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2013;927:3-21
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P242
Núñez O - Gallart-Ayala H - Martins CPB - Lucci P - Busquets R
State-of-the-art in fast liquid chromatography–mass spectrometry for bio-analytical applications.
Journal of Chromatography B
There is an increasing need of new bio-analytical methodologies with enough sensitivity, robustness and resolution to cope with the analysis of a large number of analytes in complex matrices in short analysis time. For this purpose, all steps included in any bio-analytical method (sampling, extraction, clean-up, chromatographic analysis and detection) must be taken into account to achieve good and reliable results with cost-effective methodologies. The purpose of this review is to describe the state-of-the-art of the most employed technologies in the period 2009–2012 to achieve fast analysis with liquid chromatography coupled to mass spectrometry (LC–MS) methodologies for bio-analytical applications. Current trends in fast liquid chromatography involve the use of several column technologies and this review will focus on the two most frequently applied: sub-2 µm particle size packed columns to achieve ultra high pressure liquid chromatography (UHPLC) separations and porous-shell particle packed columns to attain high efficiency separations with reduced column back-pressures. Additionally, recent automated sample extraction and clean-up methodologies to reduce sample manipulation, variability and total analysis time in bio-analytical applications such as on-line solid phase extraction coupled to HPLC or UHPLC methods, or the use of other approaches such as molecularly imprinted polymers, restricted access materials, and turbulent flow chromatography will also be addressed. The use of mass spectrometry and high or even ultra-high resolution mass spectrometry to reduce sample manipulation and to solve ion suppression or ion enhancement and matrix effects will also be presented. The advantages and drawbacks of all these methodologies for fast and sensitive analysis of biological samples are going to be discussed by means of relevant applications. © 2013 Elsevier B.V. All rights reserved.

Sous presse, 2013
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P241
Kaabia Z - Dervilly-Pinel G - Popot MA - Bailly-Chouriberry F - Plou P - Bonnaire Y - Le Bizec B
Monitoring the endogenous steroid profile disruption in urine and blood upon nandrolone administration: An efficient and innovative strategy to screen for nandrolone abuse in horses.
Drug Testing and Analysis
Nandrolone (17β-hydroxy-4-estren-3-one) is amongst the most misused endogenous steroid hormones in entire male horses. The detection of such a substance is challenging with regard to its endogenous presence. The current international threshold level for nandrolone misuse is based on the urinary concentration ratio of 5α-estrane-3β,17α-diol (EAD) to 5(10)-estrene-3β,17α-diol (EED). This ratio, however, can be influenced by a number of factors due to existing intra- and inter-variability standing, respectively, for the variation occurring in endogenous steroids concentration levels in a single subject and the variation in those same concentration levels observed between different subjects. Targeting an efficient detection of nandrolone misuse in entire male horses, an analytical strategy was set up in order to profile a group of endogenous steroids in nandrolone-treated and non-treated equines. Experiment plasma and urine samples were steadily collected over more than three months from a stallion administered with nandrolone laurate (1 mg/kg). Control plasma and urine samples were collected monthly from seven non-treated stallions over a one-year period. A large panel of steroids of interest (n = 23) were extracted from equine urine and plasma samples using a C18 cartridge. Following a methanolysis step, liquid-liquid and solid-phase extractions purifications were performed before derivatization and analysis on gas chromatography-tandem mass spectrometry (GC-MS/MS) for quantification. Statistical processing of the collected data permitted to establish statistical models capable of discriminating control samples from those collected during the three months following administration. Furthermore, these statistical models succeeded in predicting the compliance status of additional samples collected from racing horses. Copyright © 2013 John Wiley & Sons, Ltd. (DOI: 10.1002/dta.1520).

2014;210:188–2013
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P240
Martin Agnoux A - Martin Agnoux A - Alexandre-Gouabau MC - Alexandre-Gouabau MC - Le Dréan G - Le Dréan G - Antignac JP - Antignac JP - Parnet P - Parnet P
Relative contribution of fetal and postnatal nutritional periods on feeding regulation in adult rats
Acta Physiologica
Aim: The aim of this study was to assess the contribution of both foetal and/or post-natal nutritional periods on feeding regulation in adult rats. Methods: Body weight gain, adipose tissue development, food preferences and feeding pattern under regular chow or Western diets were characterized on four experimental groups of rats: pups born from protein-restricted dams (R) and weaned by control (RC) or R dams (RR) and pups born from control dams weaned by C (CC) or R dams (CR). Results: Rats born with intrauterine growth restriction (IUGR) and fed a Western diet at adulthood appeared predisposed to body weight gain and more fat accretion, whereas CR rats, despite their preference for high-fat diet and their hyperphagia for Western diet, did not show significant increase in fat tissue. Daytime food intakes, as well as their speed of ingestion, were found modified in RC and RR. Alterations in the hypothalamic appetite regulatory mechanisms were investigated through neuropeptide expression analysis. IUGR rats showed altered expression of key elements of leptin and NPY signalling, while CR rats exhibited lesser expression of enterostatin, MC4r and HT-1Br mRNA. Conclusion: Altogether, these results indicate that peri-natal nutrition has different lasting effects on feeding pattern and hypothalamic appetite regulation, depending on the time window insult. Keywords feeding regulation, high-fat diet, leptin, metabolic programming, peri-natal nutrition. © 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd, doi: 10.1111/apha.12163.

2013;191:202-209
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P239
Identification and quantification of 5α-dihydrotestosterone in the teleost fathead minnow (Pimephales promelas) by gas chromatography-tandem mass spectrometry
General and Comparative Endocrinology
The steroid hormone 5-dihydrotestosterone (DHT) is one of the most physiologically important androgens in male vertebrates, with the exception of teleost fish, in which it is generally assumed that DHT does not play any major physiological role. However, this assumption is challenged by the fact that all the components involved in DHT biosynthesis and action are present and evolutionary conserved in teleost fish. In fact, testosterone (T) is converted into DHT by two isoforms of the enzyme steroid-5-alphareductase (5R), and both 5Rs gene expression and enzymatic activity have been detected in several tissues of different teleost species, which also have an androgen receptor with high binding affinity to DHT. This body of evidence strongly suggest that DHT is synthesised by teleost fish. We investigated this hypothesis using the cyprinid fathead minnow (Pimephales promelas) as the experimental model. The study of the evolutionary and functional conservation of 5Rs in teleost fish was used to support the experimental approach, based on an ultrasensitive gas chromatography–tandem mass spectrometry (GC–MS/MS) method to identify and measure simultaneously T and DHT in fathead minnow biological fluids and tissues. The analyses were performed using plasma samples collected from both male and female adult fish and samples of testicular tissue collected from sexually mature males. Both T and DHT were identified and quantified in all the samples analysed, and in particular, the high concentrations of DHT quantified in the testes suggested that these organs are a likely site of synthesis of DHT in the teleost fathead minnow, as they are in mammals. These results may represent the basis for future studies aimed at elucidating the physiological role, if any, of DHT in teleost fish. © 2013 Elsevier Inc. All rights reserved.

2013;796:75-83
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P237
Gallart-Ayala H - Courant F - Sévère S - Antignac JP - Morio F - Abadie J - Le Bizec B
Versatile lipid profiling by Liquid Chromatography – High Resolution Mass Spectrometry using all ions fragmentation and polarity switching. Preliminary application for serum samples phenotyping related to canine mammary cancer
Analytica Chimica Acta
Lipids represent an extended class of substances characterized by such high variety and complexity that makes their unified analyses by liquid chromatography coupled to either high resolution or tandem mass spectrometry (LC–HRMS or LC–MS/MS) a real challenge. In the present study, a new versatile methodology associating ultra high performance liquid chromatography coupled to high resolution tandem mass spectrometry (UHPLC–HRMS/MS) have been developed for a comprehensive analysis of lipids. The use of polarity switching and “all ion fragmentation” (AIF) have been two action levels particularly exploited to finally permit the detection and identification of a multi-class and multi-analyte extended range of lipids in a single run. For identification purposes, both higher energy collision dissociation (HCD) and in-source CID (collision induced dissociation) fragmentation were evaluated in order to obtain information about the precursor and product ions in the same spectra. This approach provides both class-specific and lipid-specific fragments, enhancing lipid identification. Finally, the developed method was applied for differential phenotyping of serum samples collected from pet dogs developing spontaneous malignant mammary tumors and health controls. A biological signature associated with the presence of cancer was then successfully revealed from this lipidome analysis, which required to be further investigated and confirmed at larger scale. © 2013 Elsevier B.V. All rights reserved.

2013;12:2914−2920
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P236
Courant F - Antignac JP - Monteau F - Le Bizec B
Metabolomics as a potential new approach for investigating human reproductive disorders.
Journal of Proteome Research
Metabolomics has been emerging for several years as a global chemical phenotyping approach offering fascinating descriptive capabilities for addressing life complexity. It facilitates the understanding of the mechanisms of biological and biochemical processes in complex systems and promises new insights into specific research questions. The objective of this study was to use for the first time a metabolomic approach based on liquid chromatography high resolution mass spectrometry for characterizing an alteration of the testicular function, namely impaired semen quality. Metabolomic fingerprints were generated from serum samples collected from Danish young men presenting low, intermediate, or high sperm concentrations. Serum metabolic profiles were found to be significantly different among the three groups of volunteers. The developed methodology permitted to correlate the studied clinical parameter (i.e., sperm concentration) with the metabolite profiles generated. Peptides related to the Protein Complement C3f were identified as putative markers associated with this clinical parameter. The biological interpretation and further robustness linked to this observation remain to be further investigated, in particular to address the inter- and intraindividual variabilities. © 2013 American Chemical Society.

2013;141:1032–1040
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P235
Le Boucher C - Courant F - Jeanson S - Chéreau S - Maillard MB - Royer AL - Thierry A - Dervilly-Pinel G - Le Bizec B - Lortal S
First mass spectrometry metabolic fingerprinting of bacterial metabolism in a model cheese.
Food Chemistry
Metabolic fingerprinting is an untargeted approach which has not yet been undertaken to investigate cheese. This study is a proof of concept, concerning the ability of mass spectrometry (MS) metabolic fingerprinting to investigate modifications induced by bacterial metabolism in cheese over time. An ultrafiltrated milk concentrate was used to manufacture model cheeses inoculated with Lactococcus lactis LD61. Metabolic fingerprints were acquired after 0, 8 and 48 h from two different fractions of the metabolome: the water-soluble fraction using liquid chromatography–high resolution-MS and a volatile fraction using gas chromatography–MS. Metabolic fingerprints differed significantly over time. Forty-five metabolites were identified, including well-known cheese metabolites, such as 12 amino acids and 25 volatile metabolites, and less studied ones, such as four vitamins, uric acid, creatine and L-carnitine. These results showed the relevance of cheese MS fingerprinting to generate new findings and to detect even slight differences between two conditions. © 2013 Elsevier Ltd. All rights reserved.

2013;14(4):287-288
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P234
Lauby-Secretan B - Loomis D - Grosse Y - El Ghissassi F - Bouvard V - Benbrahim-Tallaa L - Guha N - Baan R - Mattock H - Straif K
On behalf of the International Agency for Research on Cancer Monograph Working Group IARC, Lyon, France. Carcinogenicity of polychlorinated biphenyls and polybrominated biphenyls.
The Lancet Oncology

2013;61(29):7156-7164
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P233
Fournier A - Rychen G - Marchand P - Toussaint H - Le Bizec B - Feidt C
Polychlorinated Biphenyl (PCB) decontamination kinetics in lactating goats (Capra hircus) following a contaminated corn silage exposure.
Journal of Agricultural and Food Chemistry
This study aimed to determine the kinetics of contamination and decontamination of PCBs and PCDD/Fs in milk of lactating goats. Four goats were fed during 39 days with corn silage collected in an area accidentally contaminated and then with uncontaminated silage during 20 days. Concentrations of DL-PCBs + PCDD/Fs in milk exceeded rapidly (<15 days) the European limit value and approached steady state after 5 weeks. The decontamination kinetics in milk included first a rapid elimination phase (<10 days) followed by a slower elimination phase of 33, 51, and 59 days for DL-PCBs, NDL-PCBs, and PCDD/Fs, respectively. Therefore, in lactating goats, PCBs and PCDD/Fs contaminated forage raises concerns in terms of food safety. The study also indicates that a decontamination process of lactating animals remains feasible; 20 days was considered to be sufficient to obtain a DL-PCBs + PCDD/Fs level in milk below the regulatory value. © 2013 American Chemical Society.

2013;91:802-808
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P232
Antignac JP - Veyrand B - Kadar H - Marchand P - Oleko A - Le Bizec B - Vandentorren S
Occurrence of perfluorinated contaminants in breast milk of French women and relation with socio-demographical and clinical parameters: results of the elfe pilot study.
Chemosphere
A previously developed and validated methodology based on liquid chromatography coupled to high resolution mass spectrometry was used for determine the concentration levels of 14 perfluoroalkylated substances (PFASs) in a set of 48 breast milk samples collected from French women in the frame of the ELFE pilot study. In accordance with other similar studies conducted at european and international levels, PFOS, PFOA, and PFHxS were detected and quantified in most of the analyzed samples (90%, 98% and 100%, respectively), and appeared as major contributors to the total PFAS exposure (38%, 37%, 25%, respectively), whereas the other targeted PFAS were very rarely, if not, found at the limits of detection of the method. Also in agreement with other published data, the concentration levels measured for the detected substances varied from <0.05 to 0.33 µg/L for PFOS (median = 0.079), from <0.05 to 0.22 µg/L for PFOA (median = 0.075), and from 0.04 to 0.07 µg/L for PFHxS (median = 0.050). On the basis of this relatively limited data set, no statistically significant relation was observed between these exposure levels and developmental outcomes, in particular the weight at birth. Similarly, no relation was observed between the measured PFAS levels and various socio-demographical parameters including the consumption of seafood, alcohol, smoking, or socio-economical level. These results suggest a need for further research and better knowledge regarding the sources, pharmacokinetics, and factors of exposure for other substances belonging to this class of emerging contaminants. © 2013 Elsevier Ltd. All rights reserved.

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