human health, in addition to the organoleptic properties they impart to fresh and processed foods. Consumer expectations such as appearance, taste, or texture influence their purchasing decisions. Thorough identification of phenolic com...

  2. Primary expectations of secondary metabolites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    My program examines the plant secondary metabolites (i.e. phenolics) important for human health, and which impart the organoleptic properties that are quality indicators for fresh and processed foods. Consumer expectations such as appearance, taste, or texture influence their purchasing decisions; a...

  3. Quinones as toxic metabolites of benzene

    SciTech Connect

    Irons, R.D.

    1985-01-01

    Occupational exposure to benzene has long been associated with toxicity to the blood and bone marrow, including lymphocytopenia, pancytopenia, aplastic anemia, acute myelogenous leukemia, and possible lymphoma. A variety of studies have established that benzene itself is not the toxic species but requires metabolism to reactive intermediates. The bioactivation of benzene is complex. Both primary and secondary oxidation of benzene and its metabolites are mediated via cytochrome P-450 in the liver, although the role of secondary metabolism in the bone marrow is not clear. Toxicity is associated with the dihydroxy metabolites, hydroquinone and catechol, which concentrate in bone marrow. Hydroquinone and its terminal oxidation product, p-benzoquinone, have been demonstrated to be potent suppressors of cell growth in culture. Suppression of lymphocyte blastogenesis by these compounds is a sulfhydryl-dependent process and occurs at concentrations that do not result in cell death, or in detectable alterations in energy metabolism, intracellular glutathione concentration, or protein synthesis. Recent studies suggest that these compounds and other membrane-penetrating sulfyhdryl alkylating agents, such as N-ethylmaleimide and cytochalasin A, and endogenous regulatory molecules, such as soluble immune response suppressor (SIRS), interfere with microtubule assembly in vitro and selectively interfere with microtubule-dependent cell functions at identical concentrations. These agents appear to react with nucleophilic sulfhydryl groups essential for guanosine triphosphate binding to tubulin that are particularly sensitive to sulfhydryl-alkylating agents.

  4. Microbial secondary metabolites in homes in association with moisture damage and asthma.

    PubMed

    Kirjavainen, P V; Täubel, M; Karvonen, A M; Sulyok, M; Tiittanen, P; Krska, R; Hyvärinen, A; Pekkanen, J

    2016-06-01

    We aimed to characterize the presence of microbial secondary metabolites in homes and their association with moisture damage, mold, and asthma development. Living room floor dust was analyzed by LC-MS/MS for 333 secondary metabolites from 93 homes of 1-year-old children. Moisture damage was present in 15 living rooms. At 6 years, 8 children had active and 15 lifetime doctor-diagnosed asthma. The median number of different metabolites per house was 17 (range 8-29) and median sum load 65 (4-865) ng/m(2) . Overall 42 different metabolites were detected. The number of metabolites present tended to be higher in homes with mold odor or moisture damage. The higher sum loads and number of metabolites with loads over 10 ng/m(2) were associated with lower prevalence of active asthma at 6 years (aOR 0.06 (95% CI <0.001-0.96) and 0.05 (<0.001-0.56), respectively). None of the individual metabolites, which presence tended (P < 0.2) to be increased by moisture damage or mold, were associated with increased risk of asthma. Microbial secondary metabolites are ubiquitously present in home floor dust. Moisture damage and mold tend to increase their numbers and amount. There was no evidence indicating that the secondary metabolites determined would explain the association between moisture damage, mold, and the development of asthma.

  5. Excretion of berberine and its metabolites in oral administration in rats.

    PubMed

    Ma, Jing-Yi; Feng, Ru; Tan, Xiang-Shan; Ma, Chao; Shou, Jia-Wen; Fu, Jie; Huang, Min; He, Chi-Yu; Chen, Shuo-Nan; Zhao, Zhen-Xiong; He, Wen-Yi; Wang, Yan; Jiang, Jian-Dong

    2013-11-01

    Berberine (BBR) has been confirmed to show extensive bioactivities for the treatments of diabetes and hypercholesterolemia in clinic. However, there are few pharmacokinetic studies to elucidate the excretions of BBR and its metabolites. Our research studied the excretions of BBR and its metabolites in rats after oral administration (200 mg/kg). Metabolites in bile, urine, and feces were detected by liquid chromatography coupled to ion trap time-of-flight mass spectrometry; meanwhile, a validated liquid chromatography coupled with tandem mass spectrometry method was developed for their quantifications. Sixteen metabolites, including 10 Phase I and six Phase II metabolites were identified and clarified after dosing in vivo. Total recovered rate of BBR was 22.83% (19.07% of prototype and 3.76% of its metabolites) with 9.2 × 10(-6) % in bile (24 h), 0.0939% in urine (48 h), and 22.74% in feces (48 h), respectively. 83% of BBR was excreted as thalifendine (M1) from bile, whereas thalifendine (M1) and berberrubine (M2) were the major metabolites occupying 78% of urine excretion. Most of BBR and its metabolites were found in feces containing 84% of prototype. In summary, we provided excretion profiles of BBR and its metabolites after oral administration in rats in vivo.

  6. High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy. I. Development of direct injection/on-line guard cartridge extraction/tandem mass spectrometry for the simultaneous detection of CYP probe substrates and their metabolites.

    PubMed

    Bu, H Z; Magis, L; Knuth, K; Teitelbaum, P

    2000-01-01

    A highly efficient direct injection/on-line guard cartridge extraction/tandem mass spectrometry (DI-GCE/MS/MS) method utilizing electrospray polarity switching was developed for the simultaneous detection of probe substrates and marker metabolites of seven human hepatic cytochrome P450 (CYP) isozymes: CYP1A2, 2A6, 3A4, 2C9, 2C19, 2D6 and 2E1. Microsomal incubations were terminated with formic acid, centrifuged, and the resulting supernatants were injected for analysis by DI-GCE/MS/MS. This method employed an extremely short C(18) cartridge (4 mm in length) which allowed rapid cleanup of sample matrices while retaining the analytes an appropriate time (2. 0-2.2 min). From 1.5 to 2.7 min the effluent was directed to the mass spectrometer for detection otherwise diverted to waste. As a result of the efficient on-line extraction, matrix (e.g., salts and proteins) suppression was minimized. In addition, no visible source contamination was observed and system performance (chromatographic and mass spectrometric) did not significantly deteriorate after 500 consecutive injections. Electrospray polarity switching was strategically executed on a Micromass Quattro II mass spectrometer by establishing dummy ion transitions to protect the analytes from the interference of the overwhelming noise which was unavoidable for the first transition scanned following each polarity switch. This unique strategy led to the simultaneous detection of seven CYP probe substrates and seven corresponding marker metabolites (12 by positive mode and 2 by negative mode).

  7. Metabolite profiling of (14)C-omacetaxine mepesuccinate in plasma and excreta of cancer patients.

    PubMed

    Nijenhuis, Cynthia M; Lucas, Luc; Rosing, Hilde; Robertson, Philmore; Hellriegel, Edward T; Schellens, Jan H M; Beijnen, And Jos H

    2016-12-01

    Omacetaxine mepesuccinate (hereafter referred to as omacetaxine) is a protein translation inhibitor approved by the US Food and Drug Administration for adult patients with chronic myeloid leukemia with resistance and/or intolerance to two or more tyrosine kinase inhibitors. The objective was to investigate the metabolite profile of omacetaxine in plasma, urine and faeces samples collected up to 72 h after a single 1.25-mg/m(2) subcutaneous dose of (14)C-omacetaxine in cancer patients. High-performance liquid chromatography mass spectrometry (MS) (high resolution) in combination with off-line radioactivity detection was used for metabolite identification. In total, six metabolites of omacetaxine were detected. The reactions represented were mepesuccinate ester hydrolysis, methyl ester hydrolysis, pyrocatechol conversion from the 1,3-dioxole ring. Unchanged omacetaxine was the most prominent omacetaxine-related compound in plasma. In urine, unchanged omacetaxine was also dominant, together with 4'-DMHHT. In feces very little unchanged omacetaxine was found and the pyrocatechol metabolite of omacetaxine, M534 and 4'-desmethyl homoharringtonine (4'-DMHHT) was the most abundant metabolites. Omacetaxine was extensively metabolized, with subsequent renal and hepatic elimination of the metabolites. The low levels of the metabolites found in plasma indicate that the metabolites are unlikely to contribute materially to the efficacy and/or toxicity of omacetaxine.

  8. Extending metabolome coverage for untargeted metabolite profiling of adherent cultured hepatic cells.

    PubMed

    García-Cañaveras, Juan Carlos; López, Silvia; Castell, José Vicente; Donato, M Teresa; Lahoz, Agustín

    2016-02-01

    MS-based metabolite profiling of adherent mammalian cells comprises several challenging steps such as metabolism quenching, cell detachment, cell disruption, metabolome extraction, and metabolite measurement. In LC-MS, the final metabolome coverage is strongly determined by the separation technique and the MS conditions used. Human liver-derived cell line HepG2 was chosen as adherent mammalian cell model to evaluate the performance of several commonly used procedures in both sample processing and LC-MS analysis. In a first phase, metabolite extraction and sample analysis were optimized in a combined manner. To this end, the extraction abilities of five different solvents (or combinations) were assessed by comparing the number and the levels of the metabolites comprised in each extract. Three different chromatographic methods were selected for metabolites separation. A HILIC-based method which was set to specifically separate polar metabolites and two RP-based methods focused on lipidome and wide-ranging metabolite detection, respectively. With regard to metabolite measurement, a Q-ToF instrument operating in both ESI (+) and ESI (-) was used for unbiased extract analysis. Once metabolite extraction and analysis conditions were set up, the influence of cell harvesting on metabolome coverage was also evaluated. Therefore, different protocols for cell detachment (trypsinization or scraping) and metabolism quenching were compared. This study confirmed the inconvenience of trypsinization as a harvesting technique, and the importance of using complementary extraction solvents to extend metabolome coverage, minimizing interferences and maximizing detection, thanks to the use of dedicated analytical conditions through the combination of HILIC and RP separations. The proposed workflow allowed the detection of over 300 identified metabolites from highly polar compounds to a wide range of lipids.

  9. Fungal metabolites with anticancer activity.

    PubMed

    Evidente, Antonio; Kornienko, Alexander; Cimmino, Alessio; Andolfi, Anna; Lefranc, Florence; Mathieu, Véronique; Kiss, Robert

    2014-05-01

    Covering: 1964 to 2013. Natural products from bacteria and plants have played a leading role in cancer drug discovery resulting in a large number of clinically useful agents. In contrast, the investigations of fungal metabolites and their derivatives have not led to a clinical cancer drug in spite of significant research efforts revealing a large number of fungi-derived natural products with promising anticancer activity. Many of these natural products have displayed notable in vitro growth-inhibitory properties in human cancer cell lines and select compounds have been demonstrated to provide therapeutic benefits in mouse models of human cancer. Many of these compounds are expected to enter human clinical trials in the near future. The present review discusses the reported sources, structures and biochemical studies aimed at the elucidation of the anticancer potential of these promising fungal metabolites.

  10. Non-targeted analysis of spatial metabolite composition in strawberry (Fragariaxananassa) flowers.

    PubMed

    Hanhineva, Kati; Rogachev, Ilana; Kokko, Harri; Mintz-Oron, Shira; Venger, Ilya; Kärenlampi, Sirpa; Aharoni, Asaph

    2008-10-01

    Formation of flower organs and the subsequent pollination process require a coordinated spatial and temporal regulation of particular metabolic pathways. In this study a comparison has been made between the metabolite composition of individual flower organs of strawberry (Fragariaxananassa) including the petal, sepal, stamen, pistil and the receptacle that gives rise to the strawberry fruit. Non-targeted metabolomics analysis of the semi-polar secondary metabolites by the use of UPLC-qTOF-MS was utilized in order to localize metabolites belonging to various chemical classes (e.g. ellagitannins, proanthocyanidins, flavonols, terpenoids, and spermidine derivatives) to the different flower organs. The vast majority of the tentatively identified metabolites were ellagitannins that accumulated in all five parts of the flower. Several metabolite classes were detected predominantly in certain flower organs, as for example spermidine derivatives were present uniquely in the stamen and pistil, and the proanthocyanidins were almost exclusively detected in the receptacle and sepals. The latter organ was also rich in terpenoids (i.e. triterpenoid and sesquiterpenoid derivatives) whereas phenolic acids and flavonols were the predominant classes of compounds detected in the petals. Furthermore, we observed extensive variation in the accumulation of metabolites from the same class in a single organ, particularly in the case of ellagitannins, and the flavonols quercetin, kaempferol and isorhamnetin. These results allude to spatially-restricted production of secondary metabolite classes and specialized derivatives in flowers that take part in implementing the unique program of individual organs in the floral life cycle.

  11. Secondary Metabolites from Polar Organisms

    PubMed Central

    Tian, Yuan; Li, Yan-Ling; Zhao, Feng-Chun

    2017-01-01

    Polar organisms have been found to develop unique defences against the extreme environment environment, leading to the biosynthesis of novel molecules with diverse bioactivities. This review covers the 219 novel natural products described since 2001, from the Arctic and the Antarctic microoganisms, lichen, moss and marine faunas. The structures of the new compounds and details of the source organism, along with any relevant biological activities are presented. Where reported, synthetic and biosynthetic studies on the polar metabolites have also been included. PMID:28241505

  12. Tear metabolite changes in keratoconus

    PubMed Central

    Karamichos, D; Zieske, JD; Sejersen, H; Sarker-Nag, A; Asara, John M; Hjortdal, J

    2015-01-01

    While efforts have been made over the years, the exact cause of keratoconus (KC) remains unknown. The aim of this study was to identify alterations in endogenous metabolites in the tears of KC patients compared with age-matched healthy subjects. Three groups were tested: 1) Age-matched controls with no eye disease (N=15), 2) KC – patients wearing Rigid Gas permeable lenses (N=16), and 3) KC – No Correction (N=14). All samples were processed for metabolomics analysis using LC-MS/MS. We identified a total of 296 different metabolites of which >40 were significantly regulated between groups. Glycolysis and gluconeogenesis had significant changes, such as 3-phosphoglycerate and 1,3 diphopshateglycerate. As a result the citric acid cycle (TCA) was also affected with notable changes in Isocitrate, aconitate, malate, and acetylphosphate, up regulated in Group 2 and/or 3. Urea cycle was also affected, especially in Group 3 where ornithine and aspartate were up-regulated by at least 3 fold. The oxidation state was also severely affected. Groups 2 and 3 were under severe oxidative stress causing multiple metabolites to be regulated when compared to Group 1. Group 2 and 3, both showed significant down regulation in GSH-to-GSSG ratio when compared to Group 1. Another indicator of oxidative stress, the ratio of lactate – pyruvate was also affected with Groups 2 and 3 showing at least a 2-fold up regulation. Overall, our data indicate that levels of metabolites related to urea cycle, TCA cycle and oxidative stress are highly altered in KC patients. PMID:25579606

  13. Identification of in vivo and in vitro metabolites of 4,5-dimethoxycanthin-6-one by HPLC-Q-TOF-MS/MS.

    PubMed

    Miao, Xiaolei; Wang, Junjun; Chen, Liang; Peng, Zhihong; Chen, Yong

    2016-05-01

    4,5-Dimthexycanthin-6-one and 5-hydroxy-4-methoxycanthin-6-one are the main active ingredients of Picrasma quassioides, which is a widely used herbal medicine for the treatment of gastroenteritis, snakebite, infection and hypertension in China. In the present study, the in vitro metabolites of 4,5-dimethoxycanthin-6-one in rat, mouse, dog and human liver microsomes, as well as the in vivo metabolites in rat plasma and urine following a single oral dose of 4,5-dimethoxycanthin-6-one, were identified by high-performance liquid chromatography combined with triple TOF mass spectrometry (HPLC-TOF/MS/MS). The metabolites were elucidated based on an accurate mass measurement, the MS/MS fragmentation patterns, the retention times of the parent drug and its metabolites, and the relevant drug biotransformation rules. After incubation in liver mcrosomes for 50 min, 4,5-dimethoxycanthin-6-one produced 8 phase I metabolites including 2 mono-demethylated metabolites (M1, M2), 3 mono-hydroxylated metabolites (M3-M5), and 3 mono-demethylated and mono-hydroxylated metabolites (M6-M8) in rat and mouse liver microsomes, 7 phase I metabolites (without M7) in dog and human liver microsomes. After a single oral administration of 4,5-dimethoxycanthin-6-one to rats, there were 3 phase I metabolites (M1, M2 and M5) detected in rat plasma and 5 phase I metabolites (M1-M5) in rat urine. Phase II metabolites were not detected in rat plasma and urine. Among these metabolites, mono-demethylated metabolites (M1 and M2) were the major metabolites of 4,5-dimethoxycanthin-6-one, mono-hydroxylated metabolites (M3-M5) were the minor metabolites of 4,5-dimethoxycanthin-6-one.

  14. Compartmentalization and antiviral effect of efavirenz metabolites in blood plasma, seminal plasma, and cerebrospinal fluid.

    PubMed

    Avery, Lindsay B; VanAusdall, Jennifer L; Hendrix, Craig W; Bumpus, Namandjé N

    2013-02-01

    Efavirenz (EFV) is one of the most commonly prescribed antiretrovirals for use in the treatment of human immunodeficiency virus (HIV) infection. EFV is extensively metabolized by cytochrome P450 to a number of oxygenated products; however, the pharmacologic activity and distribution of these metabolites in anatomic compartments have yet to be explored. The systemic distribution of EFV oxidative metabolites was examined in blood plasma, seminal plasma, and cerebrospinal fluid from subjects on an EFV-based regimen. The 8-hydroxy EFV metabolite was detected in blood plasma, seminal plasma, and cerebrospinal fluid, with median concentrations of 314.5 ng/ml, 358.5 ng/ml, and 3.37 ng/ml, respectively. In contrast, 7-hydroxy and 8,14-hydroxy EFV were only detected in blood plasma and seminal plasma with median concentrations of 8.84 ng/ml and 10.23 ng/ml, and 5.63 ng/ml and 5.43 ng/ml, respectively. Interestingly, protein-free concentrations of metabolites were only detectable in seminal plasma, where a novel dihdyroxylated metabolite of EFV was also detected. This accumulation of protein-free EFV metabolites was demonstrated to be the result of differential protein binding in seminal plasma compared with that of blood plasma. In addition, the oxidative metabolites of EFV did not present with any significant pharmacologic activity toward HIV-1 as measured using an HIV green fluorescent protein single-round infectivity assay. This study is the first to report the physiologic distribution of metabolites of an antiretroviral into biologic compartments that the virus is known to distribute and to examine their anti-HIV activity. These data suggest that the male genital tract may be a novel compartment that should be considered in the evaluation of drug metabolite exposure.

  15. Tissue Distribution of trans-Resveratrol and Its Metabolites after Oral Administration in Human Eyes

    PubMed Central

    Weinreb, Robert N.

    2017-01-01

    Purpose. This study was performed to measure the concentration of trans-resveratrol and its three metabolites in human eyes. Methods. The patients who underwent pars plana vitrectomy for rhegmatogenous retinal detachment were included. The participants were orally given trans-resveratrol-based supplement (Longevinex®). A suitable amount of conjunctiva, aqueous humor, and vitreous humor were obtained during the operation. High-performance liquid chromatography (HPLC) with mass spectrometry (LC/MS/MS) was used to detect the concentration of trans-resveratrol and its three metabolites in the various samples. Results. The average concentration of resveratrol in the conjunctiva was 17.19 ± 15.32 nmol/g (mean ± SD). The concentration of resveratrol in the aqueous humor was close to the limit of detection, but its metabolites could be quantified. The concentrations of resveratrol metabolites in the aqueous humor can be detected. In the vitreous humor, the average concentration of resveratrol-3-O-sulfate was 62.95 ± 41.97 nmol/L. The sulfate conjugations of resveratrol were recovered in the conjunctiva, aqueous humor, and vitreous humor. Conclusions. Resveratrol and its three metabolites can be detected in the ocular tissues after oral administration. Although the concentration of parent resveratrol was low in the eyes, its metabolites could be detected and may have a role in the treatment of ocular diseases.

  16. Different profiles of quercetin metabolites in rat plasma: comparison of two administration methods.

    PubMed

    Kawai, Yoshichika; Saito, Satomi; Nishikawa, Tomomi; Ishisaka, Akari; Murota, Kaeko; Terao, Junji

    2009-03-23

    The bioavailability of polyphenols in human and rodents has been discussed regarding their biological activity. We found different metabolite profiles of quercetin in rat plasma between two administration procedures. A single intragastric administration (50 mg/kg) resulted in the appearance of a variety of metabolites in the plasma, whereas only a major fraction was detected by free access (1% quercetin). The methylated/non-methylated metabolites ratio was much higher in the free access group. Mass spectrometric analyses showed that the fraction from free access contained highly conjugated quercetin metabolites such as sulfo-glucuronides of quercetin and methylquercetin. The metabolite profile of human plasma after an intake of onion was similar to that with intragastric administration in rats. In vitro oxidation of human low-density lipoprotein showed that methylation of the catechol moiety of quercetin significantly attenuated the antioxidative activity. These results might provide information about the bioavailability of quercetin when conducting animal experiments.

  17. Characterization of oxygenated metabolites of ginsenoside Rg1 in plasma and urine of rat.

    PubMed

    Wang, Jing-Rong; Tong, Tian-Tian; Yau, Lee-Fong; Chen, Cheng-Yu; Bai, Li-Ping; Ma, Jing; Hu, Ming; Liu, Liang; Jiang, Zhi-Hong

    2016-07-15

    This study describes the characterization of oxygenated metabolites of ginsenoside Rg1 in rat urine and plasma. These in vivo metabolites were profiled by using UHPLC-QTOF MS-based method. On the basis of high-resolution MS/MS data, and comparison with chemically synthesized authentic compounds, nine oxygenated metabolites of Rg1 were characterized as vinaginsenosides 21 and 22 (M1 and M2), vinaginsenoside R15 (M3), 6-O-(β-d-glucopyranosyl)-20-O-(β-d-glucopyranosyl) 3β, 6α, 12β, 20(S)-tetrahydroxy-24ξ-hydroxydammar-25-ene (M4 and M5), floralginsenoside A (M7 and M8), floralginsenoside B (M9) and epoxyginsenoside Rg1 (M13), respectively. Among these metabolites, M4, M5 and M13 are new ginsenosides and others were detected as in vivo metabolites of Rg1 for the first time. In addition, a series of oxygenated metabolites of Rh1 and deglycosylated metabolite of Rg1, were observed and characterized by comparing with compounds synthesized by us, which revealed an association between C-20 configuration and the extent of oxidation metabolism. Appearance of all these metabolites in blood stream and urine after i.v. dosing and oral administration of Rg1 was further examined, which clearly showed that mono-oxygenated metabolites of Rg1 were major circulating metabolites at the early stage after dosing. Characterization of exact chemical structures of these circulating metabolites contribute greatly to our understanding of chemical exposure after consumption of ginseng products, and provide valuable information for explaining multiple bioactivities of ginseng products.

  18. Anabolic, doping, and lifestyle drugs, and selected metabolites in wastewater--detection, quantification, and behaviour monitored by high-resolution MS and MS(n) before and after sewage treatment.

    PubMed

    Schröder, Horst Fr; Gebhardt, Wilhelm; Thevis, Mario

    2010-10-01

    Municipal wastewater has been examined for steroids, beta(2)-agonists, stimulants, diuretics, and phosphodiesterase type V inhibitors (PDE type V inhibitors), which are "dual-use-drugs" applied either as anabolic, doping, and lifestyle drugs or for treatment of diverse diseases. To identify their origin, fitness centre discharges under suspicion of being point sources and sewage-treatment plant feed and effluents were sampled and concentrations determined. Sensitive and selective methods for determination and quantification based on solid-phase extraction (SPE) followed by high-performance liquid chromatography-high resolution mass and tandem mass spectrometry (HPLC-(HR)MS and HPLC-MS-MS) were developed and established for analysis of these compounds in wastewater and to assess their effect on the environment. The methods developed enabled quantification at trace concentrations (limit of quantification (LOQ): 5 ng L(-1)). Of the steroids and stimulants under investigation, testosterone, methyltestosterone, and boldenone or ephedrine, amphetamine, and MDMA (3,4-methylendioxy-N-methylamphetamine) were observed at up to 5 microg L(-1) (ephedrine). Of the beta(2)-agonists salbutamol only, and of the diuretics furosemide and hydrochlorothiazide were confirmed in the extracts. Quite high concentrations of the PDE type V inhibitors sildenafil, tadalafil, and vardenafil and their metabolites were confirmed in fitness centre discharges (sildenafil: 1,945 ng L(-1)) whereas their concentrations in municipal wastewater did not exceed 35 ng L(-1). This study identified anabolic and doping drugs in wastewater for the first time. Results obtained from wastewater treatment plant effluents proved that these "dual-use-drugs", with the exception of hydrochlorothiazide, were mostly eliminated.

  19. Metabolite

    MedlinePlus

    Kumar V, Abbas AK, Aster JC. Cellular responses to stress and toxic insults: Adaptation, injury, and death. In: Kumar V, Abbas AK, Aster JC, eds. Robbins and Cotran Pathologic Basis of Disease . 9th ed. Philadelphia, PA: ...

  20. Associations of cord blood metabolites with early childhood obesity risk

    PubMed Central

    Isganaitis, Elvira; Rifas-Shiman, Sheryl L.; Oken, Emily; Dreyfuss, Jonathan; Gall, Walt; Gillman, Matthew W.; Patti, Mary-Elizabeth

    2015-01-01

    Background/Objective Rapid postnatal weight gain is a potentially modifiable risk factor for obesity and metabolic syndrome. To identify markers of rapid infancy weight gain and childhood obesity, we analyzed the metabolome in cord blood from infants differing in their postnatal weight trajectories. Methods We performed a nested case-control study within Project Viva, a longitudinal cohort of mothers and children. We selected cases (n=26) based on top quartile of change in weight-for-age 0-6 mo and BMI >85th percentile in mid-childhood (median 7.7 years). Controls (n=26) were age- and sex-matched, had normal postnatal weight gain (2nd or 3rd quartile of change in weight-for-age 0-6 mo) and normal mid-childhood weight (BMI 25th-75th percentile). Cord blood metabolites were measured using untargeted LC/MS; individual metabolites and pathways differing between cases vs. controls were compared in categorical analyses. We adjusted metabolites for maternal age, maternal BMI, and breastfeeding duration (linear regression), and assessed whether metabolites improved the ability to predict case-control status (logistic regression). Results Of 415 detected metabolites, 16 were altered in cases vs. controls (T-test, nominal P<0.05). 3 metabolites were related to tryptophan: serotonin, tryptophan betaine, and tryptophyl leucine (46%, 48% and 26% lower in cases, respectively, P<0.05). Mean levels of 2 methyl donors, dimethylglycine and N-acetylmethionine, were also lower in cases (18% and 16% respectively, P=0.01). Moreover, the glutamine:glutamate ratio was reduced by 33% (P<0.05) in cases. Levels of serotonin, tryptophyl leucine, and N-acetylmethionine remained significantly different after adjustment for maternal BMI, age, and breastfeeding. Adding metabolite levels to logistic regression models including only clinical covariates improved the ability to predict case vs. control status. Conclusions Several cord blood metabolites are associated with rapid postnatal weight gain

  1. Metabolites of tamoxifen in animals and man: identification, pharmacology, and significance.

    PubMed

    Jordan, V C

    1982-01-01

    Over the past decade, the non-steroidal antiestrogen tamoxifen has gained general acceptance for the palliative treatment of breast cancer. Although there has been much interest in the pharmacology of tamoxifen, our knowledge of its metabolism in laboratory animals and patients is incomplete and the precise mechanism of action within target tissue and breast tumor cells is unknown. This review briefly describes the pharmacology of tamoxifen in various laboratory species and patients. Several metabolites of tamoxifen are known and their relative potencies as estrogens and antiestrogens are compared with the parent compound. Apart from monohydroxytamoxifen, none of tamoxifen's metabolites are more potent antiestrogens, but a metabolite in the dog, Metabolite E, is fully estrogenic. Routine assays (tlc, HPLC, glc/ms) are available to detect tamoxifen, N-desmethyltamoxifen, monohydroxytamoxifen, and a newly identified metabolite, designated Metabolite Y, in biological fluids. Continuous therapy with tamoxifen (10 mg bid) produces steady-state levels (100-200 ng/ml serum) within 4 weeks. Levels of N-desmethyltamoxifen are often up to twice the levels achieved with tamoxifen, while levels of monohydroxytamoxifen and Metabolite Y are below 10 ng/ml. Although monohydroxytamoxifen has a high binding affinity for the estrogen receptor, the metabolic activation of tamoxifen is an advantage rather than a requirement for antiestrogenic activity. The action of tamoxifen in vivo is the net result of the individual actions of the parent compound and its metabolites competing for the occupation of receptors within target tissues and tumors.

  2. Reactive metabolites in early drug development: predictive in vitro tools.

    PubMed

    Pelkonen, Olavi; Pasanen, Markku; Tolonen, Ari; Koskinen, Mikko; Hakkola, Jukka; Abass, Khaled; Laine, Jaana; Hakkinen, Merja; Juvonen, Risto; Auriola, Seppo; Storvik, Markus; Huuskonen, Pasi; Rousu, Timo; Rahikkala, Maiju

    2015-01-01

    Drug metabolism can result in the formation of highly reactive metabolites that are known to play a role in toxicity resulting in a significant proportion of attrition during drug development and clinical use. Thus, the earlier such reactivity was detected, the better. This review summarizes our multi-year project, together with pertinent literature, to examine a battery of in vitro tests capable of detecting the formation of reactive metabolites. Principal prerequisites for such tests were delineated: chemicals known/not known to cause tissue injury and produce reactive metabolites, activation system (mainly human-derived), small- and large-molecular targets (small-molecular trappers, peptides, proteins), analytical techniques (mass spectrometry), and cellular toxicity biomarkers. The current status of in vitro tools to detect reactive intermediates is the following: 1. Small-molecular trapping agents such glutathione or cyanide detect the production of reactive species with high sensitivity by proper MS technique. However, it seems that also putative "negatives" give rise to corresponding adducts. 2. Results from peptide and dG (DNA targeting) trapper studies are generally in line with those of small-molecular trappers, although also important differences exist. These two trapping platforms do not overlap. 3. It is anticipated that the in vitro adduct studies could be fully interpreted only in conjunction with toxicity biomarker (such as the Nrf2 pathway) information from whole cells or tissues. However, while there are tools to characterize the chemical liability and there are correlation between individual/integrated endpoints and toxicity, there are still severe gaps in understanding the mechanisms behind the link between reactive metabolites and adverse effects.

  3. Monitoring of propofol and its metabolite during total intravenous anesthesia

    NASA Astrophysics Data System (ADS)

    Elizarov, A. Yu.; Ershov, T. D.; Levshankov, A. I.

    2011-12-01

    Intravenous hypnotic propofol and its metabolite are detected in real time during total intravenous anesthesia by an electron ionization mass spectrometer. The mass spectrometer is connected directly to the breathing circuit of an apparatus for inhalational anesthesia. Ratios between the propofol concentrations in expired air and blood serum are measured. It is concluded that real-time noninvasive monitoring of the propofol concentration in blood using electron ionization mass spectrometry is feasible.

  4. Cyclic metabolites: chemical and biological considerations.

    PubMed

    Erve, John C L

    2008-02-01

    Metabolism of xenobiotics can sometimes generate cyclic metabolites. Such metabolites are usually the result of intramolecular reactions occurring within a primary or secondary metabolite and this chemistry may lead to unexpected structures. Intramolecular chemistry is often driven by nucleophilic groups reacting with electrophilic atoms, often carbon, although radical processes also occur. Conjugation of xenobiotics or their metabolites with endogenous thiols, such as glutathione or cysteine, introduce a reactive amino group that can lead to the formation of cyclic structures. Less common than chemically driven cyclizations are enzymatically mediated ring-closures, although this may reflect our incomplete recognition of enzymatic involvement in this step of cyclic metabolite formation. While some cyclic metabolites are biologically inactive, others are biologically active. Thus, a cyclic metabolite may display desirable pharmacology, or, contribute to toxicology. When a cyclic metabolite is identified, it is important to consider the possibility that it is an artifact, i.e. metabonate, that was formed during processing of the sample, for example, through degradation or by chemical reactions with other components present in the matrix. From a medicinal chemistry perspective, a cyclic metabolite with a different chemical scaffold from the parent structure may lead to a new series of structurally novel, biologically active molecules with the same, or different, pharmacology from the parent. This review will cover a selection of cyclic metabolites from a mechanistic point of view, and when possible, discuss their biological relevance.

  5. Seasonal Variation in the Measurement of Urinary Pesticide Metabolites among Latino Farmworkers in Eastern North Carolina

    PubMed Central

    Arcury, Thomas A.; Grzywacz, Joseph G.; Isom, Scott; Whalley, Lara E.; Vallejos, Quirina M.; Chen, Haiying; Galván, Leonardo; Barr, Dana B.; Quandt, Sara A.

    2009-01-01

    This analysis describes the detection of urinary pesticide metabolites for Latino farmworkers across the agricultural season. Two hundred and eighty four farmworkers were recruited from 44 camps in eastern North Carolina in 2007. Data were collected at one month intervals for a total of 939 data points. The OP insecticide metabolites 3,5,6-trichloropyridinol (46.2%), malathion dicarboxylic acid (27.7%), and para-nitrophenol (97.4%); the pyrethroid metabolite 3-phenoxybenzoic acid (56.4%); and the herbicides 2,4-D (68.1%), acetochlor (29.2%), and metolachlor (16.9%) were found in sizable percentages of the samples. The percentage of farmworkers for whom metabolites were detected varied across the agricultural season. None of the farmworker characteristics were significantly associated with the detection of any pesticide metabolite. Seasonality overrides the effects of other farmworker characteristics in predicting detection of pesticide urinary metabolites. Future research needs to collect multiple exposure measures at frequent intervals over an extended period to characterize factors associated with exposure. PMID:19886344

  6. [Development of analytical method for determination nicotine metabolites in urine].

    PubMed

    Piekoszewski, Wojciech; Florek, Ewa; Kulza, Maksymilian; Wilimowska, Jolanta; Loba, Urszula

    2009-01-01

    The assay of biomarkers in biological material is the most popular and reliable method in estimate exposure to tobacco smoke. Nicotine and its metabolites qualify to the most specific biomarkers for tobacco smoke. Currently the most often used are cotinine and trans-3'-hydroxycotinine. The aim of this study was development of easy and quick method of determining nicotine and its main metabolites with high performance liquid chromatography--available in most laboratories. Nicotine and its metabolites in urine (cotinine, trans-3'-hydroxycotinine, nornicotine and nicotine N-oxide) was determined by means of high performance liquid chromatography with spectrometry detection (HPLC-UV). The determined compounds were extracted from urine by means of the liquid-liquid technique, before analysed by the HPLC method. Developed technique of high performance liquid chromatography proved to be useful to assessment nicotine and its four metabolites in smokers, though further research are necessary. The further modification of procedure is required, because of the interferences of cotinine N-oxide with matrix, which prevent determination. Increasing the efficiency of extraction nicotine and nornicotine could enable the determination in people exposed on environmental tobacco smoke (ETS). This study confirm other authors' observations that 3'-hydroxycotinine might be equivalent with cotinine predictor of tobacco smoke exposure, however further studies are required.

  7. Characterization of a new rat urinary metabolite of piperine by LC/NMR/MS studies.

    PubMed

    Bajad, Sunil; Coumar, Mohane; Khajuria, Ravi; Suri, Om P; Bedi, Kasturi L

    2003-08-01

    Potential of piperine, an active alkaloid of black and long peppers, to increase the bioavailability of drugs in humans is of great clinical significance owing to its omnipresence in food. In an attempt to further study the reported differences in its metabolism in rats and humans, a new major urinary metabolite was detected in rat urine and plasma using HPLC. The metabolite was partially purified using reverse phase column chromatography on Sephadex((R))-LH 20 and characterized as 5-(3, 4-methylenedioxy phenyl)-2E,4E-pentadienoic acid-N-(3-yl propionic acid)-amide with the help of LC/NMR/positive ESI-MS studies. Complete mass fragmentation pattern could be assigned with MS/MS studies. The metabolite has a unique structure compared to the previously reported metabolites in that it retains methylenedioxy ring and conjugated double bonds while the piperidine ring is modified to form propionic acid group. Mechanism of formation of the metabolite by oxidation and cleavage of piperidine ring is proposed. Kidney appears to be the major excretion route for piperine metabolites in rats as no metabolite could be detected in feces.

  8. Phthalate metabolites in the European eel (Anguilla anguilla) from Mediterranean coastal lagoons.

    PubMed

    Fourgous, C; Chevreuil, M; Alliot, F; Amilhat, E; Faliex, E; Paris-Palacios, S; Teil, M J; Goutte, A

    2016-11-01

    The levels and fate of phthalate metabolites have been poorly evaluated in fish, despite their potential ecotoxicological impacts. The present study aims to characterize the levels of phthalate metabolites in muscle tissue of yellow eels (Anguilla anguilla) from two coastal Mediterranean lagoons, during three sampling periods. Nine phthalate metabolites were detected in >70% of the samples. Slightly higher levels of phthalate metabolites were detected in March and June compared to October, suggesting possible seasonal variations in environmental release and/or phthalate metabolization process by eels. The large sample size (N=117) made it possible to explore correlations between phthalate metabolites' levels and individual parameters, such as body length, age, body condition and hepatic histo-pathologies. Body length and estimated age poorly correlated with phthalate metabolites, suggesting that eels did not accumulate phthalates during growth, contrary to persistent compounds. Eels presented different grades of hepatic fibrosis and lipidosis. A negative correlation was found between the severity of these pathologies in the liver and the sum of phthalate metabolites levels, supporting the hypothesis that eels with damaged liver are less able to metabolize xenobiotics.

  9. Seed metabolomic study reveals significant metabolite variations and correlations among different soybean cultivars.

    PubMed

    Lin, Hong; Rao, Jun; Shi, Jianxin; Hu, Chaoyang; Cheng, Fang; Wilson, Zoe A; Zhang, Dabing; Quan, Sheng

    2014-09-01

    Soybean [Glycine max (L.) Merr.] is one of the world's major crops, and soybean seeds are a rich and important resource for proteins and oils. While "omics" studies, such as genomics, transcriptomics, and proteomics, have been widely applied in soybean molecular research, fewer metabolomic studies have been conducted for large-scale detection of low molecular weight metabolites, especially in soybean seeds. In this study, we investigated the seed metabolomes of 29 common soybean cultivars through combined gas chromatography-mass spectrometry and ultra-performance liquid chromatography-tandem mass spectrometry. One hundred sixty-nine named metabolites were identified and subsequently used to construct a metabolic network of mature soybean seed. Among the 169 detected metabolites, 104 were found to be significantly variable in their levels across tested cultivars. Metabolite markers that could be used to distinguish genetically related soybean cultivars were also identified, and metabolite-metabolite correlation analysis revealed some significant associations within the same or among different metabolite groups. Findings from this work may potentially provide the basis for further studies on both soybean seed metabolism and metabolic engineering to improve soybean seed quality and yield.

  10. Identification of novel circulating coffee metabolites in human plasma by liquid chromatography-mass spectrometry.

    PubMed

    Redeuil, Karine; Smarrito-Menozzi, Candice; Guy, Philippe; Rezzi, Serge; Dionisi, Fabiola; Williamson, Gary; Nagy, Kornél; Renouf, Mathieu

    2011-07-22

    This study reports a liquid chromatography-mass spectrometry method for the detection of polyphenol-derived metabolites in human plasma without enzymatic treatment after coffee consumption. Separation of available standards was achieved by reversed-phase ultra performance liquid chromatography and detection was performed by high resolution mass spectrometry in negative electrospray ionization mode. This analytical method was then applied for the identification and relative quantification of circulating coffee metabolites. A total of 34 coffee metabolites (mainly reduced, sulfated and methylated forms of caffeic acid, coumaric acid, caffeoylquinic acid and caffeoylquinic acid lactone) were identified based on mass accuracy (<4 ppm for most metabolites), specific fragmentation pattern and co-chromatography (when standard available). Among them, 19 circulating coffee metabolites were identified for the first time in human plasma such as feruloylquinic acid lactone, sulfated and glucuronidated forms of feruloylquinic acid lactone and sulfated forms of coumaric acid. Phenolic acid derivatives such as dihydroferulic acid, dihydroferulic acid 4'-O-sulfate, caffeic acid 3'-O-sulfate, dimethoxycinnamic acid, dihydrocaffeic acid and coumaric acid O-sulfate appeared to be the main metabolites circulating in human plasma after coffee consumption. The described method is a sensitive and reliable approach for the identification of coffee metabolites in biological fluids. In future, this analytical method will give more confidence in compound identification to provide a more comprehensive assessment of coffee polyphenol bioavailability studies in humans.

  11. Screening botanical extracts for quinoid metabolites.

    PubMed

    Johnson, B M; Bolton, J L; van Breemen, R B

    2001-11-01

    Botanical dietary supplements represent a significant share of the growing market for alternative medicine in the USA, where current regulations do not require assessment of their safety. To help ensure the safety of such products, an in vitro assay using pulsed ultrafiltration and LC-MS-MS has been developed to screen botanical extracts for the formation of electrophilic and potentially toxic quinoid species upon bioactivation by hepatic cytochromes P450. Rat liver microsomes were trapped in a flow-through chamber by an ultrafiltration membrane, and samples containing botanical extracts, GSH and NADP(H), were flow-injected into the chamber. Botanical compounds that were metabolized to reactive intermediates formed stable GSH adducts mimicking a common in vivo detoxification pathway. If present in the ultrafiltrate, GSH conjugates were detected using LC-MS-MS with precursor ion scanning followed by additional characterization using product ion scanning and comparison to standard compounds. As expected, no GSH adducts of reactive metabolites were found in extracts of Trifolium pratense L. (red clover), which are under investigation as botanical dietary supplements for the management of menopause. However, extracts of Sassafras albidum (Nutt.) Nees (sassafras), Symphytum officinale L. (comfrey), and Rosmarinus officinalis L. (rosemary), all of which are known to contain compounds that are either carcinogenic or toxic to mammals, produced GSH adducts during this screening assay. Several compounds that formed GSH conjugates including novel metabolites of rosmarinic acid were identified using database searching and additional LC-MS-MS studies. This assay should be useful as a preliminary toxicity screen during the development of botanical dietary supplements. A positive test suggests that additional toxicological studies are warranted before human consumption of a botanical product.

  12. Pharmacokinetics of chlorpromazine and key metabolites.

    PubMed

    Yeung, P K; Hubbard, J W; Korchinski, E D; Midha, K K

    1993-01-01

    A study was carried out in 11 healthy young men to investigate the pharmacokinetics of chlorpromazine (CPZ) after a bolus intravenous (i.v.) dose (10 mg) and three single oral doses (25, 50 and 100 mg), with a washout period of two weeks between doses. Plasma levels of CPZ, CPZ N-oxide (CPZNO), CPZ sulfoxide (CPZSO) and both free and conjugated 7-hydroxy-CPZ (7-HOCPZ) were measured by extraction radioimmunoassays. CPZ exhibited multicompartmental pharmacokinetics in most subjects. There was wide between-subject variability in half life (11.05 h), volume of distribution (1215 l), volume of distribution at steady state (642 l) and mean residence time (8.88 h), whereas systemic clearance was somewhat less variable (76.6 l.h-1). All metabolites were present in measurable concentrations in the plasma of 9 of 11 subjects after i.v. CPZ, whereas free 7-HOCPZ was not detected in the other 2 individuals. With the exception of CPZNO, the biological half lives of the primary metabolites were longer than the half life of CPZ. After oral administration, the percentage of CPZ reaching the systemic circulation intact (F%) was very low (4-38%) and dose dependent. Moreover, both within-subject and between-subject variances were very high. The maximum plasma concentration (Cmax) and area under the plasma concentration versus time curve extrapolated to infinite time (AUC) showed evidence of nonlinearity, whereas half life did not appear to be dose dependent. These data suggest that the high degree of variability in the pharmacokinetics of CPZ is a result of extensive first pass metabolism rather than variation in half life.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Exposure measurement of aflatoxins and aflatoxin metabolites in human body fluids. A short review.

    PubMed

    Leong, Yin-Hui; Latiff, Aishah A; Ahmad, Nurul Izzah; Rosma, Ahmad

    2012-05-01

    Aflatoxins are highly toxic secondary fungal metabolites mainly produced by Aspergillus flavus and A. parasiticus. Human exposure to aflatoxins may result directly from ingestion of contaminated foods, or indirectly from consumption of foods from animals previously exposed to aflatoxins in feeds. This paper focuses on exposure measurement of aflatoxins and aflatoxin metabolites in various human body fluids. Research on different metabolites present in blood, urine, breast milk, and other human fluids or tissues including their detection techniques is reviewed. The association between dietary intake of aflatoxins and biomarker measurement is also highlighted. Finally, aspects related to the differences between aflatoxin determination in food versus the biomarker approach are discussed.

  14. In Vivo and Real-time Monitoring of Secondary Metabolites of Living Organisms by Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Hu, Bin; Wang, Lei; Ye, Wen-Cai; Yao, Zhong-Ping

    2013-07-01

    Secondary metabolites are compounds that are important for the survival and propagation of animals and plants. Our current understanding on the roles and secretion mechanism of secondary metabolites is limited by the existing techniques that typically cannot provide transient and dynamic information about the metabolic processes. In this manuscript, by detecting venoms secreted by living scorpion and toad upon attack and variation of alkaloids in living Catharanthus roseus upon stimulation, which represent three different sampling methods for living organisms, we demonstrated that in vivo and real-time monitoring of secondary metabolites released from living animals and plants could be readily achieved by using field-induced direct ionization mass spectrometry.

  15. Measuring fecal testosterone metabolites in spotted hyenas: Choosing the wrong assay may lead to erroneous results.

    PubMed

    Pribbenow, Susanne; Shrivastav, Tulsidas G; Dehnhard, Martin

    2016-11-17

    Enzyme-immunoassays (EIA) that detect fecal testosterone metabolites (fTM) are powerful tools to monitor gonadal activity non-invasively. However, a challenge with testosterone EIAs might be their potential for cross-reactivities with structurally similar glucocorticoid metabolites. Therefore, we aimed to verify the capability of four different testosterone EIAs to monitor fTM without reflecting changes in adrenocortical activity in spotted hyenas by analyzing fecal samples following testosterone and ACTH challenge tests. We demonstrated that none of the testosterone EIAs is appropriate to measure fTM as all of them showed substantial cross-reactivities to unknown metabolites. Our study underlines the importance of validating androgen EIAs.

  16. Novel rapid liquid chromatography tandem masspectrometry method for vemurafenib and metabolites in human plasma, including metabolite concentrations at steady state.

    PubMed

    Vikingsson, Svante; Strömqvist, Malin; Svedberg, Anna; Hansson, Johan; Höiom, Veronica; Gréen, Henrik

    2016-08-01

    A novel, rapid and sensitive liquid chromatography tandem-mass spectrometry method for quantification of vemurafenib in human plasma, that also for the first time allows for metabolite semi-quantification, was developed and validated to support clinical trials and therapeutic drug monitoring. Vemurafenib was analysed by precipitation with methanol followed by a 1.9 min isocratic liquid chromatography tandem masspectrometry analysis using an Acquity BEH C18 column with methanol and formic acid using isotope labelled internal standards. Analytes were detected in multireaction monitoring mode on a Xevo TQ. Semi-quantification of vemurafenib metabolites was performed using the same analytical system and sample preparation with gradient elution. The vemurafenib method was successfully validated in the range 0.5-100 μg/mL according to international guidelines. The metabolite method was partially validated owing to the lack of commercially available reference materials. For the first time concentration levels at steady state for melanoma patients treated with vemurafenib is presented. The low abundance of vemurafenib metabolites suggests that they lack clinical significance. Copyright © 2016 John Wiley & Sons, Ltd.

  17. Qualitative detection of diuretics and acidic metabolites of other doping agents in human urine by high-performance liquid chromatography-tandem mass spectrometry: comparison between liquid-liquid extraction and direct injection.

    PubMed

    Deventer, K; Pozo, O J; Van Eenoo, P; Delbeke, F T

    2009-07-31

    Direct injection of urine has gained interest in the field of analytical toxicology, including doping control analysis. However, implementation of a direct urinalysis method for the LC-MS/MS detection of 34 diuretics and 9 other doping agents yielded several analytical problems, which were not observed using a traditional liquid-liquid extraction. Therefore a comparative study was made between liquid-liquid extraction and direct injection. Comparison of validation results showed that the liquid-liquid extraction at pH 7 allows to analyze samples without major drawbacks regarding matrix effects. Hence, good sensitivity was observed and detection limits ranged between 1 and 250 ng/mL for all compounds. In the direct injection approach shifted retention times were observed for several acidic and basic compounds due to unwanted matrix effects. This shift was reduced by a 25-fold dilution of the urine samples. Besides the improved retention time stability the diluted samples also exhibited lower ion suppression than the undiluted ones. After 25-fold dilution, detection limits ranged between 10 and 250 ng/mL for all compounds. Since these detection limits are at or below the minimum required performance level, imposed by the World Anti-Doping Agency, the method could be applied to routine anti-doping analysis. Samples, previously declared positive, were reanalysed using both the liquid-liquid extraction and direct injection. With both techniques all 26 samples were found to be positive, showing the applicability of direct injection for the analysis of diuretics.

  18. [The research of the heroin and its metabolites analysis in clinical samples].

    PubMed

    Lin, Lan; Liao, Lin-chuan; Yan, You-yi

    2003-01-01

    Heroin can be metabolized easily in body and the mail metabolites are 6-MAM, morphine and so on. At present, there are urine, blood, hair and so on as specimens for detection, while the analytical technology conclude TLC, GC, HPLC, GC/MS, LC/MS, IA, CE etc. In this paper, these technologies used for heroin's metabolites were viewed in order to provide some reference to the study in relative field.

  19. Intra- and Inter-Individual Variability of Urinary Phthalate Metabolite Concentrations in Hmong Women of Reproductive Age

    PubMed Central

    Peck, Jennifer David; Sweeney, Anne M; Symanski, Elaine; Gardiner, Joseph; Silva, Manori J.; Calafat, Antonia M.; Schantz, Susan L

    2010-01-01

    The reproducibility of urinary phthalate metabolite concentrations has not been well characterized in nonpregnant women of reproductive age. Our primary study objectives were to describe the distribution of urinary phthalate metabolites concentrations among a population of Hmong women of reproductive age, and to evaluate intra- and inter-individual variability of phthalate metabolite concentrations. Ten phthalate metabolites were measured in first morning urine samples collected from 45 women and 20 of their spouses who were members of the Fox River Environment and Diet Study cohort in Green Bay, Wisconsin. Repeated first morning urine samples were collected and analyzed from 25 women who provided up to three samples over approximately one month. Measurement variability was assessed using intraclass correlations (ICCs) and surrogate category analysis. Linear mixed models were used to evaluate the associations between participant characteristics and phthalate metabolite concentrations. Nine of the 10 phthalate metabolites were detected in > 80% of all samples analyzed, of which seven were detected in all samples. As a measure of reliability, ICCs were strongest for monobenzyl phthalate (0.64) and weakest for the metabolites of di(2-ethylhexyl)phthalate (DEHP) (ranging from 0.13 to 0.22). Similarly, surrogate category analysis suggested that a single urine sample characterized average one-month exposure with reasonable accuracy across low, medium and high tertiles for all metabolites except the DEHP metabolites. Geometric mean concentrations of monoethyl phthalate increased with age, but patterns by education, income, body mass index, environmental tobacco smoke or season were not observed when measures were adjusted for urinary dilution. Our results suggest that the participant characteristics assessed in this study have limited influence on inter-individual variability of phthalate metabolite concentrations. With regard to intra-individual variability, our results

  20. Stable isotope coded derivatizing reagents as internal standards in metabolite profiling.

    PubMed

    Bruheim, Per; Kvitvang, Hans Fredrik Nyvold; Villas-Boas, Silas G

    2013-06-28

    Gas chromatography (GC) and liquid chromatography (LC) coupled to mass spectrometric (MS) detection have become the two main techniques for the analysis of metabolite pools (i.e. Metabolomics). These technologies are especially suited for Metabolite Profiling analysis of various metabolite groups due to high separation capabilities of the chromatographs and high sensitivity of the mass analysers. The trend in quantitative Metabolite Profiling is to add more metabolites and metabolite groups in a single method. This should not be done by compromising the analytical precision. Mass spectrometric detection comes with certain limitations, especially in the quantitative aspects as standards are needed for conversion of ion abundance to concentration and ionization efficiencies are directly dependent on eluent conditions. This calls for novel strategies to counteract all variables that can influence the quantitative precision. Usually, internal standards are used to correct any technical variation. For quantitation of single or just a few analytes this can be executed with spiking isotopically labeled standards. However, for more comprehensive analytical tasks, e.g. profiling tens or hundreds of analytes simultaneously, this strategy becomes expensive and in many cases isotopically labeled standards are not available. An alternative is to introduce a derivatizing step where the sample is derivatized with naturally labeled reagent, while a standard solution is separately derivatized with isotopically labeled reagent and spiked into the sample solution prior to analysis. This strategy, named isotope coded derivatization - ICD, is attractive in the emerging field of quantitative Metabolite Profiling where current protocols can easily comprise over hundred metabolites. This review provides an overview of isotopically labeled derivatizing reagents that have been developed for important metabolite groups with the aim to improve analytical performance and precision.

  1. Synchronous fluorometric measurement of metabolites of polycyclic aromatic hydrocarbons in the bile of brown bullhead

    SciTech Connect

    Lin, E.L.C.; Cormier, S.M. . Environmental Monitoring Systems Lab.); Racine, R.N.

    1994-05-01

    A synchronous fluorescent spectroscopy (SFS) method was developed to measure pyrene-type metabolites in the bile of brown bullhead (Ameiurus nebulosus) and to estimate the exposure of fish to PAHs in four Lake Erie tributaries collected in the spring and fall of 1990 and 1991. For comparison, fish biliary benzo[a]pyrene (B[a]P) metabolites were also measured by HPLC/fluorescent detection (HPLC/F). Both methods showed that concentrations of biliary PAH metabolites of fish collected in polluted rivers were significantly higher than those collected from reference rivers. Concentrations biliary metabolites of fish caught in the Black River were five to 20 times greater than those collected in Old Woman Creek by SFS and three to five times greater by HPLC/F. Fish from the Cuyahoga River had four to 24 times more biliary PAH metabolites than fish from Old Woman Creek, measured by SFS, and five to 10 times more, measured by HPLC/F. Brown bullhead from the Toussaint River had fewer PAH metabolites than fish from Old Woman Creek. Correlation analyses of the two sets of data obtained by SFS and HPLC/F showed significance by both Pearson's sample correlation and Spearman's rank correlation. This study indicates that pyrene-type metabolites determined by SFS can be used to estimate B[a]P-type metabolites in fish bile. SFS appears to be a highly sensitive method for detecting PAH metabolites and, because of its simplicity, a cost-efficient method for screening large numbers of samples for exposure to PAHs in fish.

  2. Atrazine and its metabolites as indicators of stream-aquifer interaction in Kansas, USA

    USGS Publications Warehouse

    Townsend, M.A.; Young, D.P.

    2000-01-01

    A survey of atrazine and its metabolites in Kansas ground water indicated that ground-water quality was impacted by stream-aquifer interaction between rivers in the Kansas River basin and their adjacent alluvial aquifers. Atrazine was detected in 19 of the 78 samples. The most common metabolite, deethylatrazine, was detected in 25 samples, 18 of which also had atrazine. The deethylatrazine/atrazine ratio (DAR) of < 1.0 indicates rapid movement of agricultural chemicals to ground water. In this study, 12 of 18 samples had DAR values < 1.0, suggesting rapid recharge to the aquifers. Hydroxyatrazine is seldom detected in ground water. In this study hydroxyatrazine was detected primarily in wells sited in alluvium of rivers. These rivers contain atrazine in varying concentrations. Results of the study suggest that stream-aquifer interaction is a process contributing to the presence of both atrazine and its metabolites in ground water in these areas.A survey of atrazine and its metabolites in Kansas ground water indicated that ground water quality was impacted by stream-aquifer interaction between rivers in the Kansas River basin and their adjacent alluvial aquifers. Atrazine was detected in 19 of the 78 samples. The most common metabolite, deethylatrazine, was detected in 25 samples, 18 of which also had atrazine. The deethylatrazine/attrazine ratio (DAR) of < 1.0 indicates rapid movement of agricultural chemicals to ground water. In this study, 12 of 18 samples had DAR values < 1.0, suggesting rapid recharge to the aquifers. Hydroxyatrazine is seldom detected in ground water. In this study hydroxyatrazine was detected primarily in wells sited in alluvium of rivers. These rivers contain atrazine in varying concentration. Results of the study suggest that stream-aquifer interaction is a process contributing to the presence of both attrazine and its metabolites in ground water in these areas.

  3. Gas chromatography – mass spectrometry of JWH-018 metabolites in urine samples with direct comparison to analytical standards

    PubMed Central

    Emerson, Beth; Durham, Bill; Gidden, Jennifer; Lay, Jackson O.

    2013-01-01

    JWH-018 (1-pentyl-3-(1-naphthoyl)indole) is one of numerous potential aminoalkylindoles contained in products marketed as ‘K2’ or ‘Spice’. Investigation of the urinary metabolites from consumption of these compounds is important because they are banned in the United States and many European countries. An efficient extraction procedure and gas chromatography – mass spectrometry (GC-MS) method were developed for detection of ‘K2’ metabolites in urine from individuals suspected of using these products. Analytical standards were used to elucidate the structure-specific mass spectral fragmentations and retention properties to confirm proposed identifications and support quantitative studies. A procedure for the synthesis of one of these metabolites (5-hydroxypentyl JWH-018) was also developed. Results are comparable to existing LC-MS/MS methods, with the same primary metabolites detected. The specific metabolite hydrolysis products include 4-hydroxpentyl, 5-hydroxypentyl, and N-pentanoic acid derivatives. PMID:23683902

  4. Structure elucidation and identification of a common metabolite for naphthoylindole-based synthetic cannabinoids using LC-TOF and comparison to a synthetic reference standard.

    PubMed

    Lovett, Dennis P; Yanes, Enrique G; Herbelin, Travis W; Knoerzer, Timm A; Levisky, Joseph A

    2013-03-10

    The identification of a predominate metabolite found in urine specimens which test positive for naphthoylindole-based synthetic cannabinoids is reported. The presence of this new metabolite was detected at the Air Force Drug Testing Lab Investigations Division during screening analysis for metabolites of JWH-018 and JWH-073, because it shares the same MRM transitions as the JWH-073 N-(3-hydroxybutyl) metabolite. However, the detected peak is chromatographically distinguished from other metabolites due to differences in retention time. This metabolite appears to be a common metabolite for select naphthoylindole-based synthetic cannabinoids that could potentially be used as a common biomarker for their qualitative and quantitative analyses. The new metabolite has been successfully identified as 3-(3-(1-naphthoyl)-1H-indol-1-yl) propanoic acid (1, JWH 072 N-propanoic acid metabolite, Fig. 1) by using various mass spectrometric and liquid chromatographic techniques as well as chemical derivatization. The metabolite identity was confirmed through the comparison of authentic positive urine and a chemically synthesized metabolite standard. Both materials shared the same chromatographic retention time on two separate chromatographic systems, mass fragmentation pattern and exact mass. Full characterization of the synthetic reference material and intermediates by (1)H NMR, (13)C NMR, IR and HRMS was also conducted.

  5. Synthetic cannabinoids: analysis and metabolites.

    PubMed

    Elsohly, Mahmoud A; Gul, Waseem; Wanas, Amira S; Radwan, Mohamed M

    2014-02-27

    Cannabimimetics (commonly referred to as synthetic cannabinoids), a group of compounds encompassing a wide range of chemical structures, have been developed by scientists with the hope of achieving selectivity toward one or the other of the cannabinoid receptors CB1 and CB2. The goal was to have compounds that could possess high therapeutic activity without many side effects. However, underground laboratories have used the information generated by the scientific community to develop these compounds for illicit use as marijuana substitutes. This chapter reviews the different classes of these "synthetic cannabinoids" with particular emphasis on the methods used for their identification in the herbal products with which they are mixed and identification of their metabolites in biological specimens.

  6. Active Metabolites of Isoxazolylpenicillins in Humans

    PubMed Central

    Thijssen, H. H. W.; Mattie, H.

    1976-01-01

    Metabolites of the isoxazolylpenicillins that still possessed antibacterial activity were shown to be present in urine and serum. In healthy subjects, the amounts excreted in urine were low; 10 to 23% of the excreted penicillin activities represented the metabolites. The highest amount of metabolite in urine was found for oxacillin, and the lowest was found for flucloxacillin. No extreme differences in the amounts of metabolite excreted were observed when the compounds were administered orally or intravenously. In one healthy subject metabolite levels were estimated for cloxacillin in serum. Very low levels were found, i.e., about 9% of the activity. In subjects with highly impaired renal function, the metabolite may represent up to 50% of the total level of penicillin in serum. The antibacterial activities of the different metabolites were of the same order of magnitude as those of the respective parent compounds. Also, the activity against benzylpenicillin-resistant staphylococci was retained. It is not likely that the formation of the active metabolites should influence therapeutic results. PMID:825029

  7. Pharmacokinetic properties of the synthetic cannabinoid JWH-018 and of its metabolites in serum after inhalation.

    PubMed

    Toennes, Stefan W; Geraths, Anna; Pogoda, Werner; Paulke, Alexander; Wunder, Cora; Theunissen, Eef L; Ramaekers, Johannes G

    2017-03-24

    Each year, synthetic cannabinoids are occurring in high numbers in the illicit drug market, but data on their pharmacology and toxicology are scarcely available. Therefore, a pilot study was performed to assess adverse effects of JWH-018, which is one of the oldest and best known synthetic cannabinoids. Six subjects inhaled smoke from 2 and 3mg JWH-018. The drug and nine of its metabolites were analyzed in their blood samples taken during the following 12h by liquid chromatography-mass spectrometry (LC-MSMS). The maximum concentration of JWH-018 reached 2.9-9.9ng/ml after inhalation and markedly decreased during the next 1.5h, followed by a multiexponential decline (t1/2 in median 1.3h and 5.7h). The concentration of the pentanoic acid metabolite was slightly higher than that of the 3-, 4- and 5-hydroxypentyl metabolites and of the 6-hydroxyindol metabolite. The data also suggest a multiexponential decline and slow terminal elimination of JWH-018 and all metabolites. The detection of JWH-018 and of its metabolites in serum requires high analytical sensitivity. The pharmacokinetic properties of inhaled JWH-018 are similar to that of THC. A slow terminal elimination of drug and metabolites may lead to accumulation in chronic users.

  8. Quantification of Water-Soluble Metabolites in Medicinal Mushrooms Using Proton NMR Spectroscopy.

    PubMed

    Lo, Yu-Chang; Chien, Shih-Chang; Mishchuk, Darya O; Slupsky, Carolyn M; Mau, Jeng-Leun

    2016-01-01

    The water-soluble metabolites in 5 mushrooms were identified and quantified using proton nuclear magnetic resonance (NMR) spectroscopy and software for targeted metabolite detection and quantification. In total, 35 compounds were found in Agaricus brasiliensis, 25 in Taiwanofungus camphoratus, 23 in Ganoderma lucidum (Taiwan) and Lentinus edodes, and 16 in G. lucidum (China). Total amounts of all identified metabolites in A. brasiliensis, T. camphoratus, G. lucidum, G. lucidum (China), and L. edodes were 149,950.51, 12,834.18, 9,549.09, 2,788.41, and 111,726.51 mg/kg dry weight, respectively. These metabolites were categorized into 4 groups: free amino acids and derivatives, carbohydrates, carboxylic acids, and nucleosides. Carbohydrates were the most abundant metabolites among all 4 groups, with mannitol having the highest concentration among all analyzed metabolites (848-94,104 mg/kg dry weight). Principal components analysis (PCA) showed obvious distinction among the metabolites of the 5 different kinds of mushrooms analyzed in this study. Thus PCA could provide an optional analytical way of identifying and recognizing the compositions of flavor products. Furthermore, the results of this study demonstrate that NMRbased metabolomics is a powerful tool for differentiating between various medicinal mushrooms.

  9. High resolution mass spectrometry to investigate omeprazole and venlafaxine metabolites in wastewater.

    PubMed

    Boix, Clara; Ibáñez, María; Bagnati, Renzo; Zuccato, Ettore; Sancho, Juan V; Hernández, Félix; Castiglioni, Sara

    2016-01-25

    This study reports an investigation of omeprazole and venlafaxine parent substances and metabolites in Italian municipal influent wastewaters (IWWs). These pharmaceuticals were selected because they are widely consumed in Italy, but are poorly detected in waste and surface water. The aim of the study was to identify the most relevant pharmaceuticals metabolites in wastewater in order to improve the prioritization step and choose priority pollutants for environmental monitoring campaigns. This was done by investigating omeprazole, venlafaxine and their main metabolites in 30 IWWs from ten Italian cities and by comparing results with information from pharmacokinetic studies. Analysis was performed by solid phase extraction (SPE) and high-performance liquid chromatography (HPLC) coupled to high resolution mass spectrometry (HRMS). We searched for 23 omeprazole and four venlafaxine metabolites using data-dependent and MS/MS methods. Parent omeprazole was never present in the samples. Six omeprazole metabolites were found in IWWs. Venlafaxine and two metabolites were present in all the samples. The metabolic profiles in Italian IWW agreed with results in IWW from Spain and with urinary excretion profiles from pharmacokinetic studies. Comparing results from different sources was useful to improve the identification of pharmaceuticals metabolites in environmental samples and to focus the attention of future studies on the most relevant compounds.

  10. Identification of hydroxylated metabolites of hexabromocyclododecane in wildlife and 28-days exposed Wistar rats.

    PubMed

    Brandsma, Sicco H; Van der Ven, Leo T M; De Boer, Jacob; Leonards, Pim E G

    2009-08-01

    We studied the presence of hydroxylated metabolites of hexabromocyclododecane (HBCD) in three wildlife species (tern egg, seal, and flounder) and in Wistar rats exposed to 30 and 100 mg HBCD/kg bw/day for 28 days. A nondestructive extraction, fractionation, and cleanup method was developed to separate the hydroxylated HBCD metabolites from the biotic sample matrix. Four different groups of hydroxylated HBCD metabolites were identified in rat adipose, liver, lung, and muscle tissues by liquid and gas chromatography (LC and GC) combined with mass spectrometry (MS): monohydroxy metabolites of HBCD, pentabromocyclododecene (PBCDe), tetrabromocyclododecene (TBCDe), and dihydroxy-HBCD. Dihydroxy-PBCDe was identified by GC-MS but could not be confirmed by LCMS. Debromination of HBCD to PBCDe was another metabolic pathway observed. In tern eggs from the Western Scheldt the monohydroxy-HBCD was found and in the blubber of harbor seal (Wadden Sea) the monohydroxy metabolites of HBCD and PBCDe were found. No hydroxylated metabolites were detected in the tissue of flounder (Wadden Sea). To our knowledge, this is the first study to identify different hydroxylated metabolite groups of HBCD in rat and wildlife samples.

  11. [Biologically active metabolites of the marine actinobacteria].

    PubMed

    Sobolevskaia, M P; Kuznetsova, T A

    2010-01-01

    This review systematically data on the chemical structure and biological activity of metabolites of obligate and facultative marine actinobacteria, published from 2000 to 2007. We discuss some structural features of the five groups of metabolites related to macrolides and compounds containing lactone, quinone and diketopiperazine residues, cyclic peptides, alkaloids, and compounds of mixed biosynthesis. Survey shows a large chemical diversity of metabolites actinobacteria isolated from marine environment. It is shown that, along with metabolites, identical to previously isolated from terrestrial actinobacteria, marine actinobacteria synthesize unknown compounds not found in other natural sources, including micro organisms. Perhaps the biosynthesis of new chemotypes bioactive compounds in marine actinobacteria is one manifestation of chemical adaptation of microorganisms to environmental conditions at sea. Review stresses the importance of the chemical study of metabolites of marine actinobacteria. These studies are aimed at obtaining new data on marine microorganisms producers of biologically active compounds and chemical structure and biological activity of new low-molecular bioregulators of natural origin.

  12. [Pharmacological research on bonnecor and its metabolites].

    PubMed

    Poppe, H; Heer, S; Barch, R

    1990-01-01

    The antiarrhythmic and local anesthetic effects of 4 metabolites (G 491, ABD 19-200, ABD 19-199, ABD 19-205) of a new antiarrhythmic drug bonnecor (GS-015) were studied on the models of arrhythmias induced by aconitine (rats), barium chloride (rabbits), electrical fibrillation (cats), ouabain (dogs) as well as surface anesthesia (rabbit cornea). The side effects on the cardiovascular system were investigated on anesthetized cats. As compared with the original compound (bonnecor) metabolites G 491 and ABD 19-200 on different test models exhibited the action which on the antiarrhythmic terms was 2-14 times less weak than that of bonnecor but the metabolites were less toxic. Metabolites ABD 19-199 and ABD 19-205 reach the degree of effectiveness of bonnecor but their toxicity is higher. It follows from the above that the beneficial effect of bonnecor is not achieved by its metabolites.

  13. Compound to Extract to Formulation: a knowledge-transmitting approach for metabolites identification of Gegen-Qinlian Decoction, a traditional Chinese medicine formula.

    PubMed

    Qiao, Xue; Wang, Qi; Wang, Shuang; Miao, Wen-Juan; Li, Yan-Jiao; Xiang, Cheng; Guo, De-An; Ye, Min

    2016-12-20

    Herbal medicines usually contain a large group of chemical components, which may be transformed into more complex metabolites in vivo. In this study, we proposed a knowledge-transmitting strategy for metabolites identification of compound formulas. Gegen-Qinlian Decoction (GQD) is a classical formula in traditional Chinese medicine (TCM). It is widely used to treat diarrhea and diabetes in clinical practice. However, only tens of metabolites could be detected using conventional approaches. To comprehensively identify the metabolites of GQD, a "compound to extract to formulation" strategy was established in this study. The metabolic pathways of single representative constituents in GQD were studied, and the metabolic rules were transmitted to chemically similar compounds in herbal extracts. After screening diversified metabolites from herb extracts, the knowledge was summarized to identify the metabolites of GQD. Tandem mass spectrometry (MS(n)), fragment-based scan (NL, PRE), and selected reaction monitoring (SRM) were employed to identify, screen, and monitor the metabolites, respectively. Using this strategy, we detected 131 GQD metabolites (85 were newly generated) in rats biofluids. Among them, 112 metabolites could be detected when GQD was orally administered at a clinical dosage (12.5 g/kg). This strategy could be used for systematic metabolites identification of complex Chinese medicine formulas.

  14. Compound to Extract to Formulation: a knowledge-transmitting approach for metabolites identification of Gegen-Qinlian Decoction, a traditional Chinese medicine formula

    PubMed Central

    Qiao, Xue; Wang, Qi; Wang, Shuang; Miao, Wen-juan; Li, Yan-jiao; Xiang, Cheng; Guo, De-an; Ye, Min

    2016-01-01

    Herbal medicines usually contain a large group of chemical components, which may be transformed into more complex metabolites in vivo. In this study, we proposed a knowledge-transmitting strategy for metabolites identification of compound formulas. Gegen-Qinlian Decoction (GQD) is a classical formula in traditional Chinese medicine (TCM). It is widely used to treat diarrhea and diabetes in clinical practice. However, only tens of metabolites could be detected using conventional approaches. To comprehensively identify the metabolites of GQD, a “compound to extract to formulation” strategy was established in this study. The metabolic pathways of single representative constituents in GQD were studied, and the metabolic rules were transmitted to chemically similar compounds in herbal extracts. After screening diversified metabolites from herb extracts, the knowledge was summarized to identify the metabolites of GQD. Tandem mass spectrometry (MSn), fragment-based scan (NL, PRE), and selected reaction monitoring (SRM) were employed to identify, screen, and monitor the metabolites, respectively. Using this strategy, we detected 131 GQD metabolites (85 were newly generated) in rats biofluids. Among them, 112 metabolites could be detected when GQD was orally administered at a clinical dosage (12.5 g/kg). This strategy could be used for systematic metabolites identification of complex Chinese medicine formulas. PMID:27996040

  15. Complicating factors in safety testing of drug metabolites: Kinetic differences between generated and preformed metabolites

    SciTech Connect

    Prueksaritanont, Thomayant . E-mail: thomayant_prueksaritanont@merck.com; Lin, Jiunn H.; Baillie, Thomas A.

    2006-12-01

    This paper aims to provide a scientifically based perspective on issues surrounding the proposed toxicology testing of synthetic drug metabolites as a means of ensuring adequate nonclinical safety evaluation of drug candidates that generate metabolites considered either to be unique to humans or are present at much higher levels in humans than in preclinical species. We put forward a number of theoretical considerations and present several specific examples where the kinetic behavior of a preformed metabolite given to animals or humans differs from that of the corresponding metabolite generated endogenously from its parent. The potential ramifications of this phenomenon are that the results of toxicity testing of the preformed metabolite may be misleading and fail to characterize the true toxicological contribution of the metabolite when formed from the parent. It is anticipated that such complications would be evident in situations where (a) differences exist in the accumulation of the preformed versus generated metabolites in specific tissues, and (b) the metabolite undergoes sequential metabolism to a downstream product that is toxic, leading to differences in tissue-specific toxicity. Owing to the complex nature of this subject, there is a need to treat drug metabolite issues in safety assessment on a case-by-case basis, in which a knowledge of metabolite kinetics is employed to validate experimental paradigms that entail administration of preformed metabolites to animal models.

  16. A new paradigm for known metabolite identification in metabonomics/metabolomics: metabolite identification efficiency.

    PubMed

    Everett, Jeremy R

    2015-01-01

    A new paradigm is proposed for assessing confidence in the identification of known metabolites in metabonomics studies using NMR spectroscopy approaches. This new paradigm is based upon the analysis of the amount of metabolite identification information retrieved from NMR spectra relative to the molecular size of the metabolite. Several new indices are proposed including: metabolite identification efficiency (MIE) and metabolite identification carbon efficiency (MICE), both of which can be easily calculated. These indices, together with some guidelines, can be used to provide a better indication of known metabolite identification confidence in metabonomics studies than existing methods. Since known metabolite identification in untargeted metabonomics studies is one of the key bottlenecks facing the science currently, it is hoped that these concepts based on molecular spectroscopic informatics, will find utility in the field.

  17. Occurrence of selected pesticides and their metabolites in near-surface aquifers of the midwestern United States

    USGS Publications Warehouse

    Kolpin, D.W.; Michael, Thurman E.; Goolsby, D.A.

    1996-01-01

    The occurrence and distribution of selected pesticides and their metabolites were investigated through the collection of 837 water-quality samples from 303 wells across the Midwest. Results of this study showed that five of the six most frequently detected compounds were pesticide metabolites. Thus, it was common for a metabolite to be found more frequently in groundwater than its parent compound. The metabolite alachlor ethanesulfonic acid (alachlor-ESA; 2-[(2,6-diethylphenyl)(methoxymethyl)amino]-2-oxoethanesulfonic acid) was detected almost 10 times as frequently and at much higher concentrations than its parent compound alachlor (2-chloro-2‘,6‘-diethyl-N-(methoxymethyl)acetamide). The median detectable atrazine (2-chloro-4-ethylamino-6- isopropylamino-s-triazine) concentration was almost half that of atrazine residue (atrazine plus the two atrazine metabolites analyzed). Cyanazine amide [2-chloro-4-(1-carbamoyl-1-methylethylamino)-6-ethylamino-s-triazine] was detected almost twice as frequently as cyanazine (2-chloro-4-ethylamino-6-methylpropionitrileamino-s-triazine). Results show that information on pesticide metabolites is necessary to understand the environmental fate of pesticides. Consequently, if pesticide metabolites are not quantified, the effects of chemical use on groundwater quality would be substantially underestimated. Thus, continued research is needed to identify major degradation pathways for all pesticides and to develop analytical methods to determine their concentrations in water and other environmental media.

  18. A new sulphate metabolite as a long-term marker of metandienone misuse.

    PubMed

    Gómez, C; Pozo, O J; Garrostas, L; Segura, J; Ventura, R

    2013-12-11

    Metandienone is one of the most frequently detected anabolic androgenic steroids in sports drug testing. Metandienone misuse is commonly detected by monitoring different metabolites excreted free or conjugated with glucuronic acid using gas chromatography mass spectrometry (GC-MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS) after hydrolysis with β-glucuronidase and liquid-liquid extraction. It is known that several metabolites are the result of the formation of sulphate conjugates in C17, which are converted to their 17-epimers in urine. Therefore, sulphation is an important phase II metabolic pathway of metandienone that has not been comprehensively studied. The aim of this work was to evaluate the sulphate fraction of metandienone metabolism by LC-MS/MS. Seven sulphate metabolites were detected after the analysis of excretion study samples by applying different neutral loss scan, precursor ion scan and SRM methods. One of the metabolites (M1) was identified and characterised by GC-MS/MS and LC-MS/MS as 18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one sulphate. M1 could be detected up to 26 days after the administration of a single dose of metandienone (5 mg), thus improving the period in which the misuse can be reported with respect to the last long-term metandienone metabolite described (18-nor-17β-hydroxymethyl-17α-methylandrost-1,4,13-triene-3-one excreted in the glucuronide fraction).

  19. PAH Metabolites in Bile of European Eel (Anguilla anguilla) from Morocco.

    PubMed

    Wariaghli, Fatima; Kammann, Ulrike; Hanel, Reinhold; Yahyaoui, Ahmed

    2015-12-01

    Environmental pollution of fish with organic contaminants is a topic of rising attention in Morocco. Polycyclic aromatic hydrocarbons (PAH) are prominent organic contaminants which are rapidly metabolized in fish. Their metabolites are accumulated in the bile fluid and can be used to assess PAH exposure. The two PAH metabolites 1-hydroxypyrene and 1-hydroxyphenanthrene were quantified in European eels (Anguilla anguilla) from two Moroccan river systems by high-performance liquid chromatography with fluorescence detection. Mean values ranged from 52 to 210 ng/mL 1-hydroxypyrene and from 61 to 73 ng/mL 1-hydroxyphenanthrene. The overall concentrations of PAH metabolites in eel from Morocco appeared moderate compared to eel from European rivers and coastal sites. The present study provides first information on concentrations of PAH metabolites in fish from Morocco.

  20. Temperament type specific metabolite profiles of the prefrontal cortex and serum in cattle.

    PubMed

    Brand, Bodo; Hadlich, Frieder; Brandt, Bettina; Schauer, Nicolas; Graunke, Katharina L; Langbein, Jan; Repsilber, Dirk; Ponsuksili, Siriluk; Schwerin, Manfred

    2015-01-01

    In the past decade the number of studies investigating temperament in farm animals has increased greatly because temperament has been shown not only to affect handling but also reproduction, health and economically important production traits. However, molecular pathways underlying temperament and molecular pathways linking temperament to production traits, health and reproduction have yet to be studied in full detail. Here we report the results of metabolite profiling of the prefrontal cortex and serum of cattle with distinct temperament types that were performed to further explore their molecular divergence in the response to the slaughter procedure and to identify new targets for further research of cattle temperament. By performing an untargeted comprehensive metabolite profiling, 627 and 1097 metabolite features comprising 235 and 328 metabolites could be detected in the prefrontal cortex and serum, respectively. In total, 54 prefrontal cortex and 51 serum metabolite features were indicated to have a high relevance in the classification of temperament types by a sparse partial least square discriminant analysis. A clear discrimination between fearful/neophobic-alert, interested-stressed, subdued/uninterested-calm and outgoing/neophilic-alert temperament types could be observed based on the abundance of the identified relevant prefrontal cortex and serum metabolites. Metabolites with high relevance in the classification of temperament types revealed that the main differences between temperament types in the response to the slaughter procedure were related to the abundance of glycerophospholipids, fatty acyls and sterol lipids. Differences in the abundance of metabolites related to C21 steroid metabolism and oxidative stress indicated that the differences in the metabolite profiles of the four extreme temperament types could be the result of a temperament type specific regulation of molecular pathways that are known to be involved in the stress and fear response.

  1. Temperament Type Specific Metabolite Profiles of the Prefrontal Cortex and Serum in Cattle

    PubMed Central

    Brand, Bodo; Hadlich, Frieder; Brandt, Bettina; Schauer, Nicolas; Graunke, Katharina L.; Langbein, Jan; Repsilber, Dirk; Ponsuksili, Siriluk; Schwerin, Manfred

    2015-01-01

    In the past decade the number of studies investigating temperament in farm animals has increased greatly because temperament has been shown not only to affect handling but also reproduction, health and economically important production traits. However, molecular pathways underlying temperament and molecular pathways linking temperament to production traits, health and reproduction have yet to be studied in full detail. Here we report the results of metabolite profiling of the prefrontal cortex and serum of cattle with distinct temperament types that were performed to further explore their molecular divergence in the response to the slaughter procedure and to identify new targets for further research of cattle temperament. By performing an untargeted comprehensive metabolite profiling, 627 and 1097 metabolite features comprising 235 and 328 metabolites could be detected in the prefrontal cortex and serum, respectively. In total, 54 prefrontal cortex and 51 serum metabolite features were indicated to have a high relevance in the classification of temperament types by a sparse partial least square discriminant analysis. A clear discrimination between fearful/neophobic-alert, interested-stressed, subdued/uninterested-calm and outgoing/neophilic-alert temperament types could be observed based on the abundance of the identified relevant prefrontal cortex and serum metabolites. Metabolites with high relevance in the classification of temperament types revealed that the main differences between temperament types in the response to the slaughter procedure were related to the abundance of glycerophospholipids, fatty acyls and sterol lipids. Differences in the abundance of metabolites related to C21 steroid metabolism and oxidative stress indicated that the differences in the metabolite profiles of the four extreme temperament types could be the result of a temperament type specific regulation of molecular pathways that are known to be involved in the stress and fear response

  2. Human hydroxylated metabolites of BDE-47 and BDE-99 are glucuronidated and sulfated in vitro.

    PubMed

    Erratico, Claudio; Zheng, Xiaobo; Ryden, Andreas; Marsh, Goran; Maho, Walid; Covaci, Adrian

    2015-07-16

    Polybrominated diphenyl ethers (PBDEs) were used worldwide as additive flame retardants and are classified as persistent, bioaccumulable and toxic environmental pollutants. In humans, the hydroxylated metabolites of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) formed in vitro have also been detected in vivo. To further characterize the metabolism of BDE-47 and BDE-99 and to identify candidate markers for monitoring the human exposure to PBDEs using non-invasive approaches, glucuronidation and sulfation of hydroxylated metabolites of BDE-47 and BDE-99 were investigated using human liver microsomes and cytoplasm, respectively. The formed Phase II metabolites were analyzed by liquid chromatography-tandem mass spectrometry using a novel approach to develop analytical methods in absence of authentic standards. All available standards for hydroxylated metabolites of BDE-47 and BDE-99 were glucuronidated and sulfated, showing that glucuronidation and sulfation are part of the metabolism pathway of BDE-47 and BDE-99 in vitro. The major glucuronidated and sulfated analogs of hydroxylated metabolites of BDE-47 were (a) 2,4-DBP-Gluc and 5-Gluc-BDE-47, and (b) 2'-Sulf-BDE-28, 4-Sulf-BDE-42 and 3-Sulf-BDE-47, respectively. The major glucuronidated and sulfated analogs of hydroxylated metabolites of BDE-99 were (a) 2,4,5-TBP-Gluc and 6'-Gluc-BDE-99, and (b) 3'-Sulf-BDE-99 and 5'-Sulf-BDE-99, respectively. Apparent Km values associated with the formation of sulfated metabolites of BDE-47 and BDE-99 were ten times lower than those of the corresponding glucuronidated metabolites, suggesting that sulfated rather than glucuronidated metabolites of OH-PBDEs might be used as markers of human exposure to PBDEs using a non-invasive approach based on urine sample collection.

  3. Highly sensitive simultaneous quantification of estrogenic tamoxifen metabolites and steroid hormones by LC-MS/MS.

    PubMed

    Johänning, Janina; Heinkele, Georg; Precht, Jana C; Brauch, Hiltrud; Eichelbaum, Michel; Schwab, Matthias; Schroth, Werner; Mürdter, Thomas E

    2015-09-01

    Tamoxifen is a mainstay in the treatment of estrogen receptor-positive breast cancer and is metabolized to more than 30 different compounds. Little is known about in vivo concentrations of estrogenic metabolites E-metabolite E, Z-metabolite E, and bisphenol and their relevance for tamoxifen efficacy. Therefore, we developed a highly sensitive HPLC-ESI-MS/MS quantification method for tamoxifen metabolites bisphenol, E-metabolite E, and Z-metabolite E as well as for the sex steroid hormones estradiol, estrone, testosterone, androstenedione, and progesterone. Plasma samples were subjected to protein precipitation followed by solid phase extraction. Upon derivatization with 3-[(N-succinimide-1-yl)oxycarbonyl]-1-methylpyridinium iodide, all analytes were separated on a sub-2-μm column with a gradient of acetonitrile in water with 0.1 % of formic acid. Analytes were detected on a triple-quadrupole mass spectrometer with positive electrospray ionization in the multiple reaction monitoring mode. Our method demonstrated high sensitivity, accuracy, and precision. The lower limits of quantification were 12, 8, and 25 pM for bisphenol, E-metabolite E, and Z-metabolite E, respectively, and 4 pM for estradiol and estrogen, 50 pM for testosterone and androstenedione, and 25 pM for progesterone. The method was applied to plasma samples of postmenopausal patients taken at baseline and under tamoxifen therapy. Graphical Abstract Sample preparation and derivatization for highly sensitive quantification of estrogenic tamoxifen metabolites and steroid hormones by HPLC-MS/MS.

  4. The in silico prediction of human-specific metabolites from hepatotoxic drugs.

    PubMed

    Valerio, Luis G; Long, Anthony

    2010-09-01

    In this study we employed the use of the Meteor computational software program to perform predictions in silico on 17 hepatotoxic drugs for determining human-specific drug metabolites. Congruence of the in silico predictions from a qualitative standpoint of drug metabolite structures was established by comparison to human in vivo drug metabolic profiles characterized in publically available clinical studies. A total of 87 human-specific metabolites were identified from the 17 drugs. We found that Meteor's positive predictions included 4 out of the 9 reported major metabolites (detected in excreta at a level of >10% of the administered p.o. dose) and 10 out of the 15 major phase II metabolites giving a total of 14 correctly predicted drug metabolite structures out of 23 major metabolites. A significant level of unconfirmed positive predictions resulted and discussion on reasons for this is presented. An example is given whereby the in silico metabolism prediction succeeded to predict the putative toxic pathway of one of the drugs whilst conventional rodent liver microsomal assays failed to predict the pathway. Overall, we describe a reasonable simulation of human metabolic profiling using this in silico method with this data set of hepatotoxic drugs now withdrawn from the market. We provide an in-depth and objective discussion of this first of its kind validation test using clinical study data with interest in the prediction human-specific metabolism. Further research is discussed on what areas need to be investigated to improve upon the predictive data. The strong potential of this method to predict human-specific drug metabolites suggests the utility of this computational tool to help support not only the discovery development of therapeutics but also the safety assessment in identifying drug metabolites early to protect patients prior to initiating clinical studies.

  5. Expanding the Limits of Human Blood Metabolite Quantitation Using NMR Spectroscopy

    PubMed Central

    2015-01-01

    A current challenge in metabolomics is the reliable quantitation of many metabolites. Limited resolution and sensitivity combined with the challenges associated with unknown metabolite identification have restricted both the number and the quantitative accuracy of blood metabolites. Focused on alleviating this bottleneck in NMR-based metabolomics, investigations of pooled human serum combining an array of 1D/2D NMR experiments at 800 MHz, database searches, and spiking with authentic compounds enabled the identification of 67 blood metabolites. Many of these (∼1/3) are new compared with those reported previously as a part of the Human Serum Metabolome Database. In addition, considering both the high reproducibility and quantitative nature of NMR as well as the sensitivity of NMR chemical shifts to altered sample conditions, experimental protocols and comprehensive peak annotations are provided here as a guide for identification and quantitation of the new pool of blood metabolites for routine applications. Further, investigations focused on the evaluation of quantitation using organic solvents revealed a surprisingly poor performance for protein precipitation using acetonitrile. One-third of the detected metabolites were attenuated by 10–67% compared with methanol precipitation at the same solvent-to-serum ratio of 2:1 (v/v). Nearly 2/3 of the metabolites were further attenuated by up to 65% upon increasing the acetonitrile-to-serum ratio to 4:1 (v/v). These results, combined with the newly established identity for many unknown metabolites in the NMR spectrum, offer new avenues for human serum/plasma-based metabolomics. Further, the ability to quantitatively evaluate nearly 70 blood metabolites that represent numerous classes, including amino acids, organic acids, carbohydrates, and heterocyclic compounds, using a simple and highly reproducible analytical method such as NMR may potentially guide the evaluation of samples for analysis using mass spectrometry

  6. Determination of proline in human serum by a robust LC-MS/MS method: application to identification of human metabolites as candidate biomarkers for esophageal cancer early detection and risk stratification.

    PubMed

    Liang, Su; Sanchez-Espiridion, Beatriz; Xie, Huan; Ma, Jing; Wu, Xifeng; Liang, Dong

    2015-04-01

    Altered serum proline levels are related to cancer metabolism. This study developed and validated a LC-MS/MS method to analyze proline in human serum. Surrogate blank serum, coupled with stable isotope l-proline-(13) C5 ,(15)  N as internal standard, was used for generating standard curves ranging from 2.5 to 100 μg/mL. Proline was extracted from serum samples using methanol. A Phenomenex Lux 5u Cellulose-1 column (250 × 4.6 mm) was used for chromatographic separation with 40% methanol in 0.05% formic acid aqueous solution as a mobile phase. Mass detection was performed under positive ionization electrospray. Intra- and inter-day accuracy and precision were <10%. The extraction recovery and matrix factor were 99.17 and 1.47%, respectively. Our study showed that the chiral column had high specificity and selectivity for separating proline from serum components. The assay was successfully applied for the quantification of human serum proline levels from 30 esophageal cancer patients and 30 healthy volunteers. Statistical analyses showed significantly lower levels of serum proline in the patients as compared with the healthy volunteers (p-value = 0.011). We report here a simple, specific and reproducible LC-MS/MS method for the quantification of proline in human serum as a potential screening biomarker for esophageal cancer.

  7. NMR metabolomics profiling of blood plasma mimics shows that medium- and long-chain fatty acids differently release metabolites from human serum albumin

    NASA Astrophysics Data System (ADS)

    Jupin, M.; Michiels, P. J.; Girard, F. C.; Spraul, M.; Wijmenga, S. S.

    2014-02-01

    Metabolite profiling by NMR of body fluids is increasingly used to successfully differentiate patients from healthy individuals. Metabolites and their concentrations are direct reporters of body biochemistry. However, in blood plasma the NMR-detected free-metabolite concentrations are also strongly affected by interactions with the abundant plasma proteins, which have as of yet not been considered much in metabolic profiling. We previously reported that many of the common NMR-detected metabolites in blood plasma bind to human serum albumin (HSA) and many are released by fatty acids present in fatted HSA. HSA is the most abundant plasma protein and main transporter of endogenous and exogenous metabolites. Here, we show by NMR how the two most common fatty acids (FAs) in blood plasma - the long-chain FA, stearate (C18:0) and medium-chain FA, myristate (C14:0) - affect metabolite-HSA interaction. Of the set of 18 common NMR-detected metabolites, many are released by stearate and/or myristate, lactate appearing the most strongly affected. Myristate, but not stearate, reduces HSA-binding of phenylalanine and pyruvate. Citrate signals were NMR invisible in the presence of HSA. Only at high myristate-HSA mole ratios 11:1, is citrate sufficiently released to be detected. Finally, we find that limited dilution of blood-plasma mimics releases HSA-bound metabolites, a finding confirmed in real blood plasma samples. Based on these findings, we provide recommendations for NMR experiments for quantitative metabolite profiling.

  8. NMR metabolomics profiling of blood plasma mimics shows that medium- and long-chain fatty acids differently release metabolites from human serum albumin.

    PubMed

    Jupin, M; Michiels, P J; Girard, F C; Spraul, M; Wijmenga, S S

    2014-02-01

    Metabolite profiling by NMR of body fluids is increasingly used to successfully differentiate patients from healthy individuals. Metabolites and their concentrations are direct reporters of body biochemistry. However, in blood plasma the NMR-detected free-metabolite concentrations are also strongly affected by interactions with the abundant plasma proteins, which have as of yet not been considered much in metabolic profiling. We previously reported that many of the common NMR-detected metabolites in blood plasma bind to human serum albumin (HSA) and many are released by fatty acids present in fatted HSA. HSA is the most abundant plasma protein and main transporter of endogenous and exogenous metabolites. Here, we show by NMR how the two most common fatty acids (FAs) in blood plasma - the long-chain FA, stearate (C18:0) and medium-chain FA, myristate (C14:0) - affect metabolite-HSA interaction. Of the set of 18 common NMR-detected metabolites, many are released by stearate and/or myristate, lactate appearing the most strongly affected. Myristate, but not stearate, reduces HSA-binding of phenylalanine and pyruvate. Citrate signals were NMR invisible in the presence of HSA. Only at high myristate-HSA mole ratios 11:1, is citrate sufficiently released to be detected. Finally, we find that limited dilution of blood-plasma mimics releases HSA-bound metabolites, a finding confirmed in real blood plasma samples. Based on these findings, we provide recommendations for NMR experiments for quantitative metabolite profiling.

  9. Application of mass spectrometry for metabolite identification.

    PubMed

    Ma, Shuguang; Chowdhury, Swapan K; Alton, Kevin B

    2006-06-01

    Metabolism studies play a pivotal role in drug discovery and development. Characterization of metabolic "hot-spots" as well as reactive and pharmacologically active metabolites is critical to designing new drug candidates with improved metabolic stability, toxicological profile and efficacy. Metabolite identification in the preclinical species used for safety evaluation is required in order to determine whether human metabolites have been adequately tested during non-clinical safety assessment. From an instrumental standpoint, high performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) dominates all analytical tools used for metabolite identification. The general strategies employed for metabolite identification in both drug discovery and drug development settings together with sample preparation techniques are reviewed herein. These include a discussion of the various ionization methods, mass analyzers, and tandem mass spectrometry (MS/MS) techniques that are used for structural characterization in a modern drug metabolism laboratory. Mass spectrometry-based techniques, such as stable isotope labeling, on-line H/D exchange, accurate mass measurement to enhance metabolite identification and recent improvements in data acquisition and processing for accelerating metabolite identification are also described. Rounding out this review, we offer additional thoughts about the potential of alternative and less frequently used techniques such as LC-NMR/MS, CRIMS and ICPMS.

  10. Profiling a gut microbiota-generated catechin metabolite's fate in human blood cells using a metabolomic approach.

    PubMed

    Mülek, Melanie; Fekete, Agnes; Wiest, Johannes; Holzgrabe, Ulrike; Mueller, Martin J; Högger, Petra

    2015-10-10

    The microbial catechin metabolite δ-(3,4-dihydroxy-phenyl)-γ-valerolactone (M1) has been found in human plasma samples after intake of maritime pine bark extract (Pycnogenol). M1 has been previously shown to accumulate in endothelial and blood cells in vitro after facilitated uptake and to exhibit anti-inflammatory activity. The purpose of the present research approach was to systematically and comprehensively analyze the metabolism of M1 in human blood cells in vitro and in vivo. A metabolomic approach that had been successfully applied for drug metabolite profiling was chosen to detect 19 metabolite peaks of M1 which were subsequently further analyzed and validated. The metabolites were categorized into three levels of identification according to the Metabolomics Standards Initiative with six compounds each confirmed at levels 1 and 2 and seven putative metabolites at level 3. The predominant metabolites were glutathione conjugates which were rapidly formed and revealed prolonged presence within the cells. Although a formation of an intracellular conjugate of M1 and glutathione (M1-GSH) was already known two GSH conjugate isomers, M1-S-GSH and M1-N-GSH were observed in the current study. Additionally detected organosulfur metabolites were conjugates with oxidized glutathione and cysteine. Other biotransformation products constituted the open-chained ester form of M1 and a methylated M1. Six of the metabolites determined in in vitro assays were also detected in blood cells in vivo after ingestion of the pine bark extract by two volunteers. The present study provides the first evidence that multiple and structurally heterogeneous polyphenol metabolites can be generated in human blood cells. The bioactivity of the M1 metabolites and their contribution to the previously determined anti-inflammatory effects of M1 now need to be elucidated.

  11. Discovery of novel metabolites from marine actinomycetes.

    PubMed

    Lam, Kin S

    2006-06-01

    Recent findings from culture-dependent and culture-independent methods have demonstrated that indigenous marine actinomycetes exist in the oceans and are widely distributed in different marine ecosystems. There is tremendous diversity and novelty among the marine actinomycetes present in marine environments. Progress has been made to isolate novel actinomycetes from samples collected at different marine environments and habitats. These marine actinomycetes produce different types of new secondary metabolites. Many of these metabolites possess biological activities and have the potential to be developed as therapeutic agents. Marine actinomycetes are a prolific but underexploited source for the discovery of novel secondary metabolites.

  12. Metabolism and metabolites of polychlorinated biphenyls.

    PubMed

    Grimm, Fabian A; Hu, Dingfei; Kania-Korwel, Izabela; Lehmler, Hans-Joachim; Ludewig, Gabriele; Hornbuckle, Keri C; Duffel, Michael W; Bergman, Åke; Robertson, Larry W

    2015-03-01

    Abstract The metabolism of polychlorinated biphenyls (PCBs) is complex and has an impact on toxicity, and thereby on the assessment of PCB risks. A large number of reactive and stable metabolites are formed in the processes of biotransformation in biota in general, and in humans in particular. The aim of this document is to provide an overview of PCB metabolism, and to identify the metabolites of concern and their occurrence. Emphasis is given to mammalian metabolism of PCBs and their hydroxyl, methylsulfonyl, and sulfated metabolites, especially those that persist in human blood. Potential intracellular targets and health risks are also discussed.

  13. Metabolism and metabolites of polychlorinated biphenyls (PCBs)

    PubMed Central

    Grimm, FA; Hu, D; Kania-Korwel, I; Lehmler, HJ; Ludewig, G; Hornbuckle, KC; Duffel, MW; Bergman, A; Robertson, LW

    2015-01-01

    The metabolism of polychlorinated biphenyls (PCBs) is complex and has an impact on toxicity and thereby assessment of PCB risks. A large number of reactive and stable metabolites are formed in the processes of biotransformation in biota in general and in humans in particular. The aim of this document is to provide an overview of PCB metabolism and to identify metabolites of concern and their occurrence. Emphasis is given to mammalian metabolism of PCBs and their hydroxyl, methylsulfonyl, and sulfated metabolites, especially those that persist in human blood. Potential intracellular targets and health risks are also discussed. PMID:25629923

  14. Biosynthesis of human diazepam and clonazepam metabolites.

    PubMed

    de Paula, Núbia C; Araujo Cordeiro, Kelly C F; de Melo Souza, Paula L; Nogueira, Diogo F; da Silva e Sousa, Diego B; Costa, Maísa B; Noël, François; de Oliveira, Valéria

    2015-03-01

    A screening of fungal and microbial strains allowed to select the best microorganisms to produce in high yields some of the human metabolites of two benzodiazepine drugs, diazepam and clonazepam, in order to study new pharmacological activities and for chemical standard proposes. Among the microorganisms tested, Cunninghamella echinulata ATCC 9244 and Rhizopus arrhizus ATCC 11145 strains, were the most active producers of the mains metabolites of diazepam which included demethylated, hydroxylated derivatives. Beauveria bassiana ATCC 7159 and Chaetomium indicum LCP 984200 produced the 7 amino-clonazepam metabolite and a product of acid hydrolysis of this benzodiazepine.

  15. Metabolite fingerprinting in transgenic lettuce.

    PubMed

    Garratt, Lee C; Linforth, Robert; Taylor, Andrew J; Lowe, Kenneth C; Power, J Brian; Davey, Michael R

    2005-03-01

    Metabolite fingerprinting has been achieved using direct atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) and linked gas chromatography (GC-APCI/EI-MS) for transgenic lettuce (Lactuca sativa L. cv. Evola) plants expressing an IPT gene under the control of the senescence-specific SAG12 promoter from Arabidopsis thaliana (P(SAG12)-IPT). Mature heads of transgenic lettuce and their azygous controls were maintained under defined conditions to assess their shelf life. Transgenic lettuce plants exhibited delayed senescence and significant increases (up to a maximum of threefold) in the concentrations of three volatile organic compounds (VOCs), corresponding to molecular masses of 45, 47 and 63, when compared with heads from azygous plants. These VOCs were identified as acetaldehyde (45), ethanol (47) and dimethyl sulphide (63). The increase in dimethyl sulphide was paralleled by an accumulation of reactive oxygen species (ROS) in the heads of transgenic plants. These results demonstrate the applicability of metabolic fingerprinting techniques to elucidate the underlying pleiotropic responses of plants to transgene expression.

  16. Proficiency study for the determination of nitrofuran metabolites in shrimps.

    PubMed

    Hurtaud-Pessel, D; Verdon, E; Blot, J; Sanders, P

    2006-06-01

    A proficiency test for the determination of nitrofuran metabolites in shrimp tissue was organized in the first half of 2003. This test was intended to allow the participants to use their routine method and to assess their competence on this specific analysis. The participation in this proficiency test was offered to all the National Reference Laboratories (NRLs) of the European Union (EU) in charge of the analysis of nitrofurans, to Official Laboratories of the then 10 Candidate Countries for entry in EU and to some countries exporting food to the EU. The participants (20) analysed nitrofuran metabolites in eight randomly coded frozen samples including three blank samples. All participants performed a confirmatory method using liquid chromatography/mass spectrometry to detect total nitrofuran metabolite residues. Both qualitative and quantitative analyses of the results were investigated. Qualitatively, 16 laboratories out of 20 gave the correct interpretation of the results in term of compliant/non-compliant sample. Quantitatively, laboratory performance was evaluated by calculating the z-scores.

  17. Analysis of Serum Metabolites to Diagnose Bicuspid Aortic Valve

    PubMed Central

    Wang, Wenshuo; Maimaiti, Aikebaier; Zhao, Yun; Zhang, Lingfei; Tao, Hongyue; Nian, Hui; Xia, Limin; Kong, Biao; Wang, Chunsheng; Liu, Mofang; Wei, Lai

    2016-01-01

    Bicuspid aortic valve (BAV) is the most common congenital heart disease. The current study aims to construct a diagnostic model based on metabolic profiling as a non-invasive tool for BAV screening. Blood serum samples were prepared from an estimation group and a validation group, each consisting of 30 BAV patients and 20 healthy individuals, and analyzed by liquid chromatography-mass spectrometry (LC-MS). In total, 2213 metabolites were detected and 41 were considered different. A model for predicting BAV in the estimation group was constructed using the concentration levels of monoglyceride (MG) (18:2) and glycerophospho-N-oleoyl ethanolamine (GNOE). A novel model named Zhongshan (ZS) was developed to amplify the association between BAV and the two metabolites. The area under curve (AUC) of ZS for BAV prediction was 0.900 (0.782–0.967) and was superior to all single-metabolite models when applied to the estimation group. Using optimized cutoff (−0.1634), ZS model had a sensitivity score of 76.7%, specificity score of 90.0%, positive predictive value of 80% and negative predictive value of 85.0% for the validation group. These results support the use of serum-based metabolomics profiling method as a complementary tool for BAV screening in large populations. PMID:27845433

  18. The profiling and identification of the metabolites of (+)-catechin and study on their distribution in rats by HPLC-DAD-ESI-IT-TOF-MS(n) technique.

    PubMed

    Liang, Jing; Xu, Feng; Zhang, Ya-Zhou; Zang, Xin-Yu; Wang, Dan; Shang, Ming-Ying; Wang, Xuan; Chui, De-Hua; Cai, Shao-Qing

    2014-03-01

    (+)-Catechin, a potential beneficial compound to human health, is widely distributed in plants and foods. A high-performance liquid chromatography with diode array detector and combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry method was applied to profile and identify the metabolites of (+)-catechin in rats and to study the distribution of these metabolites in rat organs for the first time. In total, 51 phase II metabolites (44 new) and three phase I metabolites were tentatively identified, comprising 16 (+)-catechin conjugates, 14 diarylpropan-2-ol metabolites, 6 phenyl valerolactone metabolites and 18 aromatic acid metabolites. Further, 19 phase II metabolites were new compounds. The in vivo metabolic reactions of (+)-catechin in rats were found to be ring-cleavage, sulfation, glucuronidation, methylation, dehydroxylation and dehydrogenation. The numbers of detected metabolites in urine, plasma, small intestine, kidney, liver, lung, heart, brain and spleen were 53, 23, 27, 9, 7, 5, 3, 2 and 1, respectively. This indicated that small intestine, kidney and liver were the major organs for the distribution of (+)-catechin metabolites. In addition, eight metabolites were found to possess bioactivities according to literature. These results are very helpful for better comprehension of the in vivo metabolism of (+)-catechin and its pharmacological actions, and also can give strong indications on the effective forms of (+)-catechin in vivo.

  19. A novel HPLC-MRM strategy to discover unknown and long-term metabolites of stanozolol for expanding analytical possibilities in doping-control.

    PubMed

    Wang, Zhe; Zhou, Xinmiao; Liu, Xin; Dong, Ying; Zhang, Jinlan

    2017-01-01

    Stanozolol is one of the most commonly abused anabolic androgenic steroids (AAS) by athletes and usually detected by its parent drug and major metabolites. However, its metabolic pathway is complex, varied and individually different, it is important to characterize its overall metabolic profiles and discover new and long-term metabolites for the aims of expanding detection windows. High performance liquid chromatography coupled with triple quadrupole mass spectrometer (HPLC-MS/MS) was used to analyze the human urine after oral administration of stanozolol. Multiple reaction monitoring (MRM), one of the scan modes of triple quadrupole mass spectrometer showing extremely high sensitivity was well used to develop a strategy for metabolic profiles characterization and long-term metabolites detection based on typical precursor to product ion transitions of parent drug and its major metabolites. Utilizing the characteristic fragment ions of stanozolol and its major metabolites as the product ions, and speculating unknown precursor ions based on the possible phase I and phase II metabolic reactions in human body, the metabolite profiles of stanozolol could be comprehensively discovered, especially for those unknown and low concentration metabolites in human urine. Then these metabolites were further well structure identified by targeted high resolution MS/MS scan of quadrupole-time of flight mass spectrometry (Q-TOF). Applying this strategy, 27 phase I and 21 phase II metabolites of stanozolol were identified, in which 13 phase I and 14 phase II metabolites have not been reported previously. The 9 out of 48 metabolites could be detected over 15days post drug administration. This strategy could be employed effectively to characterize AAS metabolic profiles and discover unknown and long-term metabolites in sports drug testing.

  20. Disposition of xenobiotic chemicals and metabolites in marine organisms

    SciTech Connect

    Varanasi, U.; Stein, J.E. )

    1991-01-01

    Studies with several bottom fish species from urban waterways show that of the identified xenobiotic chemicals in bottom sediments, polycyclic aromatic hydrocarbons (PAHs) are the most strongly associated with the prevalence of liver lesions, including neoplasms. Accordingly, there is concern about the transfer of contaminants, such as PAHs, from aquatic species to humans. Because PAHs exert their toxicity only after being biotransformed, increasing attention has been focused on the ability of aquatic organisms to metabolize these chemicals. Overall, the results of both laboratory and field studies show that generally low levels of a few low molecular weight PAHs may be present in edible tissue of fish from contaminated areas and that high molecular weight PAHs, such as the carcinogen benzo(a)pyrene, will rarely be detected because of extensive metabolism. Additionally, the results from a few studies suggest that even though interactions between xenobiotics can affect both biochemical and physiological systems to alter the disposition of PAHs in fish, these interactions do not markedly change the relative proportions of metabolites to parent PAH in tissues. Thus, these studies clearly demonstrate that to obtain some insight into the questions of whether there is any risk to human health from consuming fish and crustaceans from urban areas, techniques must be developed that measure metabolites of carcinogens, such as PAHs, in edible tissue. Initial attempts may focus on semiquantitative methods that permit rapid assessment of the level of metabolites in edible tissues of fish and crustaceans from many urban areas. Based on information from such screening studies, further refinement in methodology leading to identification of specific compounds may be needed because certain metabolites may not be as toxic or carcinogenic as others.

  1. Identification and quantification of metabolites of AB-CHMINACA in a urine specimen of an abuser.

    PubMed

    Wurita, Amin; Hasegawa, Koutaro; Minakata, Kayoko; Gonmori, Kunio; Nozawa, Hideki; Yamagishi, Itaru; Suzuki, Osamu; Watanabe, Kanako

    2016-03-01

    We experienced an autopsy case in which the cause of death was judged as poisoning by multiple new psychoactive substances, including AB-CHMINACA, 5-fluoro-AMB and diphenidine [Forensic Toxicol. 33 (2015): 45-53]. Although unchanged AB-CHMINACA could be detected from 8 solid tissues, it could neither be detected from blood nor urine specimens. In this article, we obtained eight kinds of reference standards of AB-CHMINACA metabolites from a commercial source. The AB-CHMINACA metabolites from the urine specimen of the abuser were extracted by a modified QuEChERS method and analyzed by liquid chromatography-tandem mass spectrometry before and after hydrolysis with β-glucuronidase. Among the eight AB-CHMINACA metabolites tested, only 2 metabolites could be identified in the urine specimen of the deceased. After hydrolysis with β-glucuronidase, the concentrations of the two metabolites were not increased, suggesting that the metabolites were not in the conjugated forms. The metabolites detected were 4-hydroxycyclohexylmethyl AB-CHMINACA (M1), followed by N-[[1-(cyclohexylmethyl)-1H-indazol-3-yl]carbonyl]-l-valine (M3). Their concentrations were 52.8 ± 3.44 and 41.3 ± 5.04 ng/ml (n=10) for M1 and M3, respectively. Although there is one preceding report showing the estimations of metabolism of AB-CHMINACA without reference standards, this is the first report dealing with exact identification using reference standards, and quantification of M1 and M3 in an authentic urine specimen.

  2. Identification and characterization of novel long term metabolites of oxymesterone and mesterolone in human urine by application of SRM GC-CI-MS/MS.

    PubMed

    Polet, Michael; Van Gansbeke, Wim; Geldof, Lore; Deventer, Koen; Van Eenoo, Peter

    2017-03-11

    The search for metabolites with longer detection times remains an important task in for example toxicology and doping control. The impact of these long term metabolites is highlighted by the high number of positive cases after reanalysis of samples that were stored for several years, e.g. samples of previous Olympic Games. A substantial number of previously alleged negative samples have now been declared positive due to the detection of various long term steroid metabolites of which the existence was unknown during the Olympic Games of 2008 and 2012. In this work, the metabolism of oxymesterone and mesterolone, two anabolic androgenic steroids (AAS), was investigated by application of a selected reaction monitoring (SRM) gas chromatography - chemical ionization - triple quadrupole mass spectrometry (GC-CI-MS/MS) protocol for metabolite detection and identification. Correlations between AAS structure and GC-CI-MS/MS fragmentation behaviour enabled the search for previously unknown but expected AAS metabolites by selection of theoretical transitions for expected metabolites. Use of different hydrolysis protocols allowed for evaluation of the detection window of both phase I and phase II metabolites. For oxymesterone, a new metabolite 18-nor-17β-hydroxymethyl-17α-methyl-4-hydroxy-androst-4,13-diene-3-one was identified. It was detectable up to 46 days by using GC-CI-MS/MS, whereas with a traditional screening (detection of metabolite 17-epioxymesterone with electron ionization GC-MS/MS) the oxymesterone administration was only detectable for 3.5 days. A new metabolite was also found for mesterolone. It was identified as 1α-methyl-5α-androstan-3,6,16-triol-17-one and its sulfate form after hydrolysis with Helix pomatia resulted in a prolonged detection time (up to 15 days) for mesterolone abuse.

  3. LC-MS/MS-based metabolites of Eurycoma longifolia (Tongkat Ali) in Malaysia (Perak and Pahang).

    PubMed

    Chua, Lee Suan; Amin, Nor Amaiza Mohd; Neo, Jason Chun Hong; Lee, Ting Hun; Lee, Chew Tin; Sarmidi, Mohamad Roji; Aziz, Ramlan Abdul

    2011-12-15

    A number of three LC-MS/MS hybrid systems (QTof, TripleTof and QTrap) has been used to profile small metabolites (m/z 100-1000) and to detect the targeted metabolites such as quassinoids, alkaloids, triterpene and biphenylneolignans from the aqueous extracts of Eurycoma longifolia. The metabolite profiles of small molecules showed four significant clusters in the principle component analysis for the aqueous extracts of E. longifolia, which had been collected from different geographical terrains (Perak and Pahang) and processed at different extraction temperatures (35°C and 100°C). A small peptide of leucine (m/z 679) and a new hydroxyl methyl β-carboline propionic acid have been identified to differentiate E. longifolia extracts that prepared at 35°C and 100°C, respectively. From the targeted metabolites identification, it was found that 3,4ɛ-dihydroeurycomanone (quassinoids) and eurylene (squalene-type triterpene) could only be detected in the Pahang extract, whereas canthin-6-one-3N-oxide could only be detected in the Perak extract. Overall, quassinoids were present in the highest concentration, particularly eurycomanone and its derivatives compared to the other groups of metabolites. However, the concentration of canthin-6-one and β-carboline alkaloids was significantly increased when the roots of the plant samples were extracted at 100°C.

  4. Untargeted metabolomics of colonic digests reveals kynurenine pathway metabolites, dityrosine and 3-dehydroxycarnitine as red versus white meat discriminating metabolites.

    PubMed

    Rombouts, Caroline; Hemeryck, Lieselot Y; Van Hecke, Thomas; De Smet, Stefaan; De Vos, Winnok H; Vanhaecke, Lynn

    2017-02-14

    Epidemiological research has demonstrated that the consumption of red meat is an important risk factor for the development of colorectal cancer (CRC), diabetes mellitus and cardiovascular diseases. However, there is no holistic insight in the (by-) products of meat digestion that may contribute to disease development. To address this hiatus, an untargeted mass spectrometry (MS)-based metabolomics approach was used to create red versus white meat associated metabolic fingerprints following in vitro colonic digestion using the fecal inocula of ten healthy volunteers. Twenty-two metabolites were unequivocally associated with simulated colonic digestion of red meat. Several of these metabolites could mechanistically be linked to red meat-associated pathways including N'-formylkynurenine, kynurenine and kynurenic acid (all involved in tryptophan metabolism), the oxidative stress marker dityrosine, and 3-dehydroxycarnitine. In conclusion, the used MS-based metabolomics platform proved to be a powerful platform for detection of specific metabolites that improve the understanding of the causal relationship between red meat consumption and associated diseases.

  5. Untargeted metabolomics of colonic digests reveals kynurenine pathway metabolites, dityrosine and 3-dehydroxycarnitine as red versus white meat discriminating metabolites

    PubMed Central

    Rombouts, Caroline; Hemeryck, Lieselot Y.; Van Hecke, Thomas; De Smet, Stefaan; De Vos, Winnok H.; Vanhaecke, Lynn

    2017-01-01

    Epidemiological research has demonstrated that the consumption of red meat is an important risk factor for the development of colorectal cancer (CRC), diabetes mellitus and cardiovascular diseases. However, there is no holistic insight in the (by-) products of meat digestion that may contribute to disease development. To address this hiatus, an untargeted mass spectrometry (MS)-based metabolomics approach was used to create red versus white meat associated metabolic fingerprints following in vitro colonic digestion using the fecal inocula of ten healthy volunteers. Twenty-two metabolites were unequivocally associated with simulated colonic digestion of red meat. Several of these metabolites could mechanistically be linked to red meat-associated pathways including N’-formylkynurenine, kynurenine and kynurenic acid (all involved in tryptophan metabolism), the oxidative stress marker dityrosine, and 3-dehydroxycarnitine. In conclusion, the used MS-based metabolomics platform proved to be a powerful platform for detection of specific metabolites that improve the understanding of the causal relationship between red meat consumption and associated diseases. PMID:28195169

  6. Coping with shrub secondary metabolites by ruminants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rangelands throughout the world contain varying but often substantial proportions of shrubs. Shrubs are generally heavily chemically defended, and herbivores must either contend with their plant secondary metabolites (PSM) or avoid a significant component of the available forage. Browsing ruminants ...

  7. Catechol conjugates are in vivo metabolites of Salicis cortex.

    PubMed

    Knuth, Susanne; Abdelsalam, Rania M; Khayyal, Mohamed T; Schweda, Frank; Heilmann, Jörg; Kees, Martin Georg; Mair, Georg; Kees, Frieder; Jürgenliemk, Guido

    2013-11-01

    After oral administration of 100 mg/kg b. w. (235.8 µmol/kg) salicortin to Wistar rats, peak serum concentrations of 1.43 mg/L (13.0 µM) catechol were detected after 0.5 h in addition to salicylic acid by HPLC-DAD after serum processing with β-glucuronidase and sulphatase. Both metabolites could also be detected in the serum of healthy volunteers following oral administration of a willow bark extract (Salicis cortex, Salix spec., Salicaceae) corresponding to 240 mg of salicin after processing with both enzymes. In humans, the cmax (1.46 mg/L, 13.3 µM) of catechol was reached after 1.2 h. The predominant phase-II metabolite in humans and rats was catechol sulphate, determined by HPLC analysis of serum samples processed with only one kind of enzyme. Without serum processing with glucuronidase and sulphatase, no unconjugated catechol could be detected in human and animal serum samples. As catechol is described as an anti-inflammatory compound, these results may contribute to the elucidation of the mechanism of the action of willow bark extract.

  8. Hydrophobicity and Charge Shape Cellular Metabolite Concentrations

    PubMed Central

    Bar-Even, Arren; Noor, Elad; Flamholz, Avi; Buescher, Joerg M.; Milo, Ron

    2011-01-01

    What governs the concentrations of metabolites within living cells? Beyond specific metabolic and enzymatic considerations, are there global trends that affect their values? We hypothesize that the physico-chemical properties of metabolites considerably affect their in-vivo concentrations. The recently achieved experimental capability to measure the concentrations of many metabolites simultaneously has made the testing of this hypothesis possible. Here, we analyze such recently available data sets of metabolite concentrations within E. coli, S. cerevisiae, B. subtilis and human. Overall, these data sets encompass more than twenty conditions, each containing dozens (28-108) of simultaneously measured metabolites. We test for correlations with various physico-chemical properties and find that the number of charged atoms, non-polar surface area, lipophilicity and solubility consistently correlate with concentration. In most data sets, a change in one of these properties elicits a ∼100 fold increase in metabolite concentrations. We find that the non-polar surface area and number of charged atoms account for almost half of the variation in concentrations in the most reliable and comprehensive data set. Analyzing specific groups of metabolites, such as amino-acids or phosphorylated nucleotides, reveals even a higher dependence of concentration on hydrophobicity. We suggest that these findings can be explained by evolutionary constraints imposed on metabolite concentrations and discuss possible selective pressures that can account for them. These include the reduction of solute leakage through the lipid membrane, avoidance of deleterious aggregates and reduction of non-specific hydrophobic binding. By highlighting the global constraints imposed on metabolic pathways, future research could shed light onto aspects of biochemical evolution and the chemical constraints that bound metabolic engineering efforts. PMID:21998563

  9. Secondary metabolites in bryophytes: an ecological aspect.

    PubMed

    Xie, Chun-Feng; Lou, Hong-Xiang

    2009-03-01

    Bryophytes frequently grow in an unfavorable environment as the earliest land plants, and inevitably biosynthesize secondary metabolites against biotic or abiotic stress. They not only defend against the plant competition, microbial attack, and insect or animal predation, but also function in UV protection, drought tolerance, and freezing survival. This review covers the ecological aspect of secondary metabolites in bryophytes and is taxonomically presented according to the ecological significances.

  10. The Significance of Lichens and Their Metabolites

    NASA Astrophysics Data System (ADS)

    Huneck, S.

    Lichens, symbiontic organisms of fungi and algae, synthesize numerous metabolites, the "lichen substances," which comprise aliphatic, cycloaliphatic, aromatic, and terpenic compounds. Lichens and their metabolites have a manifold biological activity: antiviral, antibiotic, antitumor, allergenic, plant growth inhibitory, antiherbivore, and enzyme inhibitory. Usnic acid, a very active lichen substance is used in pharmaceutical preparations. Large amounts of Pseudevernia furfuracea and Evernia prunastri are processed in the perfume industry, and some lichens are sensitive reagents for the evaluation of air pollution.

  11. Tailoring specialized metabolite production in streptomyces.

    PubMed

    Hiltner, Jana K; Hunter, Iain S; Hoskisson, Paul A

    2015-01-01

    Streptomycetes are prolific producers of a plethora of medically useful metabolites. These compounds are made by complex secondary (specialized) metabolic pathways, which utilize primary metabolic intermediates as building blocks. In this review we discuss the evolution of specialized metabolites and how expansion of gene families in primary metabolism has lead to the evolution of diversity in these specialized metabolic pathways and how developing a better understanding of expanded primary metabolic pathways can help enhance synthetic biology approaches to industrial pathway engineering.

  12. Cellular toxicity of nicotinamide metabolites.

    PubMed

    Rutkowski, Bolesław; Rutkowski, Przemysław; Słomińska, Ewa; Smolenski, Ryszard T; Swierczyński, Julian

    2012-01-01

    There are almost 100 different substances called uremic toxins. Nicotinamide derivatives are known as new family of uremic toxins. These uremic compounds play a role in an increased oxidative stress and disturbances in cellular repair processes by inhibiting poly (ADP-ribose) polymerase activity. New members of this family were discovered and described. Their toxic properties were a subject of recent studies. This study evaluated the concentration of 4-pyridone-3-carboxamid-1-β-ribonucleoside-triphosphate (4PYTP) and 4-pyridone-3-carboxamid-1-β-ribonucleoside-monophosphate (4PYMP) in erythrocytes of patients with chronic renal failure. Serum and red blood cells were collected from chronic renal failure patients on conservative treatment, those treated with hemodialysis, and at different times from those who underwent kidney transplantation. Healthy volunteers served as a control group. Nicotinamide metabolites were determined using liquid chromatography with mass spectrometry based on originally discovered and described method. Three novel compounds were described: 4-pyridone-3-carboxamid-1-β-ribonucleoside (4PYR), 4PYMP, and 4PYTP. 4PYR concentration was elevated in the serum, whereas 4PYMP and 4PYTP concentrations were augmented in erythrocytes of dialysis patients. Interestingly, concentrations of these compounds were less elevated during the treatment with erythropoietin-stimulating agents (ESAs). After successful kidney transplantation, concentrations of 4PYR and 4PYMP normalized according to the graft function, whereas that of 4PYTP was still elevated. During the incubation of erythrocytes in the presence of 4PYR, concentration of 4PYMP rose very rapidly while that of 4PYTP increased slowly. Therefore, we hypothesized that 4PYR, as a toxic compound, was actively absorbed by erythrocytes and metabolized to the 4PYMP and 4PYTP, which may interfere with function and life span of these cells.

  13. Modules of co-regulated metabolites in turmeric (Curcuma longa) rhizome suggest the existence of biosynthetic modules in plant specialized metabolism.

    PubMed

    Xie, Zhengzhi; Ma, Xiaoqiang; Gang, David R

    2009-01-01

    Turmeric is an excellent example of a plant that produces large numbers of metabolites from diverse metabolic pathways or networks. It is hypothesized that these metabolic pathways or networks contain biosynthetic modules, which lead to the formation of metabolite modules-groups of metabolites whose production is co-regulated and biosynthetically linked. To test whether such co-regulated metabolite modules do exist in this plant, metabolic profiling analysis was performed on turmeric rhizome samples that were collected from 16 different growth and development treatments, which had significant impacts on the levels of 249 volatile and non-volatile metabolites that were detected. Importantly, one of the many co-regulated metabolite modules that were indeed readily detected in this analysis contained the three major curcuminoids, whereas many other structurally related diarylheptanoids belonged to separate metabolite modules, as did groups of terpenoids. The existence of these co-regulated metabolite modules supported the hypothesis that the 3-methoxyl groups on the aromatic rings of the curcuminoids are formed before the formation of the heptanoid backbone during the biosynthesis of curcumin and also suggested the involvement of multiple polyketide synthases with different substrate selectivities in the formation of the array of diarylheptanoids detected in turmeric. Similar conclusions about terpenoid biosynthesis could also be made. Thus, discovery and analysis of metabolite modules can be a powerful predictive tool in efforts to understand metabolism in plants.

  14. The silvering process of European eel (Anguilla anguilla) influences PAH metabolite concentrations in bile fluid: consequences for monitoring.

    PubMed

    Nagel, Florian; Wagner, Carola; Hanel, Reinhold; Kammann, Ulrike

    2012-03-01

    The stock of the catadromous European eel (Anguilla anguilla L.) continues to decline and there is growing evidence that poor health status due to contaminants might be a key element in this decrease. Organic contaminants such as polycyclic aromatic hydrocarbons (PAHs) belong to the major threats to yellow eel in their growth habitat and their metabolites are detectable in the bile. Starting the silvering process eels undergo physiological and morphological changes including cessation of feeding and downstream migration back to their spawning grounds. Reduced feed intake results in a diminishment of bile production and induces accumulation of e.g. PAH-metabolites in bile. Therefore, the aim of the present study was to demonstrate the impact of silvering on biliary PAH metabolite concentrations and to utilize normalization procedures to overcome silvering related accumulation effects of PAH-metabolites in eel bile. We investigated the hydroxyl-metabolites of pyrene (1-OH Pyr) and phenantrene (1-OH Phen) in the bile of different maturation stages of eels (silvering index I-V) from nine German rivers. We detected increasing absolute PAH metabolite levels in bile during the silvering process. The highest rise could be observed at the transition from pre migration stage III to the migrating stage IV, suggesting the onset of cessation of feeding at this stage. A cessation bias in PAH metabolite measurement could be diminished by normalization of absolute values against bile pigments (A(380), biliverdin). In conclusion, we demonstrated the impact of silvering on PAH metabolite concentrations in eel bile and present suitable normalization procedures to overcome silvering related accumulation effects. Thus, for a future eel monitoring we recommend (1) to regularly monitor PAH metabolites in bile, (2) to determine silvering index of eel and (3) to normalize PAH metabolite values in bile based on maturation/silvering stages. The knowledge of the silvering stage is mandatory

  15. Identification of metabolites associated with water stress responses in Solanum tuberosum L. clones.

    PubMed

    Drapal, M; Farfan-Vignolo, E R; Gutierrez, O R; Bonierbale, M; Mihovilovich, E; Fraser, P D

    2017-03-01

    Water deficiency has become a major issue for modern agriculture as its effects on crop yields and tuber quality have become more pronounced. Potato genotypes more tolerant to water shortages have been identified through assessment of yield and dry matter. In the present study, a combination of metabolite profiling and physiological/agronomical measurements has been used to explore complex system level responses to non-lethal water restriction. The metabolites identified were associated with physiological responses in three different plant tissues (leaf, root and tuber) of five different potato genotypes varying in susceptibility/tolerance to drought. This approach explored the potential of metabolite profiling as a tool to unravel sectors of metabolism that react to stress conditions and could mirror the changes in the plant physiology. The metabolite results showed different responses of the three plant tissues to the water deficit, resulting either in different levels of the metabolites detected or different metabolites expressed. The leaf material displayed the most changes to drought as reported in literature. The results highlighted genotype-specific signatures to water restriction over all three plant tissues suggesting that the genetics can predominate over the environmental conditions. This will have important implications for future breeding approaches.

  16. Elucidation of Drug Metabolite Structural Isomers Using Molecular Modeling Coupled with Ion Mobility Mass Spectrometry.

    PubMed

    Reading, Eamonn; Munoz-Muriedas, Jordi; Roberts, Andrew D; Dear, Gordon J; Robinson, Carol V; Beaumont, Claire

    2016-02-16

    Ion mobility-mass spectrometry (IM-MS) in combination with molecular modeling offers the potential for small molecule structural isomer identification by measurement of their gas phase collision cross sections (CCSs). Successful application of this approach to drug metabolite identification would facilitate resource reduction, including animal usage, and may benefit other areas of pharmaceutical structural characterization including impurity profiling and degradation chemistry. However, the conformational behavior of drug molecules and their metabolites in the gas phase is poorly understood. Here the gas phase conformational space of drug and drug-like molecules has been investigated as well as the influence of protonation and adduct formation on the conformations of drug metabolite structural isomers. The use of CCSs, measured from IM-MS and molecular modeling information, for the structural identification of drug metabolites has also been critically assessed. Detection of structural isomers of drug metabolites using IM-MS is demonstrated and, in addition, a molecular modeling approach has been developed offering rapid conformational searching and energy assessment of candidate structures which agree with experimental CCSs. Here it is illustrated that isomers must possess markedly dissimilar CCS values for structural differentiation, the existence and extent of CCS differences being ionization state and molecule dependent. The results present that IM-MS and molecular modeling can inform on the identity of drug metabolites and highlight the limitations of this approach in differentiating structural isomers.

  17. Changes in cerebral metabolites in obstructive sleep apnea: a systemic review and meta-analysis

    PubMed Central

    Xia, Yunyan; Fu, Yiqun; Xu, Huajun; Guan, Jian; Yi, Hongliang; Yin, Shankai

    2016-01-01

    Cognitive impairment is associated with changes in cerebral metabolites in patients with obstructive sleep apnea (OSA). Several studies have used magnetic resonance spectroscopy (MRS) to detect variations in cerebral metabolites; however, the results have been inconsistent. This meta-analysis summarizes the differences in cerebral metabolites between patients with OSA and controls. Two electronic databases, PubMed and Embase, were searched for articles (published before March 31, 2016) describing studies that used MRS to evaluate the cerebral metabolite changes. The overall effects were measured using the weighted mean difference with a 95% confidence interval. Subgroup analysis and sensitivity analysis were used to explore the sources of between-study heterogeneity and the stability of the results. Publication bias was also evaluated. Thirteen studies were ultimately included. In the hippocampus, the N-acetylaspartate (NAA)/creatine ratio was lower in patients with OSA. In the frontal lobe, only the NAA/choline ratio was lower in patients with OSA. Cerebral metabolites are significantly altered in the hippocampus in patients with OSA. Further clinical studies are needed to explore the underlying mechanisms between OSA and the changes in cerebral metabolites in the brain. PMID:27349417

  18. MSM, an Efficient Workflow for Metabolite Identification Using Hybrid Linear Ion Trap Orbitrap Mass Spectrometer

    NASA Astrophysics Data System (ADS)

    Cho, Robert; Huang, Yingying; Schwartz, Jae C.; Chen, Yan; Carlson, Timothy J.; Ma, Ji

    2012-05-01

    Identification of drug metabolites can often yield important information regarding clearance mechanism, pharmacologic activity, or toxicity for drug candidate molecules. Additionally, the identification of metabolites can provide beneficial structure-activity insight to help guide lead optimization efforts towards molecules with optimal metabolic profiles. There are challenges associated with detecting and identifying metabolites in the presence of complex biological matrices, and new LC-MS technologies have been developed to meet these challenges. In this report, we describe the development of an experimental approach that applies unique features of the hybrid linear ion trap Orbitrap mass spectrometer to streamline in vitro and in vivo metabolite identification experiments. The approach, referred to as MSM, utilizes multiple collision cells, dissociation methods, mass analyzers, and detectors. With multiple scan types and different dissociation modes built into one experimental method, along with flexible post-acquisition analysis options, the MSM workflow offers an attractive option to fast and reliable identification of metabolites in different kinds of in vitro and in vivo samples. The MSM workflow was successfully applied to metabolite identification analysis of verapamil in both in vitro rat hepatocyte incubations and in vivo rat bile samples.

  19. Elevated cytokinin content in ipt transgenic creeping bentgrass promotes drought tolerance through regulating metabolite accumulation.

    PubMed

    Merewitz, Emily B; Du, Hongmei; Yu, Wenjuan; Liu, Yimin; Gianfagna, Thomas; Huang, Bingru

    2012-02-01

    Increased endogenous plant cytokinin (CK) content through transformation with an adenine isopentyl transferase (ipt) gene has been associated with improved plant drought tolerance. The objective of this study is to determine metabolic changes associated with elevated CK production in ipt transgenic creeping bentgrass (Agrostis stolonifera L.) with improved drought tolerance. Null transformants (NTs) and plants transformed with ipt controlled by a stress- or senescence-activated promoter (SAG12-ipt) were exposed to well-watered conditions or drought stress by withholding irrigation in an environmental growth chamber. Physiological analysis confirmed that the SAG12-ipt line (S41) had improved drought tolerance compared with the NT plants. Specific metabolite changes over the course of drought stress and differential accumulation of metabolites in SAG12-ipt plants compared with NT plants at the same level of leaf relative water content (47% RWC) were identified using gas chromatography-mass spectroscopy. The metabolite profiling analysis detected 45 metabolites differentially accumulated in response to ipt expression or drought stress, which included amino acids, carbohydrates, organic acids, and organic alcohols. The enhanced drought tolerance of SAG12-ipt plants was associated with the maintenance of accumulation of several metabolites, particularly amino acids (proline, γ-aminobutyric acid, alanine, and glycine) carbohydrates (sucrose, fructose, maltose, and ribose), and organic acids that are mainly involved in the citric acid cycle. The accumulation of these metabolites could contribute to improved drought tolerance due to their roles in the stress response pathways such as stress signalling, osmotic adjustment, and respiration for energy production.

  20. Metabolites of MDMA induce oxidative stress and contractile dysfunction in adult rat left ventricular myocytes.

    PubMed

    Shenouda, Sylvia K; Varner, Kurt J; Carvalho, Felix; Lucchesi, Pamela A

    2009-03-01

    Repeated administration of 3,4-methylenedioxymethamphetamine (MDMA) (ecstasy) produces eccentric left ventricular (LV) dilation and diastolic dysfunction. While the mechanism(s) underlying this toxicity are unknown, oxidative stress plays an important role. MDMA is metabolized into redox cycling metabolites that produce superoxide. In this study, we demonstrated that metabolites of MDMA induce oxidative stress and contractile dysfunction in adult rat left ventricular myocytes. Metabolites of MDMA used in this study included alpha-methyl dopamine, N-methyl alpha-methyl dopamine and 2,5-bis(glutathion-S-yl)-alpha-MeDA. Dihydroethidium was used to detect drug-induced increases in reactive oxygen species (ROS) production in ventricular myocytes. Contractile function and changes in intracellular calcium transients were measured in paced (1 Hz), Fura-2 AM loaded, myocytes using the IonOptix system. Production of ROS in ventricular myocytes treated with MDMA was not different from control. In contrast, all three metabolites of MDMA exhibited time- and concentration-dependent increases in ROS that were prevented by N-acetyl-cysteine (NAC). The metabolites of MDMA, but not MDMA alone, significantly decreased contractility and impaired relaxation in myocytes stimulated at 1 Hz. These effects were prevented by NAC. Together, these data suggest that MDMA-induced oxidative stress in the left ventricle can be due, at least in part, to the metabolism of MDMA to redox active metabolites.

  1. Metabolite profiling of wheat grains (Triticum aestivum L.) from organic and conventional agriculture.

    PubMed

    Zörb, Christian; Langenkämper, Georg; Betsche, Thomas; Niehaus, Karsten; Barsch, Aiko

    2006-10-18

    In some European community countries up to 8% of the agricultural area is managed organically. The aim was to obtain a metabolite profile for wheat (Triticum aestivum L.) grains grown under comparable organic and conventional conditions. These conditions cannot be found in plant material originating from different farms or from products purchased in supermarkets. Wheat grains from a long-term biodynamic, bioorganic, and conventional farming system from the harvest 2003 from Switzerland were analyzed. The presented data show that using a high throughput GC-MS technique, it was possible to determine relative levels of a set of 52 different metabolites including amino acids, organic acids, sugars, sugar alcohols, sugar phosphates, and nucleotides from wheat grains. Within the metabolites from all field trials, there was at the most a 50% reduction comparing highest and lowest mean values. The statistical analysis of the data shows that the metabolite status of the wheat grain from organic and mineralic farming did not differ in concentrations of 44 metabolites. This result indicates no impact or a small impact of the different farming systems. In consequence, we did not detect extreme differences in metabolite composition and quality of wheat grains.

  2. Genome-wide association study identifies novel genetic variants contributing to variation in blood metabolite levels.

    PubMed

    Draisma, Harmen H M; Pool, René; Kobl, Michael; Jansen, Rick; Petersen, Ann-Kristin; Vaarhorst, Anika A M; Yet, Idil; Haller, Toomas; Demirkan, Ayşe; Esko, Tõnu; Zhu, Gu; Böhringer, Stefan; Beekman, Marian; van Klinken, Jan Bert; Römisch-Margl, Werner; Prehn, Cornelia; Adamski, Jerzy; de Craen, Anton J M; van Leeuwen, Elisabeth M; Amin, Najaf; Dharuri, Harish; Westra, Harm-Jan; Franke, Lude; de Geus, Eco J C; Hottenga, Jouke Jan; Willemsen, Gonneke; Henders, Anjali K; Montgomery, Grant W; Nyholt, Dale R; Whitfield, John B; Penninx, Brenda W; Spector, Tim D; Metspalu, Andres; Eline Slagboom, P; van Dijk, Ko Willems; 't Hoen, Peter A C; Strauch, Konstantin; Martin, Nicholas G; van Ommen, Gert-Jan B; Illig, Thomas; Bell, Jordana T; Mangino, Massimo; Suhre, Karsten; McCarthy, Mark I; Gieger, Christian; Isaacs, Aaron; van Duijn, Cornelia M; Boomsma, Dorret I

    2015-06-12

    Metabolites are small molecules involved in cellular metabolism, which can be detected in biological samples using metabolomic techniques. Here we present the results of genome-wide association and meta-analyses for variation in the blood serum levels of 129 metabolites as measured by the Biocrates metabolomic platform. In a discovery sample of 7,478 individuals of European descent, we find 4,068 genome- and metabolome-wide significant (Z-test, P < 1.09 × 10(-9)) associations between single-nucleotide polymorphisms (SNPs) and metabolites, involving 59 independent SNPs and 85 metabolites. Five of the fifty-nine independent SNPs are new for serum metabolite levels, and were followed-up for replication in an independent sample (N = 1,182). The novel SNPs are located in or near genes encoding metabolite transporter proteins or enzymes (SLC22A16, ARG1, AGPS and ACSL1) that have demonstrated biomedical or pharmaceutical importance. The further characterization of genetic influences on metabolic phenotypes is important for progress in biological and medical research.

  3. Metabolomics reveals significant variations in metabolites and correlations regarding the maturation of walnuts (Juglans regia L.)

    PubMed Central

    Rao, Guodong; Sui, Jinkai

    2016-01-01

    ABSTRACT The content of walnut metabolites is related to its nutritive value and physiological characteristics, however, comprehensive information concerning the metabolome of walnut kernels is limited. In this study we analyzed the metabolites of walnut kernels at five developmental stages from filling to ripening using GC-MS-based untargeted metabolomics; of a total 252 peaks identified, 85 metabolites were positively identified. Further statistical analysis revealed that these 85 metabolites covered different types of metabolism pathways. PCA scores revealed that the metabolic compositions of the embryo are different at each stage, while the metabolic composition of the endotesta could not be significantly separated into distinct groups. Additionally, 7225 metabolite-metabolite correlations were detected in walnut kernel by a Pearson correlation coefficient approach; during screening of the calculated correlations, 463 and 1047 were determined to be significant with r2≥0.49 and had a false discovery rate (FDR) ≤0.05 in endotesta and embryo, respectively. This work provides the first comprehensive metabolomic study of walnut kernels and reveals that most of the carbohydrate and protein-derived carbon was transferred into other compounds, such as fatty acids, during the maturation of walnuts, which may potentially provide the basis for further studies on walnut kernel metabolism. PMID:27215321

  4. Bacterial dynamics and metabolite changes in solid-state acetic acid fermentation of Shanxi aged vinegar.

    PubMed

    Li, Sha; Li, Pan; Liu, Xiong; Luo, Lixin; Lin, Weifeng

    2016-05-01

    Solid-state acetic acid fermentation (AAF), a natural or semi-controlled fermentation process driven by reproducible microbial communities, is an important technique to produce traditional Chinese cereal vinegars. Highly complex microbial communities and metabolites are involved in traditional Chinese solid-state AAF, but the association between microbiota and metabolites during this process are still poorly understood. In this study, we performed amplicon 16S rRNA gene sequencing on the Illumina MiSeq platform, PCR-denaturing gradient gel electrophoresis, and metabolite analysis to trace the bacterial dynamics and metabolite changes under AAF process. A succession of bacterial assemblages was observed during the AAF process. Lactobacillales dominated all the stages. However, Acetobacter species in Rhodospirillales were considerably accelerated during AAF until the end of fermentation. Quantitative PCR results indicated that the biomass of total bacteria showed a "system microbe self-domestication" process in the first 3 days, and then peaked at the seventh day before gradually decreasing until the end of AAF. Moreover, a total of 88 metabolites, including 8 organic acids, 16 free amino acids, and 66 aroma compounds were detected during AAF. Principal component analysis and cluster analyses revealed the high correlation between the dynamics of bacterial community and metabolites.

  5. 40 CFR 159.179 - Metabolites, degradates, contaminants, and impurities.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Metabolites, degradates, contaminants.../Benefit Information § 159.179 Metabolites, degradates, contaminants, and impurities. (a) Metabolites and degradates. Information which shows the existence of any metabolite or degradate of a pesticide product...

  6. 40 CFR 159.179 - Metabolites, degradates, contaminants, and impurities.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Metabolites, degradates, contaminants.../Benefit Information § 159.179 Metabolites, degradates, contaminants, and impurities. (a) Metabolites and degradates. Information which shows the existence of any metabolite or degradate of a pesticide product...

  7. 40 CFR 159.179 - Metabolites, degradates, contaminants, and impurities.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Metabolites, degradates, contaminants.../Benefit Information § 159.179 Metabolites, degradates, contaminants, and impurities. (a) Metabolites and degradates. Information which shows the existence of any metabolite or degradate of a pesticide product...

  8. 40 CFR 159.179 - Metabolites, degradates, contaminants, and impurities.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 24 2014-07-01 2014-07-01 false Metabolites, degradates, contaminants.../Benefit Information § 159.179 Metabolites, degradates, contaminants, and impurities. (a) Metabolites and degradates. Information which shows the existence of any metabolite or degradate of a pesticide product...

  9. 40 CFR 159.179 - Metabolites, degradates, contaminants, and impurities.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Metabolites, degradates, contaminants.../Benefit Information § 159.179 Metabolites, degradates, contaminants, and impurities. (a) Metabolites and degradates. Information which shows the existence of any metabolite or degradate of a pesticide product...

  10. High-performance liquid chromatography-mass spectrometry analysis of plant metabolites in brassicaceae.

    PubMed

    De Vos, Ric C H; Schipper, Bert; Hall, Robert D

    2012-01-01

    The Brassicaceae family comprises a variety of plant species that are of high economic importance as -vegetables or industrial crops. This includes crops such as Brassica rapa (turnip, Bok Choi), B. oleracea (cabbages, broccoli, cauliflower, etc.), and B. napus (oil seed rape), and also includes the famous genetic model of plant research, Arabidopsis thaliana (thale cress). Brassicaceae plants contain a large variety of interesting secondary metabolites, including glucosinolates, hydroxycinnamic acids, and flavonoids. These metabolites are also of particular importance due to their proposed positive effects on human health. Next to these well-known groups of phytochemicals, many more metabolites are of course also present in crude extracts prepared from Brassica and Arabidopsis plant material.High-pressure liquid chromatography coupled to mass spectrometry (HPLC-MS), especially if combined with a high mass resolution instrument such as a QTOF MS, is a powerful approach to separate, detect, and annotate metabolites present in crude aqueous-alcohol plant extracts. Using an essentially unbiased procedure that takes into account all metabolite mass signals from the raw data files, detailed information on the relative abundance of hundreds of both known and, as yet, unknown semipolar metabolites can be obtained. These comprehensive metabolomics data can then be used to, for instance, identify genetic markers regulating metabolic composition, determine effects of (a)biotic stress or specific growth conditions, or establish metabolite changes occurring upon food processing or storage.This chapter describes in detail a procedure for preparing crude extracts and performing comprehensive HPLC-QTOF MS-based profiling of semi-polar metabolites in Brassicaceae plant material. Compounds present in the extract can be (partially or completely) annotated based on their accurate mass, their MS/MS fragments and on other specific chemical characteristics such as retention time and UV

  11. Microbial secondary metabolites in school buildings inspected for moisture damage in Finland, The Netherlands and Spain.

    PubMed

    Peitzsch, Mirko; Sulyok, Michael; Täubel, Martin; Vishwanath, Vinay; Krop, Esmeralda; Borràs-Santos, Alicia; Hyvärinen, Anne; Nevalainen, Aino; Krska, Rudolf; Larsson, Lennart

    2012-08-01

    Secondary metabolites produced by fungi and bacteria are among the potential agents that contribute to adverse health effects observed in occupants of buildings affected by moisture damage, dampness and associated microbial growth. However, few attempts have been made to assess the occurrence of these compounds in relation to moisture damage and dampness in buildings. This study conducted in the context of the HITEA project (Health Effects of Indoor Pollutants: Integrating microbial, toxicological and epidemiological approaches) aimed at providing systematic information on the prevalence of microbial secondary metabolites in a large number of school buildings in three European countries, considering both buildings with and without moisture damage and/or dampness observations. In order to address the multitude and diversity of secondary metabolites a large number of more than 180 analytes was targeted in settled dust and surface swab samples using liquid chromatography/mass spectrometry (LC/MS) based methodology. While 42%, 58% and 44% of all samples collected in Spanish, Dutch and Finnish schools, respectively, were positive for at least one of the metabolites analyzed, frequency of detection for the individual microbial secondary metabolites - with the exceptions of emodin, certain enniatins and physcion - was low, typically in the range of and below 10% of positive samples. In total, 30 different fungal and bacterial secondary metabolites were found in the samples. Some differences in the metabolite profiles were observed between countries and between index and reference school buildings. A major finding in this study was that settled dust derived from moisture damaged, damp schools contained larger numbers of microbial secondary metabolites at higher levels compared to respective dust samples from schools not affected by moisture damage and dampness. This observation was true for schools in each of the three countries, but became statistically significant only

  12. Novel and Distinct Metabolites Identified Following a Single Oral Dose of α- or γ-Hexabromocyclododecane in Mice

    PubMed Central

    Szabo, David T.; Huwe, Janice; Diliberto, Janet; Birnbaum, Linda S.

    2013-01-01

    The metabolism of α- and γ-hexabromocyclododecane (HBCD) was investigated in adult C57BL/6 female mice. α- or γ-[14C]HBCD (3 mg/kg bw) was orally administered with subsequent urine and feces collection for 4 consecutive days; a separate group of mice were dosed and sacrificed 3 hours post-exposure to investigate tissue metabolite levels. Extractable and non-extractable HBCD metabolites were quantitated in liver, blood, fat, brain, bile, urine and feces and characterized by LC/MS (ESI-). Metabolites identified were distinct between the two stereoisomers. In mice exposed to α-HBCD, four hydroxylated metabolites were detected in fecal extracts, and one of these metabolite isomers was consistently characterized in liver, brain, and adipose tissue extracts. In contrast, mice exposed to γ-HBCD contained multiple isomers of monohydroxy-pentabromocyclododecene, dihydroxy-pentabromocyclododecene, and dihydroxy-pentabromocyclododecadiene in the feces while only a single monohydroxy-pentabromocyclododecane metabolite was measured in liver and adipose tissue. Both stereoisomers were transformed to metabolites which formed covalent bonds to proteins and/or lipids in the gut as evidenced by high fecal non-extractables. Although the potential toxicity of these free and bound metabolites remains to be determined, the presence of distinct metabolic products from the two main HBCD stereoisomers should allow biomarkers to be selected that may aid in characterizing sources of HBCD exposure. PMID:23171393

  13. The networks of human gut microbe-metabolite associations are different between health and irritable bowel syndrome.

    PubMed

    Shankar, Vijay; Homer, Daniel; Rigsbee, Laura; Khamis, Harry J; Michail, Sonia; Raymer, Michael; Reo, Nicholas V; Paliy, Oleg

    2015-08-01

    The goal of this study was to determine if fecal metabolite and microbiota profiles can serve as biomarkers of human intestinal diseases, and to uncover possible gut microbe-metabolite associations. We employed proton nuclear magnetic resonance to measure fecal metabolites of healthy children and those diagnosed with diarrhea-predominant irritable bowel syndrome (IBS-D). Metabolite levels were associated with fecal microbial abundances. Using several ordination techniques, healthy and irritable bowel syndrome (IBS) samples could be distinguished based on the metabolite profiles of fecal samples, and such partitioning was congruent with the microbiota-based sample separation. Measurements of individual metabolites indicated that the intestinal environment in IBS-D was characterized by increased proteolysis, incomplete anaerobic fermentation and possible change in methane production. By correlating metabolite levels with abundances of microbial genera, a number of statistically significant metabolite-genus associations were detected in stools of healthy children. No such associations were evident for IBS children. This finding complemented the previously observed reduction in the number of microbe-microbe associations in the distal gut of the same cohort of IBS-D children.

  14. Unambiguous Metabolite Identification in High-Throughput Metabolomics by Hybrid 1H-NMR/ESI-MS1 Approach

    SciTech Connect

    2016-10-18

    The invention improves accuracy of metabolite identification by combining direct infusion ESI-MS with one-dimensional 1H-NMR spectroscopy. First, we apply a standard 1H-NMR metabolite identification protocol by matching the chemical shift, J-coupling and intensity information of experimental NMR signals against the NMR signals of standard metabolites in a metabolomics reference libraries. This generates a list of candidate metabolites. The list contains both false positive and ambiguous identifications. The software tool (the invention) takes the list of candidate metabolites, generated from NMRbased metabolite identification, and then calculates, for each of the candidate metabolites, the monoisotopic mass-tocharge (m/z) ratios for each commonly observed ion, fragment and adduct feature. These are then used to assign m/z ratios in experimental ESI-MS spectra of the same sample. Detection of the signals of a given metabolite in both NMR and MS spectra resolves the ambiguities, and therefore, significantly improves the confidence of the identification.

  15. Detection of Rare Drug Resistance Mutations by Digital PCR in a Human Influenza A Virus Model System and Clinical Samples.

    PubMed

    Whale, Alexandra S; Bushell, Claire A; Grant, Paul R; Cowen, Simon; Gutierrez-Aguirre, Ion; O'Sullivan, Denise M; Žel, Jana; Milavec, Mojca; Foy, Carole A; Nastouli, Eleni; Garson, Jeremy A; Huggett, Jim F

    2016-02-01

    Digital PCR (dPCR) is being increasingly used for the quantification of sequence variations, including single nucleotide polymorphisms (SNPs), due to its high accuracy and precision in comparison with techniques such as quantitative PCR (qPCR) and melt curve analysis. To develop and evaluate dPCR for SNP detection using DNA, RNA, and clinical samples, an influenza virus model of resistance to oseltamivir (Tamiflu) was used. First, this study was able to recognize and reduce off-target amplification in dPCR quantification, thereby enabling technical sensitivities down to 0.1% SNP abundance at a range of template concentrations, a 50-fold improvement on the qPCR assay used routinely in the clinic. Second, a method was developed for determining the false-positive rate (background) signal. Finally, comparison of dPCR with qPCR results on clinical samples demonstrated the potential impact dPCR could have on clinical research and patient management by earlier (trace) detection of rare drug-resistant sequence variants. Ultimately this could reduce the quantity of ineffective drugs taken and facilitate early switching to alternative medication when available. In the short term such methods could advance our understanding of microbial dynamics and therapeutic responses in a range of infectious diseases such as HIV, viral hepatitis, and tuberculosis. Furthermore, the findings presented here are directly relevant to other diagnostic areas, such as the detection of rare SNPs in malignancy, monitoring of graft rejection, and fetal screening.

  16. KNApSAcK Metabolite Activity Database for retrieving the relationships between metabolites and biological activities.

    PubMed

    Nakamura, Yukiko; Afendi, Farit Mochamad; Parvin, Aziza Kawsar; Ono, Naoaki; Tanaka, Ken; Hirai Morita, Aki; Sato, Tetsuo; Sugiura, Tadao; Altaf-Ul-Amin, Md; Kanaya, Shigehiko

    2014-01-01

    Databases (DBs) are required by various omics fields because the volume of molecular biology data is increasing rapidly. In this study, we provide instructions for users and describe the current status of our metabolite activity DB. To facilitate a comprehensive understanding of the interactions between the metabolites of organisms and the chemical-level contribution of metabolites to human health, we constructed a metabolite activity DB known as the KNApSAcK Metabolite Activity DB. It comprises 9,584 triplet relationships (metabolite-biological activity-target species), including 2,356 metabolites, 140 activity categories, 2,963 specific descriptions of biological activities and 778 target species. Approximately 46% of the activities described in the DB are related to chemical ecology, most of which are attributed to antimicrobial agents and plant growth regulators. The majority of the metabolites with antimicrobial activities are flavonoids and phenylpropanoids. The metabolites with plant growth regulatory effects include plant hormones. Over half of the DB contents are related to human health care and medicine. The five largest groups are toxins, anticancer agents, nervous system agents, cardiovascular agents and non-therapeutic agents, such as flavors and fragrances. The KNApSAcK Metabolite Activity DB is integrated within the KNApSAcK Family DBs to facilitate further systematized research in various omics fields, especially metabolomics, nutrigenomics and foodomics. The KNApSAcK Metabolite Activity DB could also be utilized for developing novel drugs and materials, as well as for identifying viable drug resources and other useful compounds.

  17. Pharmacokinetic profiles of the active metamizole metabolites in healthy horses.

    PubMed

    Giorgi, M; Aupanun, S; Lee, H-K; Poapolathep, A; Rychshanova, R; Vullo, C; Faillace, V; Laus, F

    2017-04-01

    Metamizole (MT) is an analgesic and antipyretic drug labelled for use in humans, horses, cattle, swine and dogs. MT is rapidly hydrolysed to the active primary metabolite 4-methylaminoantipyrine (MAA). MAA is formed in much larger amounts compared with other minor metabolites. Among the other secondary metabolites, 4-aminoantipyrine (AA) is also relatively active. The aim of this research was to evaluate the pharmacokinetic profiles of MAA and AA after dose of 25 mg/kg MT by intravenous (i.v.) and intramuscular (i.m.) routes in healthy horses. Six horses were randomly allocated to two equally sized treatment groups according to a 2 × 2 crossover study design. Blood was collected at predetermined times within 24 h, and plasma was analysed by a validated HPLC-UV method. No behavioural changes or alterations in health parameters were observed in the i.v. or i.m. groups of animals during or after (up to 7 days) drug administration. Plasma concentrations of MAA after i.v. and i.m. administrations of MT were detectable from 5 min to 10 h in all the horses. Plasma concentrations of AA were detectable in the same range of time, but in smaller amounts. Maximum concentration (Cmax ), time to maximum concentration (Tmax ) and AUMC0-last of MAA were statistically different between the i.v. and i.m. groups. The AUCIM /AUCIV ratio of MAA was 1.06. In contrast, AUC0-last of AA was statistically different between the groups (P < 0.05) with an AUCIM /AUCIV ratio of 0.54. This study suggested that the differences in the MAA and AA plasma concentrations found after i.m. and i.v. administrations of MT might have minor consequences on the pharmacodynamics of the drug.

  18. Polyamines and Their Metabolites as Diagnostic Markers of Human Diseases

    PubMed Central

    Park, Myung Hee; Igarashi, Kazuei

    2013-01-01

    Polyamines, putrescine, spermidine and spermine, are ubiquitous in living cells and are essential for eukaryotic cell growth. These polycations interact with negatively charged molecules such as DNA, RNA, acidic proteins and phospholipids and modulate various cellular functions including macromolecular synthesis. Dysregulation of the polyamine pathway leads to pathological conditions including cancer, inflammation, stroke, renal failure and diabetes. Increase in polyamines and polyamine synthesis enzymes is often associated with tumor growth, and urinary and plasma contents of polyamines and their metabolites have been investigated as diagnostic markers for cancers. Of these, diacetylated derivatives of spermidine and spermine are elevated in the urine of cancer patients and present potential markers for early detection. Enhanced catabolism of cellular polyamines by polyamine oxidases (PAO), spermine oxidase (SMO) or acetylpolyamine oxidase (AcPAO), increases cellular oxidative stress and generates hydrogen peroxide and a reactive toxic metabolite, acrolein, which covalently incorporates into lysine residues of cellular proteins. Levels of protein-conjuagated acrolein (PC-Acro) and polyamine oxidizing enzymes were increased in the locus of brain infarction and in plasma in a mouse model of stroke and also in the plasma of stroke patients. When the combined measurements of PC-Acro, interleukin 6 (IL-6), and C-reactive protein (CRP) were evaluated, even silent brain infarction (SBI) was detected with high sensitivity and specificity. Considering that there are no reliable biochemical markers for early stage of stroke, PC-Acro and PAOs present promising markers. Thus the polyamine metabolites in plasma or urine provide useful tools in early diagnosis of cancer and stroke. PMID:24009852

  19. Multifaceted Investigation of Metabolites During Nitrogen Fixation in Medicago via High Resolution MALDI-MS Imaging and ESI-MS

    NASA Astrophysics Data System (ADS)

    Gemperline, Erin; Jayaraman, Dhileepkumar; Maeda, Junko; Ané, Jean-Michel; Li, Lingjun

    2015-01-01

    Legumes have developed the unique ability to establish a symbiotic relationship with soil bacteria known as rhizobia. This interaction results in the formation of root nodules in which rhizobia thrive and reduce atmospheric dinitrogen into plant-usable ammonium through biological nitrogen fixation (BNF). Owing to the availability of genetic information for both of the symbiotic partners, the Medicago truncatula- Sinorhizobium meliloti association is an excellent model for examining the BNF process. Although metabolites are important in this symbiotic association, few studies have investigated the array of metabolites that influence this process. Of these studies, most target only a few specific metabolites, the roles of which are either well known or are part of a well-characterized metabolic pathway. Here, we used a multifaceted mass spectrometric (MS) approach to detect and identify the key metabolites that are present during BNF using the Medicago truncatula- Sinorhizobium meliloti association as the model system. High mass accuracy and high resolution matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) Orbitrap instruments were used in this study and provide complementary results for more in-depth characterization of the nitrogen-fixation process. We used well-characterized plant and bacterial mutants to highlight differences between the metabolites that are present in functional versus nonfunctional nodules. Our study highlights the benefits of using a combination of mass spectrometric techniques to detect differences in metabolite composition and the distributions of these metabolites in plant biology.

  20. Degradation of chloroacetanilide herbicides: The prevalence of sulfonic and oxanilic acid metabolites in Iowa groundwaters and surface waters

    USGS Publications Warehouse

    Kalkhoff, S.J.; Kolpin, D.W.; Thurman, E.M.; Ferrer, I.; Barcelo, D.

    1998-01-01

    Water samples were collected from 88 municipal wells throughout Iowa during the summer and were collected monthly at 12 stream sites in eastern Iowa from March to December 1996 to study the occurrence of the sulfonic and oxanilic metabolites of acetochlor, alachlor, and metolachlor. The sulfonic and oxanilic metabolites were present in almost 75% of the groundwater samples and were generally present from 3 to 45 times more frequently than their parent compounds. In groundwater, the median value of the summed concentrations of acetochlor, alachlor, and metolachlor was less than 0.05 μg/L, and the median value of the summed concentrations of the six metabolites was 1.2 μg/L. All surface water samples contained at least one detectable metabolite compound. Individual metabolites were detected from 2 to over 100 times more frequently than the parent compounds. In surface water, the median value of the summed concentrations of the three parent compounds was 0.13 μg/L, and the median value of the summed concentrations of the six metabolites was 6.4 μg/L. These data demonstrate the importance of analyzing both parent compounds and metabolites to more fully understand the environmental fate and transport of herbicides in the hydrologic system.

  1. Multifaceted Investigation of Metabolites During Nitrogen Fixation in Medicago via High Resolution MALDI-MS Imaging and ESI-MS

    PubMed Central

    Gemperline, Erin; Jayaraman, Dhileepkumar; Maeda, Junko; Ané, Jean-Michel; Li, Lingjun

    2014-01-01

    Legumes have developed the unique ability to establish a symbiotic relationship with soil bacteria known as rhizobia. This interaction results in the formation of root nodules in which rhizobia thrive and reduce atmospheric dinitrogen into plant-usable ammonium through biological nitrogen fixation (BNF). Due to the availability of genetic information for both of the symbiotic partners, the Medicago truncatula–Sinorhizobium meliloti association is an excellent model for examining the BNF process. Although metabolites are important in this symbiotic association, few studies have investigated the array of metabolites that influence this process. Of these studies, most target only a few specific metabolites, the roles of which are either well known or are part of a well-characterized metabolic pathway. Here, we used a multifaceted mass spectrometric (MS) approach to detect and identify the key metabolites that are present during BNF using the Medicago truncatula–Sinorhizobium meliloti association as the model system. High mass accuracy and high resolution matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) Orbitrap instruments were used in this study and provide complementary results for more in-depth characterization of the nitrogen-fixation process. We used well-characterized plant and bacterial mutants to highlight differences between the metabolites that are present in functional vs. non-functional nodules. Our study highlights the benefits of using a combination of mass spectrometric techniques to detect differences in metabolite composition and the distributions of these metabolites in plant biology. PMID:25323862

  2. Cortical metabolites as biomarkers in the R6/2 model of Huntington's disease.

    PubMed

    Zacharoff, Lori; Tkac, Ivan; Song, Qingfeng; Tang, Chuanning; Bolan, Patrick J; Mangia, Silvia; Henry, Pierre-Gilles; Li, Tongbin; Dubinsky, Janet M

    2012-03-01

    To improve the ability to move from preclinical trials in mouse models of Huntington's disease (HD) to clinical trials in humans, biomarkers are needed that can track similar aspects of disease progression across species. Brain metabolites, detectable by magnetic resonance spectroscopy (MRS), have been suggested as potential biomarkers in HD. In this study, the R6/2 transgenic mouse model of HD was used to investigate the relative sensitivity of the metabolite profiling and the brain volumetry to anticipate the disease progression. Magnetic resonance imaging (MRI) and (1)H MRS data were acquired at 9.4 T from the R6/2 mice and wild-type littermates at 4, 8, 12, and 15 weeks. Brain shrinkage was detectable in striatum, cortex, thalamus, and hypothalamus by 12 weeks. Metabolite changes in cortex paralleled and sometimes preceded those in striatum. The entire set of metabolite changes was compressed into principal components (PCs) using Partial Least Squares-Discriminant Analysis (PLS-DA) to increase the sensitivity for monitoring disease progression. In comparing the efficacy of volume and metabolite measurements, the cortical PC1 emerged as the most sensitive single biomarker, distinguishing R6/2 mice from littermates at all time points. Thus, neurochemical changes precede volume shrinkage and become potential biomarkers for HD mouse models.

  3. Simultaneous quantification of acetanilide herbicides and their oxanilic and sulfonic acid metabolites in natural waters.

    PubMed

    Heberle, S A; Aga, D S; Hany, R; Müller, S R

    2000-02-15

    This paper describes a procedure for simultaneous enrichment, separation, and quantification of acetanilide herbicides and their major ionic oxanilic acid (OXA) and ethanesulfonic acid (ESA) metabolites in groundwater and surface water using Carbopack B as a solid-phase extraction (SPE) material. The analytes adsorbed on Carbopack B were eluted selectively from the solid phase in three fractions containing the parent compounds (PCs), their OXA metabolites, and their ESA metabolites, respectively. The complete separation of the three compound classes allowed the analysis of the neutral PCs (acetochlor, alachlor, and metolachlor) and their methylated OXA metabolites by gas chromatography/mass spectrometry. The ESA compounds were analyzed by high-performance liquid chromatography with UV detection. The use of Carbopack B resulted in good recoveries of the polar metabolites even from large sample volumes (1 L). Absolute recoveries from spiked surface and groundwater samples ranged between 76 and 100% for the PCs, between 41 and 91% for the OXAs, and between 47 and 96% for the ESAs. The maximum standard deviation of the absolute recoveries was 12%. The method detection limits are between 1 and 8 ng/L for the PCs, between 1 and 7 ng/L for the OXAs, and between 10 and 90 ng/L for the ESAs.

  4. Salmonella Typhi and Salmonella Paratyphi A elaborate distinct systemic metabolite signatures during enteric fever.

    PubMed

    Näsström, Elin; Vu Thieu, Nga Tran; Dongol, Sabina; Karkey, Abhilasha; Voong Vinh, Phat; Ha Thanh, Tuyen; Johansson, Anders; Arjyal, Amit; Thwaites, Guy; Dolecek, Christiane; Basnyat, Buddha; Baker, Stephen; Antti, Henrik

    2014-06-05

    The host-pathogen interactions induced by Salmonella Typhi and Salmonella Paratyphi A during enteric fever are poorly understood. This knowledge gap, and the human restricted nature of these bacteria, limit our understanding of the disease and impede the development of new diagnostic approaches. To investigate metabolite signals associated with enteric fever we performed two dimensional gas chromatography with time-of-flight mass spectrometry (GCxGC/TOFMS) on plasma from patients with S. Typhi and S. Paratyphi A infections and asymptomatic controls, identifying 695 individual metabolite peaks. Applying supervised pattern recognition, we found highly significant and reproducible metabolite profiles separating S. Typhi cases, S. Paratyphi A cases, and controls, calculating that a combination of six metabolites could accurately define the etiological agent. For the first time we show that reproducible and serovar specific systemic biomarkers can be detected during enteric fever. Our work defines several biologically plausible metabolites that can be used to detect enteric fever, and unlocks the potential of this method in diagnosing other systemic bacterial infections.

  5. Nicotine and its metabolites in amniotic fluid at birth--assessment of prenatal tobacco smoke exposure.

    PubMed

    Köhler, E; Avenarius, S; Rabsilber, A; Gerloff, C; Jorch, G

    2010-05-01

    Amniotic fluid was collected from 78 pregnant women at birth additionally with their urine prior to delivery as well as neonatal urine and meconium. The smoking markers, nicotine and its metabolites cotinine and trans-3'-hydroxycotinine (OH-cotinine), were determined using high-performance liquid chromatography (HPLC). The self-reported smoking status during pregnancy determined by means of a questionnaire was verified by measurement of maternal urine. In all smokers, nicotine metabolites were detected in amniotic fluid and in 80% of them nicotine as well. However, the sum of the nicotine metabolites (Sum(met)) was significantly lower (p < .001) in amniotic fluid (704 +/- 464 nmol/L) than in meconium (921 +/- 588 nmol/L), neonatal urine (1139 +/- 813 nmol/L) and maternal urine (4496 +/- 3535 nmol/L). Concentrations of nicotine metabolites in amniotic fluid correlated well (p < .001) with that in the other specimen types. After environmental tobacco smoke (ETS) exposure, no nicotine or nicotine metabolites were detectable in amniotic fluid but only in maternal and neonatal urine. Analysis of amniotic fluid at birth lends itself to verifying smoking habits during pregnancy and clearly discriminating from ETS exposure, but it is not a suitable approach to differentiating between ETS exposure and non-exposure.

  6. Reference range levels of polycyclic aromatic hydrocarbons in the US population by measurement of urinary monohydroxy metabolites

    SciTech Connect

    Grainger, James . E-mail: jag2@cdc.gov; Huang, Wenlin; Patterson, Donald G.; Turner, Wayman E.; Pirkle, James; Caudill, Samuel P.; Wang, Richard Y.; Needham, Larry L.; Sampson, Eric J.

    2006-03-15

    We developed a gas chromatography isotope-dilution high-resolution mass spectrometry (GC/Id-HRMS) method for measuring 14 polycyclic aromatic hydrocarbon (PAH) metabolites representing seven parent PAHs in 3 mL of urine at low parts-per-trillion levels. PAH levels were determined in urine samples collected in 1999 and 2000 from approximately 2400 participants in the National Health and Nutrition Examination Survey, and, for the first time, reference range values were calculated for these metabolites in the US population. Using this GC/ID-HRMS method, we found detectable concentrations for monohydroxy metabolite isomers of fluorene, phenanthrene, fluoranthene, pyrene, and chrysene, benzo[c]phenanthrene, and benz[a]anthracene. Some monohydroxy metabolite isomers of benzo[c]phenanthrene, chrysene, and benz[a]anthracene exhibited low detection frequencies that did not allow for geometric mean calculations. Our study results enabled us to establish a reference range for the targeted PAHs in the general US population.

  7. [Secondary Metabolites from Marine Microorganisms. I. Secondary Metabolites from Marine Actinomycetes].

    PubMed

    Orlova, T I; Bulgakova, V G; Polin, A N

    2015-01-01

    Review represents data on new active metabolites isolated from marine actinomycetes published in 2007 to 2014. Marine actinomycetes are an unlimited source of novel secondary metabolites with various biological activities. Among them there are antibiotics, anticancer compounds, inhibitors of biochemical processes.

  8. Polycyclic aromatic hydrocarbon (PAH) metabolites in marine fishes as a specific biomarker to indicate PAH pollution in the marine coastal environment.

    PubMed

    Wang, Xin H; Hong, Hua S; Mu, Jing L; Lin, Jian Q; Wang, Shong H

    2008-02-15

    In this study, analysis methods for the PAH metabolites of naphthalene (Na), pyrene (Py) and benzo(a)pyrene (BaP) with different benzo-rings (2-4-5 rings respectively) were developed and the metabolism kinetics of Py and BaP in marine fishes were studied. Two PAH metabolites of Na and Py, namely 1-naphthol (1-OH Na) and 1-hydroxy pyrene (1-OH Py), were determined using the fixed wavelength fluorescence (FF) method, and the BaP metabolite, 3-hydroxy benzo(a)pyrene (3-OH BaP), was determined using reverse-phase HPLC with fluorescence detection. The dose- and time-response of Lateolabrax japonicus to Py metabolites and Sparus macrocephalus to BaP metabolites were studied in order to evaluate the use of PAH metabolites as a means of assessing exposure to PAHs. The results showed that both fishes could be induced to metabolize and eliminate their metabolites in vivo with increasing Py and BaP exposure concentrations in seawater. As Py and BaP concentrations increased, metabolite concentrations in the fish bile also increased. A significant dose-response of biliary PAH metabolites was observed after exposure for 1, 3 and 7 days for Py and 2, 4 and 7 days for BaP, respectively. These results provide the proof necessary for using PAH metabolites in marine fishes as a specific biomarker or early warning signal of PAH pollution in the marine coastal environment.

  9. Secondary metabolites in fungus-plant interactions

    PubMed Central

    Pusztahelyi, Tünde; Holb, Imre J.; Pócsi, István

    2015-01-01

    Fungi and plants are rich sources of thousands of secondary metabolites. The genetically coded possibilities for secondary metabolite production, the stimuli of the production, and the special phytotoxins basically determine the microscopic fungi-host plant interactions and the pathogenic lifestyle of fungi. The review introduces plant secondary metabolites usually with antifungal effect as well as the importance of signaling molecules in induced systemic resistance and systemic acquired resistance processes. The review also concerns the mimicking of plant effector molecules like auxins, gibberellins and abscisic acid by fungal secondary metabolites that modulate plant growth or even can subvert the plant defense responses such as programmed cell death to gain nutrients for fungal growth and colonization. It also looks through the special secondary metabolite production and host selective toxins of some significant fungal pathogens and the plant response in form of phytoalexin production. New results coming from genome and transcriptional analyses in context of selected fungal pathogens and their hosts are also discussed. PMID:26300892

  10. Pharmaceutically active secondary metabolites of marine actinobacteria.

    PubMed

    Manivasagan, Panchanathan; Venkatesan, Jayachandran; Sivakumar, Kannan; Kim, Se-Kwon

    2014-04-01

    Marine actinobacteria are one of the most efficient groups of secondary metabolite producers and are very important from an industrial point of view. Many representatives of the order Actinomycetales are prolific producers of thousands of biologically active secondary metabolites. Actinobacteria from terrestrial sources have been studied and screened since the 1950s, for many important antibiotics, anticancer, antitumor and immunosuppressive agents. However, frequent rediscovery of the same compounds from the terrestrial actinobacteria has made them less attractive for screening programs in the recent years. At the same time, actinobacteria isolated from the marine environment have currently received considerable attention due to the structural diversity and unique biological activities of their secondary metabolites. They are efficient producers of new secondary metabolites that show a range of biological activities including antibacterial, antifungal, anticancer, antitumor, cytotoxic, cytostatic, anti-inflammatory, anti-parasitic, anti-malaria, antiviral, antioxidant, anti-angiogenesis, etc. In this review, an evaluation is made on the current status of research on marine actinobacteria yielding pharmaceutically active secondary metabolites. Bioactive compounds from marine actinobacteria possess distinct chemical structures that may form the basis for synthesis of new drugs that could be used to combat resistant pathogens. With the increasing advancement in science and technology, there would be a greater demand for new bioactive compounds synthesized by actinobacteria from various marine sources in future.

  11. Metabolite Profiles During Oral Glucose Challenge

    PubMed Central

    Ho, Jennifer E.; Larson, Martin G.; Vasan, Ramachandran S.; Ghorbani, Anahita; Cheng, Susan; Rhee, Eugene P.; Florez, Jose C.; Clish, Clary B.; Gerszten, Robert E.; Wang, Thomas J.

    2013-01-01

    To identify distinct biological pathways of glucose metabolism, we conducted a systematic evaluation of biochemical changes after an oral glucose tolerance test (OGTT) in a community-based population. Metabolic profiling was performed on 377 nondiabetic Framingham Offspring cohort participants (mean age 57 years, 42% women, BMI 30 kg/m2) before and after OGTT. Changes in metabolite levels were evaluated with paired Student t tests, cluster-based analyses, and multivariable linear regression to examine differences associated with insulin resistance. Of 110 metabolites tested, 91 significantly changed with OGTT (P ≤ 0.0005 for all). Amino acids, β-hydroxybutyrate, and tricarboxylic acid cycle intermediates decreased after OGTT, and glycolysis products increased, consistent with physiological insulin actions. Other pathways affected by OGTT included decreases in serotonin derivatives, urea cycle metabolites, and B vitamins. We also observed an increase in conjugated, and a decrease in unconjugated, bile acids. Changes in β-hydroxybutyrate, isoleucine, lactate, and pyridoxate were blunted in those with insulin resistance. Our findings demonstrate changes in 91 metabolites representing distinct biological pathways that are perturbed in response to an OGTT. We also identify metabolite responses that distinguish individuals with and without insulin resistance. These findings suggest that unique metabolic phenotypes can be unmasked by OGTT in the prediabetic state. PMID:23382451

  12. Metabolite profiles during oral glucose challenge.

    PubMed

    Ho, Jennifer E; Larson, Martin G; Vasan, Ramachandran S; Ghorbani, Anahita; Cheng, Susan; Rhee, Eugene P; Florez, Jose C; Clish, Clary B; Gerszten, Robert E; Wang, Thomas J

    2013-08-01

    To identify distinct biological pathways of glucose metabolism, we conducted a systematic evaluation of biochemical changes after an oral glucose tolerance test (OGTT) in a community-based population. Metabolic profiling was performed on 377 nondiabetic Framingham Offspring cohort participants (mean age 57 years, 42% women, BMI 30 kg/m(2)) before and after OGTT. Changes in metabolite levels were evaluated with paired Student t tests, cluster-based analyses, and multivariable linear regression to examine differences associated with insulin resistance. Of 110 metabolites tested, 91 significantly changed with OGTT (P ≤ 0.0005 for all). Amino acids, β-hydroxybutyrate, and tricarboxylic acid cycle intermediates decreased after OGTT, and glycolysis products increased, consistent with physiological insulin actions. Other pathways affected by OGTT included decreases in serotonin derivatives, urea cycle metabolites, and B vitamins. We also observed an increase in conjugated, and a decrease in unconjugated, bile acids. Changes in β-hydroxybutyrate, isoleucine, lactate, and pyridoxate were blunted in those with insulin resistance. Our findings demonstrate changes in 91 metabolites representing distinct biological pathways that are perturbed in response to an OGTT. We also identify metabolite responses that distinguish individuals with and without insulin resistance. These findings suggest that unique metabolic phenotypes can be unmasked by OGTT in the prediabetic state.

  13. Recent Advances in Mass Spectrometry for the Identification of Neuro-chemicals and their Metabolites in Biofluids

    PubMed Central

    Kailasa, Suresh Kumar; Wu, Hui-Fen

    2013-01-01

    Recently, mass spectrometric related techniques have been widely applied for the identification and quantification of neurochemicals and their metabolites in biofluids. This article presents an overview of mass spectrometric techniques applied in the detection of neurological substances and their metabolites from biological samples. In addition, the advances of chromatographic methods (LC, GC and CE) coupled with mass spectrometric techniques for analysis of neurochemicals in pharmaceutical and biological samples are also discussed. PMID:24381533

  14. Metabolites of synthetic cannabinoids in hair--proof of consumption or false friends for interpretation?

    PubMed

    Franz, Florian; Angerer, Verena; Hermanns-Clausen, Maren; Auwärter, Volker; Moosmann, Bjoern

    2016-05-01

    The detection of drug metabolites in hair is widely accepted as a proof for systemic uptake of the drug, unless the metabolites can be formed as artefacts. However, regarding synthetic cannabinoids, not much is known about mechanisms of incorporation into hair. For a correct interpretation concerning hair findings of these compounds and their metabolites, it is necessary to identify the different routes of incorporation and to assess their contribution to analytical findings. This study presents the results of the LC-ESI-MS/MS analysis of an authentic hair sample taken from a patient with a known history of heavy consumption of synthetic cannabinoids. In the authentic hair sample, 5F-PB-22 and AB-CHMINACA as well as their main metabolites 5F-PB-22 3-carboxyindole, PB-22 5-OH-pentyl, and AB-CHMINACA valine were detected in all segments, comprising segments grown in a time period where the substances had not been distributed on the 'legal high' market. To enable interpretation of the results regarding the distribution of the detected analytes along the hair shaft, the stability of 5F-PB-22 and AB-CHMINACA in hair matrix and under thermal stress was assessed. The stability tests revealed that the three 'metabolites' are also formed in externally contaminated hair after storage of the samples under different conditions. In addition, 5F-PB-22 3-carboxyindole and AB-CHMINACA valine were identified as degradation products in smoke condensate. Therefore, interpretation of 'metabolite' findings of compounds comprising chemically labile amide/ester bonds or 5-fluoro-pentyl side chains should be carried out with utmost care, taking into account the different mechanisms of formation and incorporation into hair.

  15. [Secondary fungal metabolites (mycotoxins) in lichens of different taxonomic groups].

    PubMed

    Burkin, A A; Kononenko, G P

    2014-01-01

    Secondary fungal metabolites (mycotoxins) in 22 lichen species of the families Parmeliaceae, Nephromataceae, Umbilicariaceae, Ramalinaceae, Cladoniaceae, Peltigeraceae, and Teloschistaceae were identified determined by enzyme immunoassay enzyme-linked immunosorbent assay. The following mycotoxins were identified found in these lichens in a broad concentration range with a frequency of 70-100%: sterigmatocystin (7-2090 ng/g), alternariol (20-6460 ng/g), and emodin (45-94500 ng/g). Mycophenolic acid frequently occurred in 19 lichen species; citrinin, in 17 species; diacetoxyscirpenol, in 11 species; cyclopiazonic acid, in 10 species; and zearalenone, in 9 species. PR toxin was regularly detected in three lichen species; deoxynivalenol, fumonisins, and ochratoxin A, in two species; and T-2 toxin and ergot alkaloids, in one species. Aflatoxin B1 was detected in only six species with a frequency of 2-42%, whereas roridin A was identified present in 10% of Hypogymnia physodes samples.

  16. Monitoring microbial metabolites using an inductively coupled resonance circuit

    PubMed Central

    Karnaushenko, Daniil; Baraban, Larysa; Ye, Dan; Uguz, Ilke; Mendes, Rafael G.; Rümmeli, Mark H.; de Visser, J. Arjan G. M.; Schmidt, Oliver G.; Cuniberti, Gianaurelio; Makarov, Denys

    2015-01-01

    We present a new approach to monitor microbial population dynamics in emulsion droplets via changes in metabolite composition, using an inductively coupled LC resonance circuit. The signal measured by such resonance detector provides information on the magnetic field interaction with the bacterial culture, which is complementary to the information accessible by other detection means, based on electric field interaction, i.e. capacitive or resistive, as well as optical techniques. Several charge-related factors, including pH and ammonia concentrations, were identified as possible contributors to the characteristic of resonance detector profile. The setup enables probing the ionic byproducts of microbial metabolic activity at later stages of cell growth, where conventional optical detection methods have no discriminating power. PMID:26264183

  17. Monitoring microbial metabolites using an inductively coupled resonance circuit

    NASA Astrophysics Data System (ADS)

    Karnaushenko, Daniil; Baraban, Larysa; Ye, Dan; Uguz, Ilke; Mendes, Rafael G.; Rümmeli, Mark H.; de Visser, J. Arjan G. M.; Schmidt, Oliver G.; Cuniberti, Gianaurelio; Makarov, Denys

    2015-08-01

    We present a new approach to monitor microbial population dynamics in emulsion droplets via changes in metabolite composition, using an inductively coupled LC resonance circuit. The signal measured by such resonance detector provides information on the magnetic field interaction with the bacterial culture, which is complementary to the information accessible by other detection means, based on electric field interaction, i.e. capacitive or resistive, as well as optical techniques. Several charge-related factors, including pH and ammonia concentrations, were identified as possible contributors to the characteristic of resonance detector profile. The setup enables probing the ionic byproducts of microbial metabolic activity at later stages of cell growth, where conventional optical detection methods have no discriminating power.

  18. Analysis of cocaine and metabolites in hair: validation and application of measurement of hydroxycocaine metabolites as evidence of cocaine ingestion.

    PubMed

    Schaffer, Michael; Cheng, Chen-Chih; Chao, Oscar; Hill, Virginia; Matsui, Paul

    2016-03-01

    An LC/MS/MS method to identify and quantitate in hair the minor metabolites of cocaine-meta-, para-, and ortho-hydroxy cocaine-was developed and validated. Analysis was performed on a triple quadrupole ABSciex API 3000 MS equipped with an atmospheric pressure ionization source via an IonSpray (ESI). For LC, a series 200 micro binary pump with a Perkin Elmer Model 200 autosampler was used. The limit of detection (LOD) and limit of quantification (LOQ) were 0.02 ng/10 mg hair, with linearity from 0.02 to 10 ng/10 mg hair. Concentrations of the para isomer in extensively washed hair samples were in the range of 1-2 % of the cocaine in the sample, while the concentrations of the ortho form were considerably less. The method was used to analyze large numbers of samples from two populations: workplace and criminal justice. In vitro experiments to determine if deodorants or peroxide-containing cosmetic treatments could result in the presence of these metabolites in hair showed that this does not occur with extensively washed hair. Presence of hydroxycocaines, when detected after aggressive washing of the hair samples, provides a valuable additional indicator of ingestion of cocaine rather than mere environmental exposure.

  19. Evaluation of relative MS response factors of drug metabolites for semi-quantitative assessment of chemical liabilities in drug discovery.

    PubMed

    Blanz, Joachim; Williams, Gareth; Dayer, Jerôme; Délémonté, Thierry; Gertsch, Werner; Ramstein, Philippe; Aichholz, Reiner; Trunzer, Markus; Pearson, David

    2017-02-02

    Drug metabolism studies are performed in drug discovery to identify metabolic soft spots, detect potentially toxic or reactive metabolites and provide an early insight into potential species differences. The relative peak area approach is often used to semi-quantitatively estimate the abundance of metabolites. Differences in the LC/MS responses result in an under- or overestimation of the metabolite and misinterpretation of results. The relative MS response factors of 132 structurally diverse drug candidates and their 233 corresponding metabolites were evaluated using a capillary-LC/HRMS system. All of the synthesized metabolites discussed here were previously identified as key biotransformation products in discovery investigations or predicted to be formed. The most commonly occurring biotransformation mechanisms such as oxygenation, dealkylation and amide cleavage are represented within this dataset. However, relatively few phase II metabolites were evaluated due to the limited availability of authentic standards. 85 % of these metabolites had a relative response factor (RF) in the range between 0.2 (5 fold under-prediction) and 2.0 (2 fold over-prediction) and the median MS RF was 0.6. Exceptions to this included very small metabolites that were hardly detectable. Additional experiments performed to understand the impact of the MS platform, flow rate and concentration suggested that these parameters do not have a significant impact on the RF of the compounds tested. This indicates that the use of relative peak areas to semi-quantitatively estimate the abundance of metabolites is justified in the drug discovery setting in order to guide medicinal chemistry efforts.

  20. Ultrasensitive Profiling of Metabolites Using Tyramine-Functionalized Graphene Quantum Dots.

    PubMed

    Li, Nan; Than, Aung; Wang, Xuewan; Xu, Shaohai; Sun, Lei; Duan, Hongwei; Xu, Chenjie; Chen, Peng

    2016-03-22

    Graphene quantum dots (GQDs) are emerging fluorescence reporters attractive for optical sensing, owing to their high photostability, highly tunable photoluminescence, molecular size, atomically thin structure, biocompatibility, and ease of functionalization. Herein, we present a fluorometric sensing platform based on tyramine-functionalized GQDs, which is able to detect a spectrum of metabolites with high sensitivity and specificity. Furthermore, multiparametric blood analysis (glucose, cholesterol, L-lactate, and xanthine) is demonstrated. This convenient metabolite profiling technique could be instrumental for diagnosis, study, and management of metabolic disorders and associated diseases, such as diabetes, obesity, lactic acidosis, gout, and hypertension.

  1. Non-invasive mouthguard biosensor for continuous salivary monitoring of metabolites.

    PubMed

    Kim, Jayoung; Valdés-Ramírez, Gabriela; Bandodkar, Amay J; Jia, Wenzhao; Martinez, Alexandra G; Ramírez, Julian; Mercier, Patrick; Wang, Joseph

    2014-04-07

    The present work describes the first example of a wearable salivary metabolite biosensor based on the integration of a printable enzymatic electrode on a mouthguard. The new mouthguard enzymatic biosensor, based on an immobilized lactate oxidase and a low potential detection of the peroxide product, exhibits high sensitivity, selectivity and stability using whole human saliva samples. Such non-invasive mouthguard metabolite biosensors could tender useful real-time information regarding a wearer's health, performance and stress level, and thus hold considerable promise for diverse biomedical and fitness applications.

  2. Animal bioavailability of defined xenobiotic lignin metabolites

    SciTech Connect

    Sandermann, H. Jr.; Arjmand, M.; Gennity, I.; Winkler, R. ); Struble, C.B.; Aschbacher, P.W. )

    1990-09-01

    Lignin has been recognized as a major component of bound pesticide residues in plants and is thought to be undigestible in animals. Two defined ring-U-{sup 14}C-labeled chloroaniline/lignin metabolites have now been fed to rats, where a release of {approximately}66% of the bound xenobiotic occurred in the form of simple chloroaniline derivatives. The observed high degree of bioavailability indicates that bound pesticidal residues may possess ecotoxicological significance. In parallel studies, the white-rot fungus Phanerochaete chrysosporium was more efficient, and a soil system was much less efficient, in the degradation of the (ring-U-{sup 14}C)chloroaniline/lignin metabolites.

  3. Streptomyces metabolites in divergent microbial interactions.

    PubMed

    Takano, Hideaki; Nishiyama, Tatsuya; Amano, Sho-ichi; Beppu, Teruhiko; Kobayashi, Michihiko; Ueda, Kenji

    2016-03-01

    Streptomyces and related bacteria produce a wide variety of secondary metabolites. Of these, many compounds have industrial applications, but the question of why this group of microorganism produces such various kinds of biologically active substances has not yet been clearly answered. Here, we overview the results from our studies on the novel function and role of Streptomyces metabolites. The diverged action of negative and positive influences onto the physiology of various microorganisms infers the occurrence of complex microbial interactions due to the effect of small molecules produced by Streptomyces. The interactions may serve as a basis for the constitution of biological community.

  4. Manual therapy ameliorates delayed-onset muscle soreness and alters muscle metabolites in rats.

    PubMed

    Urakawa, Susumu; Takamoto, Kouichi; Nakamura, Tomoya; Sakai, Shigekazu; Matsuda, Teru; Taguchi, Toru; Mizumura, Kazue; Ono, Taketoshi; Nishijo, Hisao

    2015-02-01

    Delayed-onset muscle soreness (DOMS) can be induced by lengthening contraction (LC); it can be characterized by tenderness and movement-related pain in the exercised muscle. Manual therapy (MT), including compression of exercised muscles, is widely used as physical rehabilitation to reduce pain and promote functional recovery. Although MT is beneficial for reducing musculoskeletal pain (i.e. DOMS), the physiological mechanisms of MT remain unclear. In the present study, we first developed an animal model of MT in DOMS; LC was applied to the rat gastrocnemius muscle under anesthesia, which induced mechanical hyperalgesia 2-4 days after LC. MT (manual compression) ameliorated mechanical hyperalgesia. Then, we used capillary electrophoresis time-of-flight mass spectroscopy (CE-TOFMS) to investigate early effects of MT on the metabolite profiles of the muscle experiencing DOMS. The rats were divided into the following three groups; (1) normal controls, (2) rats with LC application (LC group), and (3) rats undergoing MT after LC (LC + MT group). According to the CE-TOFMS analysis, a total of 171 metabolites were detected among the three groups, and 19 of these metabolites were significant among the groups. Furthermore, the concentrations of eight metabolites, including branched-chain amino acids, carnitine, and malic acid, were significantly different between the LC + MT and LC groups. The results suggest that MT significantly altered metabolite profiles in DOMS. According to our findings and previous data regarding metabolites in mitochondrial metabolism, the ameliorative effects of MT might be mediated partly through alterations in metabolites associated with mitochondrial respiration.

  5. Manual therapy ameliorates delayed-onset muscle soreness and alters muscle metabolites in rats

    PubMed Central

    Urakawa, Susumu; Takamoto, Kouichi; Nakamura, Tomoya; Sakai, Shigekazu; Matsuda, Teru; Taguchi, Toru; Mizumura, Kazue; Ono, Taketoshi; Nishijo, Hisao

    2015-01-01

    Delayed-onset muscle soreness (DOMS) can be induced by lengthening contraction (LC); it can be characterized by tenderness and movement-related pain in the exercised muscle. Manual therapy (MT), including compression of exercised muscles, is widely used as physical rehabilitation to reduce pain and promote functional recovery. Although MT is beneficial for reducing musculoskeletal pain (i.e. DOMS), the physiological mechanisms of MT remain unclear. In the present study, we first developed an animal model of MT in DOMS; LC was applied to the rat gastrocnemius muscle under anesthesia, which induced mechanical hyperalgesia 2–4 days after LC. MT (manual compression) ameliorated mechanical hyperalgesia. Then, we used capillary electrophoresis time-of-flight mass spectroscopy (CE-TOFMS) to investigate early effects of MT on the metabolite profiles of the muscle experiencing DOMS. The rats were divided into the following three groups; (1) normal controls, (2) rats with LC application (LC group), and (3) rats undergoing MT after LC (LC + MT group). According to the CE-TOFMS analysis, a total of 171 metabolites were detected among the three groups, and 19 of these metabolites were significant among the groups. Furthermore, the concentrations of eight metabolites, including branched-chain amino acids, carnitine, and malic acid, were significantly different between the LC + MT and LC groups. The results suggest that MT significantly altered metabolite profiles in DOMS. According to our findings and previous data regarding metabolites in mitochondrial metabolism, the ameliorative effects of MT might be mediated partly through alterations in metabolites associated with mitochondrial respiration. PMID:25713324

  6. Urinary corticosterone metabolite responses to capture and captivity in the cane toad (Rhinella marina).

    PubMed

    Narayan, Edward J; Cockrem, John F; Hero, Jean-Marc

    2011-09-01

    Urinary corticosterone metabolite responses to capture have recently been shown for the first time in amphibians, and in the present study urinary corticosterone metabolite responses to capture and to confinement in captivity were measured in adult cane toads (Rhinella marina) in Queensland, Australia. An adrenocorticotropic hormone (ACTH) challenge was used to provide a biological validation for urinary corticosterone metabolite concentrations measured by radioimmunoassay (RIA). Urinary corticosterone metabolite increased 1-2 days after ACTH but not saline injection and then returned to initial values, indicating that the RIA could detect changes in corticosterone secretion in toads. Urinary corticosterone metabolite responses to short-term capture and restraint in plastic bags were first apparent 2h after capture of wild toads. Toads held communally in captivity for 5 days had elevated urinary corticosterone metabolite concentrations. Mean corticosterone concentrations declined significantly after a further 7 days in individual housing chambers. There was no sex difference in urinary corticosterone metabolite responses of toads to ACTH challenge, short-term capture or captivity. The relative amount of variation in the mean corticosterone responses was quantified by calculating coefficients of variation (CV) for each mean corticosterone response. Mean corticosterone at 0 min was more variable for captive toads than wild toads. Furthermore, initial corticosterone concentrations (0 min) were more variable than concentrations during the ACTH challenge, short-term capture and captivity. There was little change in the amount of variation of mean corticosterone levels between male and female toads with increasing time in captivity (12-29 days). This study has shown individual corticosterone responses of amphibians for the first-time, and has provided a novel method for quantifying the relative amount of variation in amphibian corticosterone responses.

  7. Human colon microbiota transform polycyclic aromatic hydrocarbons to estrogenic metabolites.

    PubMed

    Van de Wiele, Tom; Vanhaecke, Lynn; Boeckaert, Charlotte; Peru, Kerry; Headley, John; Verstraete, Willy; Siciliano, Steven

    2005-01-01

    Ingestion is an important exposure route for polycyclic aromatic hydrocarbons (PAHs) to enter the human body. Although the formation of hazardous PAH metabolites by human biotransformation enzymes is well documented, nothing is known about the PAH transformation potency of human intestinal microbiota. Using a gastrointestinal simulator, we show that human intestinal microbiota can also bioactivate PAHs, more in particular to estrogenic metabolites. PAH compounds are not estrogenic, and indeed, stomach and small intestine digestions of 62.5 nmol naphthalene, phenanthrene, pyrene, and benzo(a)pyrene showed no estrogenic effects in the human estrogen receptor bioassay. In contrast, colon digests of these PAH compounds displayed estrogenicity, equivalent to 0.31, 2.14, 2.70, and 1.48 nmol 17alpha-ethynylestradiol (EE2), respectively. Inactivating the colon microbiota eliminated these estrogenic effects. Liquid chromatography-mass spectrometry analysis confirmed the microbial PAH transformation by the detection of PAH metabolites 1-hydroxypyrene and 7-hydroxybenzo(a)pyrene in colon digests of pyrene and benzo(a)pyrene. Furthermore, we show that colon digests of a PAH-contaminated soil (simulated ingestion dose of 5 g/day) displayed estrogenic activity equivalent to 0.58 nmol EE2, whereas stomach or small intestine digests did not. Although the matrix in which PAHs are ingested may result in lower exposure concentrations in the gut, our results imply that the PAH bioactivation potency of colon microbiota is not eliminated by the presence of soil. Moreover, because PAH toxicity is also linked to estrogenicity of the compounds, the PAH bioactivation potency of colon microbiota suggests that current risk assessment may underestimate the risk from ingested PAHs.

  8. Disposition of xenobiotic chemicals and metabolites in marine organisms.

    PubMed Central

    Varanasi, U; Stein, J E

    1991-01-01

    Studies with several bottom fish species from urban waterways show that of the identified xenobiotic chemicals in bottom sediments, polycylic aromatic hydrocarbons (PAHs) are the most strongly associated with the prevalence of liver lesions, including neoplasms. Accordingly, there is concern about the transfer of contaminants, such as PAHs, from aquatic species to humans. Because PAHs exert their toxicity only after being biotransformed, increasing attention has been focused on the ability of aquatic organisms to metabolize these chemicals. Overall, the results of both laboratory and field studies show that generally low levels (nanograms per gram wet weight) of a few low molecular weight PAHs may be present in edible tissue of fish from contaminated areas and that high molecular weight PAHs, such as the carcinogen benzo(a)pyrene, will rarely be detected because of extensive metabolism. Additionally, the results from a few studies suggest that even though interactions between xenobiotics can affect both biochemical and physiological systems to alter the disposition of PAHs in fish, these interactions do not markedly change the relative proportions of metabolites to parent PAH in tissues. Thus, these studies clearly demonstrate that to obtain some insight into the questions of whether there is any risk to human health from consuming fish and crustaceans from urban areas, techniques must be developed that measure metabolites of carcinogens, such as PAHs, in edible tissue. Initial attempts may focus on semiquantitative methods that permit rapid assessment of the level of metabolites in edible tissues of fish and crustaceans from many urban areas.(ABSTRACT TRUNCATED AT 250 WORDS) Images FIGURE 4. FIGURE 4. FIGURE 4. PMID:2050086

  9. Metabolite profiling of symbiont and host during thermal stress and bleaching in the coral Acropora aspera

    NASA Astrophysics Data System (ADS)

    Hillyer, Katie E.; Dias, Daniel A.; Lutz, Adrian; Wilkinson, Shaun P.; Roessner, Ute; Davy, Simon K.

    2017-03-01

    Rising seawater temperatures pose a significant threat to the persistence of coral reefs. Despite the importance of these systems, major gaps remain in our understanding of how thermal stress and bleaching affect the metabolic networks that underpin holobiont function. We applied gas chromatography-mass spectrometry (GC-MS) metabolomics to detect changes in the intracellular free metabolite pools (polar and semi-polar compounds) of in hospite dinoflagellate symbionts and their coral hosts (and any associated microorganisms) during early- and late-stage thermal bleaching (a reduction of approximately 50 and 70% in symbiont density, respectively). We detected characteristic changes to the metabolite profiles of each symbiotic partner associated with individual cellular responses to thermal, oxidative and osmotic stress, which progressed with the severity of bleaching. Alterations were also indicative of changes to energy-generating and biosynthesis pathways in both partners, with a shift to the increased catabolism of lipid stores. Specifically, in symbiont intracellular metabolite pools, we observed accumulations of multiple free fatty acids, plus the chloroplast-associated antioxidant alpha-tocopherol. In the host, we detected a decline in the abundance of pools of multiple carbohydrates, amino acids and intermediates, in addition to the antioxidant ascorbate. These findings further our understanding of the metabolic changes that occur to symbiont and host (and its associated microorganisms) during thermal bleaching. These findings also provide further insight into the largely undescribed roles of free metabolite pools in cellular homeostasis, signalling and acclimation to thermal stress in the cnidarian-dinoflagellate symbiosis.

  10. Analysis of UR-144 and its pyrolysis product in blood and their metabolites in urine.

    PubMed

    Adamowicz, Piotr; Zuba, Dariusz; Sekuła, Karolina

    2013-12-10

    UR-144 [(1-pentyl-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone] is a synthetic cannabinoid, which has been detected in many herbal blends, resinous samples and powders seized from the Polish drug market since the beginning of 2012. This paper presents the case of intoxication by this substance. A complete picture of the symptoms observed by a witness, paramedics and medical doctors are given. In the analysis of powder residues from the plastic bag seized from the intoxicated person by gas chromatography-mass spectrometry (GC-MS), UR-144 and its major pyrolysis product [1-(1-pentyl-1H-indol-3-yl)-3-methyl-2-(propan-2-yl)but-3-en-1-one] were detected. Both substances were also identified in a blood sample collected on admission of the patient to hospital using liquid chromatography-triple quadrupole tandem mass spectrometry (LC-QqQ-MS). Blood concentration of UR-144 was 6.1 ng/mL. A urine sample collected at the same time was analyzed by liquid chromatography-quadruple time-of-flight tandem mass spectrometry (LC-QTOF-MS). The parent substance and its pyrolysis products were not detected in urine, while their five metabolites were found. The experiments allowed the location of derivative groups to be established, and thus elucidate rough structures of the metabolites; a dihydroxylated metabolite of UR-144 and mono-, dihydroxylated and carboxylated metabolites of its pyrolysis product were identified.

  11. HPLC Determination and MS Confirmation of Malachite Green, Gentian Violet, and Their Leuco Metabolites in Catfish Muscle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Residues of malachite green (MG), gentian violet (GV), and their leuco metabolites in catfish muscle were individually determined by HPLC using visible and fluorescence detectors. This detection scheme obviated a PbO2 column that converts leuco forms to chromatic forms for visible detection, thus el...

  12. Solid-phase microextraction technology for in vitro and in vivo metabolite analysis

    PubMed Central

    Zhang, Qihui; Zhou, Liandi; Chen, Hua; Wang, Chong-Zhi; Xia, Zhining; Yuan, Chun-Su

    2016-01-01

    Analysis of endogenous metabolites in biological samples may lead to the identification of biomarkers in metabolomics studies. To achieve accurate sample analysis, a combined method of continuous quick sampling and extraction is required for online compound detection. Solid-phase microextraction (SPME) integrates sampling, extraction and concentration into a single solvent-free step for chemical analysis. SPME has a number of advantages, including simplicity, high sensitivity and a relatively non-invasive nature. In this article, we reviewed SPME technology in in vitro and in vivo analyses of metabolites after the ingestion of herbal medicines, foods and pharmaceutical agents. The metabolites of microorganisms in dietary supplements and in the gastrointestinal tract will also be examined. As a promising technology in biomedical and pharmaceutical research, SPME and its future applications will depend on advances in analytical technologies and material science. PMID:27695152

  13. Mapping of Brain Metabolite Distributions by Volumetric Proton MR Spectroscopic Imaging (MRSI)

    PubMed Central

    Maudsley, A.A.; Domenig, C; Govind, V; Darkazanli, A.; Studholme, C.; Arheart, K.; Bloomer, C.

    2009-01-01

    Distributions of proton MR-detected metabolites have been mapped throughout the brain in a group of normal subjects using a volumetric MR spectroscopic imaging (MRSI) acquisition with an interleaved water reference. Data were processed with intensity and spatial normalization to enable voxel-based analysis methods to be applied across a group of subjects. Results demonstrate significant regional, tissue, and gender-dependent variations of brain metabolite concentrations, and variations of these distributions with normal aging. The greatest alteration of metabolites with age was observed for white-matter choline and creatine. An example of the utility of the normative metabolic information is then demonstrated for analysis of data acquired from a subject who suffered a traumatic brain injury. This study demonstrates the ability to obtain proton spectra from a wide region of the brain and to apply fully automated processing methods. The resultant data provide a normative reference for subsequent utilization for studies of brain injury and disease. PMID:19111009

  14. Utilizing DART Mass Spectrometry to Pinpoint Halogenated Metabolites from a Marine Invertebrate-Derived Fungus

    PubMed Central

    Watts, Katharine R.; Loveridge, Steven T.; Tenney, Karen; Media, Joseph; Valeriote, Frederick A.; Crews, Phillip

    2011-01-01

    Prenylated indole alkaloids are a diverse group of fungal secondary metabolites and represent an important biosynthetic class. In this study we have identified new halogenated prenyl-indole alkaloids from an invertebrate-derived Malbranchea graminicola strain. Using Direct Analysis in Real Time (DART) Mass Spectrometry, these compounds were initially detected from spores of the fungus grown on agar plates, without the need for any organic extraction. Subsequently, the metabolites were isolated from liquid culture in artificial seawater. The structures of two novel chlorinated metabolites, named (−)-spiromalbramide and (+)-isomalbrancheamide B, provide additional insights into the assembly of the malbrancheamide compound family. Remarkably, two new brominated analogs, (+)-malbrancheamide C and (+)-isomalbrancheamide C, were produced by enriching the growth medium with bromine salts. PMID:21682275

  15. Chemosensation of Bacterial Secondary Metabolites Modulates Neuroendocrine Signaling and Behavior of C. elegans

    PubMed Central

    Meisel, Joshua D.; Panda, Oishika; Mahanti, Parag; Schroeder, Frank C.; Kim, Dennis H.

    2014-01-01

    Summary Discrimination among pathogenic and beneficial microbes is essential for host organism immunity and homeostasis. Here, we show that chemosensory detection of two secondary metabolites produced by Pseudomonas aeruginosa modulates a neuroendocrine signaling pathway that promotes avoidance behavior in the simple animal host Caenorhabditis elegans. Secondary metabolites phenazine-1-carboxamide and pyochelin activate a G protein-signaling pathway in the ASJ chemosensory neuron pair that induces expression of the neuromodulator DAF-7/TGF-β. DAF-7, in turn, activates a canonical TGF-β signaling pathway in adjacent interneurons to modulate aerotaxis behavior and promote avoidance of pathogenic P. aeruginosa. Our data provide a chemical, genetic, and neuronal basis for how the behavior and physiology of a simple animal host can be modified by the microbial environment, and suggest that secondary metabolites produced by microbes may provide environmental cues that contribute to pathogen recognition and host survival. PMID:25303524

  16. Metabolite profiling of RCS-4, a novel synthetic cannabinoid designer drug, using human hepatocyte metabolism and TOF-MS

    PubMed Central

    Gandhi, Adarsh S; Zhu, Mingshe; Pang, Shaokun; Wohlfarth, Ariane; Scheidweiler, Karl B; Huestis, Marilyn A

    2014-01-01

    Background Since 2009, scheduling legislation of synthetic cannabinoids prompted new compound emergence to circumvent legal restrictions. 2-(4-methoxyphenyl)-1-(1-pentyl-indol-3-yl)methanone (RCS-4) is a potent cannabinoid receptor agonist sold in herbal smoking blends. Absence of parent synthetic cannabinoids in urine suggests the importance of metabolite identification for detecting RCS-4 consumption in clinical and forensic investigations. Materials & methods & Results With 1 h human hepatocyte incubation and TOF high-resolution MS, we identified 18 RCS-4 metabolites, many not yet reported. Most metabolites were hydroxylated with or without demethylation, carboxylation and dealkylation followed by glucuronidation. One additional sulfated metabolite was also observed. O-demethylation was the most common biotransformation and generated the major metabolite. Conclusion For the first time, we present a metabolic scheme of RCS-4 obtained from human hepatocytes, including Phase I and II metabolites. Metabolite structural information and associated high-resolution mass spectra can be employed for developing clinical and forensic laboratory RCS-4 urine screening methods. PMID:25046048

  17. Modulation of the cellular content of metabolites in adipocytes by insulin.

    PubMed

    Qiao, Yuhang; Tomonaga, Shozo; Matsui, Tohru; Funaba, Masayuki

    2016-03-15

    Although the insulin-mediated cell signaling pathway has been extensively examined, changes in the cellular content of metabolites currently remain unclear. We herein examined metabolite contents in 3T3-L1 adipocytes treated with insulin using a metabolomic analysis. Fifty-four compounds were detected, and the contents of metabolites from the citric acid cycle increased in response to the insulin treatment for 4 h, which was sensitive to U0126 and LY294002, inhibitors for mitogen-activated protein kinase kinase-1 and phosphoinositide 3-kinase, respectively. The cellular contents of fumaric acid and malic acid were increased more by insulin than those of citric acid and succinic acid. Time-course changes in metabolites from the citric acid cycle exhibited oscillations with a 2-h cycle. A metabolic pathway analysis also indicated that insulin affected the metabolism of alanine, aspartate and glutamate, as well as that of arginine and proline. The contents of free amino acids were slightly decreased by the insulin treatment, while the co-treatment with U0126 and LY294002 abrogated these insulin-mediated decreases. The present study revealed the unexpected accumulation of citric acid cycle metabolites in adipocytes by insulin. Our results indicate the usefulness of metabolomic analyses for obtaining a more comprehensive understanding of the regulation of metabolic pathways in cell-culture systems.

  18. Determination of hexazinone and its metabolites in groundwater by capillary electrophoresis.

    PubMed

    Kubilius, D T; Bushway, R J

    1998-01-16

    A micellar electrokinetic chromatographic method was developed to separate and quantify hexazinone and metabolites C, A1, E, B and D in groundwater. Hexazinone and its metabolites were extracted from water using Supelclean ENVI-Carb solid-phase extraction tubes. Quantitation was performed using UV photodiode detection at 220, 225, 230 and 247 nm. Intra-assay and inter-assay reproducibility studies run at 0.5, 1.0, 2.0 and 5.0 ppb indicated the procedure was reproducible. Groundwater samples collected from US Geological Survey monitoring wells were analyzed for hexazinone and its metabolites by CE. A comparison was made between CE and an established HPLC method of the hexazinone and metabolite B. The linear regression for hexazinone was y = 1.007x + 0.219 with a correlation coefficient of 0.96 while the linear regression for metabolite B was y = 1.100x-0.057 with a correlation coefficient of 0.91.

  19. Mass spectrometry-based metabolite profiling in the mouse liver following exposure to ultraviolet B radiation.

    PubMed

    Park, Hye Min; Shon, Jong Cheol; Lee, Mee Youn; Liu, Kwang-Hyeon; Kim, Jeong Kee; Lee, Sang Jun; Lee, Choong Hwan

    2014-01-01

    Although many studies have been performed on the effects of ultraviolet (UV) radiation on the skin, only a limited number of reports have investigated these effects on non-skin tissue. This study aimed to describe the metabolite changes in the liver of hairless mice following chronic exposure to UVB radiation. We did not observe significant macroscopic changes or alterations in hepatic cholesterol and triglyceride levels in the liver of UVB-irradiated mice, compared with those for normal mice. In this study, we detected hepatic metabolite changes by UVB exposure and identified several amino acids, fatty acids, nucleosides, carbohydrates, phospholipids, lysophospholipids, and taurine-conjugated cholic acids as candidate biomarkers in response to UVB radiation in the mouse liver by using various mass spectrometry (MS)-based metabolite profiling including ultra-performance liquid chromatography-quadrupole time-of-flight (TOF)-MS, gas chromatography-TOF-MS and nanomate LTQ-MS. Glutamine exhibited the most dramatic change with a 5-fold increase in quantity. The results from altering several types of metabolites suggest that chronic UVB irradiation may impact significantly on major hepatic metabolism processes, despite the fact that the liver is not directly exposed to UVB radiation. MS-based metabolomic approach for determining regulatory hepatic metabolites following UV irradiation will provide a better understanding of the relationship between internal organs and UV light.

  20. Fecal estradiol and progesterone metabolite levels in the three-toed sloth (Bradypus variegatus).

    PubMed

    Mühlbauer, M; Duarte, D P F; Gilmore, D P; Costa, C P da

    2006-02-01

    The present study was carried out to assess the possibility of measuring fecal steroid hormone metabolites as a noninvasive technique for monitoring reproductive function in the three-toed sloth, Bradypus variegatus. Levels of the estradiol (E2) and progesterone (P4) metabolites were measured by radioimmunoassay in fecal samples collected over 12 weeks from 4 captive female B. variegatus sloths. The validation of the radioimmunoassay for evaluation of fecal steroid metabolites was carried out by collecting 10 blood samples on the same day as defecation. There was a significant direct correlation between the plasma and fecal E2 and P4 levels (P < 0.05, Pearson's test), thereby validating this noninvasive technique for the study of the estrous cycle in these animals. Ovulation was detected in two sloths (SL03 and SL04) whose E2 levels reached 2237.43 and 6713.26 pg/g wet feces weight, respectively, for over four weeks, followed by an increase in P4 metabolites reaching 33.54 and 3242.68 ng/g wet feces weight, respectively. Interestingly, SL04, which presented higher levels of E2 and P4 metabolites, later gave birth to a healthy baby sloth. The results obtained indicate that this is a reliable technique for recording gonadal steroid secretion and thereby reproduction in sloths.

  1. Effects of primary metabolites of organophosphate flame retardants on transcriptional activity via human nuclear receptors.

    PubMed

    Kojima, Hiroyuki; Takeuchi, Shinji; Van den Eede, Nele; Covaci, Adrian

    2016-03-14

    Organophosphate flame retardants (OPFRs) have been used in a wide variety of applications and detected in several environmental matrices, including indoor air and dust. Continuous human exposure to these chemicals is of growing concern. In this study, the agonistic and/or antagonistic activities of 12 primary OPFR-metabolites against ten human nuclear receptors were examined using cell-based transcriptional assays, and compared to those of their parent compounds. As a result, 3-hydroxylphenyl diphenyl phosphate and 4-hydroxylphenyl diphenyl phosphate showed more potent estrogen receptor α (ERα) and ERβ agonistic activity than did their parent, triphenyl phosphate (TPHP). In addition, these hydroxylated TPHP-metabolites also showed ERβ antagonistic activity at higher concentrations and exhibited pregnane X receptor (PXR) agonistic activity as well as androgen receptor (AR) and glucocorticoid receptor (GR) antagonistic activities at similar levels to those of TPHP. Bis(2-butoxyethyl) 3'-hydroxy-2-butoxyethyl phosphate and 2-hydroxyethyl bis(2-butoxyethyl) phosphate act as PXR agonists at similar levels to their parent, tris(2-butoxyethyl) phosphate. On the other hand, seven diester OPFR-metabolites and 1-hydroxy-2-propyl bis(1-chloro-2-propyl) phosphate did not show any receptor activity. Taken together, these results suggest that hydroxylated TPHP-metabolites show increased estrogenicity compared to the parent compound, whereas the diester OPFR-metabolites may have limited nuclear receptor activity compared to their parent triester OPFRs.

  2. Biliary PAH metabolites and the hepatosomatic index of brown bullheads from Lake Erie tributaries

    USGS Publications Warehouse

    Yang, X.; Baumann, P.C.

    2006-01-01

    In studies designed to investigate the environmental exposure of fish in Lake Erie tributaries, a benthic fish, the brown bullhead (Ameiurus nebulosus), was collected from the industrially contaminated Detroit River, Ottawa River, Black River, Cuyahoga River-harbor and -upstream, Ashtabula River, Buffalo River, and Niagara River, and the non-industrialized Old Woman Creek during 1997-2000. Biliary benzo[a]pyrene (B[a]P)- and naphthalene (NAPH)-type metabolites and the hepatosomatic index (HSI) were measured in fish and compared between different sites. Fish from all of the contaminated sites except Niagara River had significantly higher concentrations of both types of polycyclic aromatic hydrocarbon (PAH) metabolites than fish from the Old Woman Creek. Concentrations of PAH metabolites in bile of fish were positively associated with concentrations of PAHs in sediments, supporting the use of bile metabolites as a measure of PAH exposure. Relatively low concentrations of PAHs detected in fish bile and sediments of the Niagara River, which had undergone extensive remediation, suggested a lowered PAH exposure for fish at this site. No apparent trend was observed in HSI between the industrialized and non-industrialized sites. This study demonstrates that biliary PAH metabolites are an effective indicator of exposure of fish to PAHs. However, because factors other than contamination could also affect the liver size of wild fish, HSI alone may be not a reliable biomarker for assessing contaminant stress. ?? 2005 Elsevier Ltd. All rights reserved.

  3. Microbial metabolism part 13 metabolites of hesperetin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fungal culture, Mucor ramannianus (ATCC 2628) transformed hesperitin to four metabolites: 4'-methoxy -5, 7, 8, 3'-tetrahydroxyflavanone (8-hydroxyhesperetin), 5, 7, 3', 4'-tetrahydroxyflavanone (eriodictyol), 4'-methoxy-5, 3'-dihydroxyflavanone 7-sulfate (hesperetin 7-sulfate) and 5, 7, 3'-tri...

  4. Eleven microbial metabolites of 6-hydroxyflavanone

    Technology Transfer Automated Retrieval System (TEKTRAN)

    6-Hydroxyflavanone (1) when fermented with fungal culture Cunninghamella blakesleeana (ATCC 8688a) yielded flavanone 6-O-ß-D-glucopyranoside (2), flavanone 6-sulfate (3), and 6-hydroxyflavanone 7-sulfate (4). Aspergillus alliaceus (ATCC 10060) also transformed 1 to metabolite 3 as well as 4'-hydrox...

  5. Biologically active secondary metabolites from Asphodelus microcarpus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bioassay guided fractionation of the ethanolic extract of Asphodelus microcarpus Salzm.et Vivi (Asphodelaceae) resulted in the isolation of one new metabolite, 1,6-dimethoxy-3-methyl-2-naphthoic acid (1) as well as nine known compounds: asphodelin (2), chrysophanol (3), 8-methoxychrysophanol (4), em...

  6. A review: trichloroethylene metabolites: potential cardiac teratogens.

    PubMed Central

    Johnson, P D; Dawson, B V; Goldberg, S J

    1998-01-01

    This review is a a series of the authors' studies designed to test the hypothesis that administration of trichloroethylene (TCE), dichloroethylene (DCE), their metabolites, and related compounds are responsible for fetal cardiac teratogenesis when given to pregnant rats during organogenesis. Identification of teratogenic compounds will allow more accurate assessment of environmental contaminants and public health risks. Epidemiologic studies and previous teratogenic studies using chick embryos and fetal rats have reported an increased number of congenital cardiac defects when exposed to TCE or DCE during fetal development. Metabolites of TCE and DCE studied in the drinking-water exposure study include trichloroacetic acid TCAA), monochloroacetic acid, trichloroethanol, carboxymethylcysteine, trichloroacetaldehyde, dichloroacetaldehyde, and dichlorovinyl cysteine. Varying doses of each were given in drinking water to pregnant rats during the period of fetal heart development. Rats receiving 2730 ppm TCAA in drinking water were the only metabolite group demonstrating a significant increase in the number of cardiac defects in fetuses on a per-litter basis (p = 0.0004 Wilcoxon test and p =0.0015 exact permutation test). Maternal and fetal variables showed no statistically significant differences between treated and untreated groups. When treated with TCAA the increased cardiac defects, as compared to controls, do not preclude the involvement of other metabolites as cardiac teratogens, but indicates TCAA as a specific cardiac teratogen. Further studies of drinking-water exposure and potential mechanisms of action on the developing heart are proceeding. Images Figure 1 PMID:9703484

  7. Simultaneous determination of eight metabolites of organophosphate and pyrethroid pesticides in urine.

    PubMed

    Guo, Xin Y; Sun, Li S; Huang, Meng Y; Xu, Wei L; Wang, Ying; Wang, Na

    2017-01-02

    A simultaneous method for quantifying eight metabolites of organophosphate pesticides and pyrethroid pesticides in urine samples has been established. The analytes were extracted using liquid-liquid extraction coupled with WCX solid phase extraction (SPE) cartridges. Eight metabolites were chemically derivatized before analysis using gas chromatography-tandem mass spectrometry (GC-MS-MS). The separation was performed on a HP-5MS capillary column (30 m × 0.25 mm × 0.25 µm) with temperature programming. The detection was performed under electro-spray ionization (ESI) in multiple reaction monitoring (MRM) mode. An internal standard method was used. The extraction solvent, types of SPE cartridges and eluents were optimized by comparing the sample recoveries under different conditions. The results showed that the calibration curves of the five organophosphorus pesticides metabolites were linear in the range of 0.2-200 μg/L (r(2) ≥ 0.992) and that of the three pyrethroid pesticides metabolites were linear in the range of 0.025-250 μg/L (r(2) ≥ 0.991). The limits of detection (LODs, S/N ≥ 3) and the limits of quantification (LOQs, S/N ≥ 10) of the eight metabolites were 0.008-0.833 μg/L and 0.25-2.5 μg/L, respectively. The recoveries of the eight metabolites ranged from 54.08% to 82.49%. This efficient, stable, and cost-effective method is adequate to handle the large number of samples required for surveying the exposure level of organophosphorus and pyrethroid pesticides in the general population.

  8. Spatio-Temporal Metabolite Profiling of the Barley Germination Process by MALDI MS Imaging

    PubMed Central

    Gorzolka, Karin; Kölling, Jan; Nattkemper, Tim W.; Niehaus, Karsten

    2016-01-01

    MALDI mass spectrometry imaging was performed to localize metabolites during the first seven days of the barley germination. Up to 100 mass signals were detected of which 85 signals were identified as 48 different metabolites with highly tissue-specific localizations. Oligosaccharides were observed in the endosperm and in parts of the developed embryo. Lipids in the endosperm co-localized in dependency on their fatty acid compositions with changes in the distributions of diacyl phosphatidylcholines during germination. 26 potentially antifungal hordatines were detected in the embryo with tissue-specific localizations of their glycosylated, hydroxylated, and O-methylated derivates. In order to reveal spatio-temporal patterns in local metabolite compositions, multiple MSI data sets from a time series were analyzed in one batch. This requires a new preprocessing strategy to achieve comparability between data sets as well as a new strategy for unsupervised clustering. The resulting spatial segmentation for each time point sample is visualized in an interactive cluster map and enables simultaneous interactive exploration of all time points. Using this new analysis approach and visualization tool germination-dependent developments of metabolite patterns with single MS position accuracy were discovered. This is the first study that presents metabolite profiling of a cereals’ germination process over time by MALDI MSI with the identification of a large number of peaks of agronomically and industrially important compounds such as oligosaccharides, lipids and antifungal agents. Their detailed localization as well as the MS cluster analyses for on-tissue metabolite profile mapping revealed important information for the understanding of the germination process, which is of high scientific interest. PMID:26938880

  9. Deposition of JWH-018, JWH-073 and their metabolites in hair and effect of hair pigmentation.

    PubMed

    Kim, Jihyun; In, Sanghwan; Park, Yuran; Park, Meejung; Kim, Eunmi; Lee, Sooyeun

    2013-12-01

    Analysis of drugs in hair is often used as a routine method to obtain detailed information about drug ingestion. However, few studies have been conducted on deposition of synthetic cannabinoids and metabolites in hair. The first purpose of this study was to establish and validate an analytical method for detection of JWH-018, JWH-073, and their metabolites in hair, by use of UHPLC-MS-MS, for forensic application. The second purpose was to investigate the distribution of synthetic cannabinoids metabolites in hair and the effect of hair pigmentation, by use of an animal model. For this, JWH-073 was chosen as a representative synthetic cannabinoid. Finally, the developed method was applied to hair samples from 18 individuals suspected of synthetic cannabinoids use. JWH-018, JWH-073, and their metabolites were extracted from hair with methanol. The extract was then filtered and analyzed by UHPLC-MS-MS with an electrospray ion source in positive-ionization mode. Validation proved the method was selective, sensitive, accurate, and precise, with acceptable linearity within the calibration ranges. No significant variations were observed when different sources of both human and rat hair were used. The animal study demonstrated that JWH-073 N-COOH M was the major metabolite of JWH-073 in rat hair, and hair pigmentation did not have a significant effect on incorporation of JWH-073 and its metabolites into hair. In the analysis of 18 authentic hair samples, only JWH-018, JWH-018 N-5-OH M, and JWH-073 were detected, with wide variation in concentrations.

  10. Pesticides and their metabolites in wells of Suffolk County, New York, 1998

    USGS Publications Warehouse

    Phillips, Patrick J.; Eckhardt, D.A.; Terracciano, S.A.; Rosenmann, Larry

    1999-01-01

    Five insecticide residues and 20 herbicide residues were detected in water samples collected from 50 shallow wells screened in the surficial sand and gravel aquifer in Suffolk County, Long Island in areas with known or suspected residues. Laboratory analytical methods with extremely low detection limits - from 0.001 to 0.2 ?g/L (micrograms per liter) - were used to analyze the samples for 60 pesticide residues. Forty-four of the samples contained at least one pesticide residue, and some samples contained as many as 11 different pesticides or pesticide metabolites. Only four water- quality standards were exceeded in the samples collected in this study. Dieldrin exceeded the New York State Class GA standard (0.004 ?g/L) in samples from eight wells. The Federal and New York State Maximum Contaminant Level for simazine (4 ?g/L) was exceeded in samples from two wells, and the State Class GA standard for simazine (0.5 ?g/L) was exceeded in samples from six wells. Federal water-quality standards have not been established for many of the compounds detected in this study, including herbicide metabolites. Maximum concentrations of four herbicide metabolites -metolachlor ESA (ethanesulfonic acid), metolachlor OA (oxanilic acid), and the alachlor metabolites alachlor ESA and alachlor OA -exceeded 20 ?g/L. The maximum concentration of one herbicide (tebuthiuron) exceeded 10 ?g/L, and the maximum concentration of three herbicides (simazine, metolachlor, and atrazine) and one herbicide metabolite (deisopropylatrazine) ranged from 1 to 10 ?g/L. The herbicide metolachlor, which is used on potato fields in