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First profiling in hydrophilic interaction liquid chromatography of intact human chorionic gonadotropin isoforms.
Camperi J, Pichon V, Fournier T, Delaunay N
J Pharm Biomed Anal - 10;174 495-499. - doi: 10.1016/j.jpba.2019.06.014 - 2019
The study of glycoproteins is a rapidly growing field, which is not surprising considering that approximately 70% of human proteins are glycosylated and that numerous biological functions are associated to the glycosylation. In this work, our interest focused on the heterodimeric human Chorionic Gonadotropin (hCG) glycoprotein that is the specific hormone of the human pregnancy, consisting of an α and a β subunit, so-called hCGα and hCGβ, respectively. This protein possesses a very high structural heterogeneity, essentially due to the presence of 8 glycosylation sites, but also other types of post-translational modifications. In this study, for the first time, the potential of hydrophilic interaction liquid chromatography (HILIC) was investigated to separate the intact hCG isoforms. Three different HILIC stationary phases were tested using an hCG-based drug as standard, a recombinant hCG. For each stationary phase, the effect of the initial mobile phase composition based on ACN/H2O mixture, the slope of the gradient, the content and nature of the acidic additive (formic acid and trifluoroacetic acid (TFA)), and the addition of a volatile salt (ammonium formate) on the retention and the resolution were studied. The best HILIC separation was obtained with the amide column and a mobile phase composed of water/ACN containing 0.1% of TFA. The repeatability in terms of retention times and peak areas was then assessed. Finally, the method was applied to the analysis of a second hCG-based drug obtained from urine of pregnant women. Both drugs gave chromatograms with more than 10 peaks. However, they were significantly different, which demonstrated the potential of HILIC method for hCG isoform fingerprinting
Specificity of the metabolic signatures of fish from cyanobacteria rich lakes
Benoît Sotton, Alain Paris, Séverine Le Manach, Alain Blond, Charlotte Duval, Qin Qiao, Arnaud Catherine, Audrey Combes, Valérie Pichon, Cécile Bernard, Benjamin Marie
Chemosphere - - DOI: 10.1016/j.chemosphere.2019.03.115 - 2019
With the increasing impact of the global warming, occurrences of cyanobacterial blooms in aquatic ecosystems are becoming a main worldwide ecological concern. Due to their capacity to produce potential toxic metabolites, interactions between the cyanobacteria, their cyanotoxins and the surrounding freshwater organisms have been investigated during the last past years. Non-targeted metabolomic analyses have the powerful capacity to study simultaneously a high number of metabolites and thus to investigate in depth the molecular signatures between various organisms encountering different environmental scenario, and potentially facing cyanobacterial blooms.

In this way, the liver metabolomes of two fish species (Perca fluviatilis and Lepomis gibbosus) colonizing various peri-urban lakes of the Île-de-France region displaying high biomass of cyanobacteria, or not, were investigated. The fish metabolome hydrophilic fraction was analyzed by 1H NMR analysis coupled with Batman peak treatment for the quantification and the annotation attempt of the metabolites. The results suggest that similar metabolome profiles occur in both fish species, for individuals collected from cyanobacterial blooming lakes compared to organism from non-cyanobacterial dominant environments. Overall, such environmental metabolomic pilot study provides new research perspectives in ecology and ecotoxicology fields, and may notably provide new information concerning the cyanobacteria/fish ecotoxicological interactions.
First characterizations by capillary electrophoresis of human Chorionic Gonadotropin at the intact level
Benoît Sotton, Alain Paris, Séverine Le Manach, Alain Blond, Charlotte Duval, Qin Qiao, Arnaud Catherine, Audrey Combes, Valérie Pichon, Cécile Bernard, Benjamin Marie
Talanta - 193 77-86 - doi: 10.1016/j.talanta.2018.09.095 - 2019
In the present work, the first characterizations by Capillary Electrophoresis of the human Chorionic Gonadotropin (hCG) hormone at the intact level were carried out. hCG is a hetero-dimeric glycoprotein, specific to the human pregnancy, consisting of an α and a β subunit, so-called hCGα and hCGβ, respectively. hCG has 8 potential glycosylation sites leading to a high number of isoforms (including glycoforms and other post-translational modifications) that we are interesting to characterize. First, Capillary Gel Electrophoresis (CGE) was used to separate the isoforms of two hCG-based drugs: Ovitrelle® (recombinant r-hCG) and Pregnyl (hCG isolated from the urine of pregnant women, u-hCG). As expected, CGE led to a better resolution than SDS-PAGE and confirmed the large heterogeneity of hCG. Different CGE profiles were obtained for the two hCG-based drugs, varying in number of peaks, migration times, and peak intensities, thus demonstrating that the drugs contain isoforms, different in nature and proportion. This result was confirmed by Capillary IsoElectrophoretic Focusing (CIEF). The pI ranges of the hCG isoforms were found between 3.4 and 4.7, and 4.5 and 5.2 for r-hCG and u-hCG, respectively. This information was further used to develop the separation of the hCG isoforms by Capillary Zone Electrophoresis (CZE). The pH, the nature, and the concentration of the background electrolyte as well as the nature and the content of its organic modifier were optimized. The use of a coated capillary to avoid protein adsorption was also evaluated. The final CZE-UV method allowed distinguishing at least 6 peaks, corresponding to different hCG isoforms. To significantly improve the level of information obtained, the CZE instrument was then coupled by means of an electrospray ionization source to a triple quadrupole (TQ) mass spectrometer. Two detection strategies were used, one focusing on the lower m/z values (100-1000) in order to identify some sugar residues as diagnostic ions to confirm the presence of glycan chains, and the second focusing on the higher m/z values (1000-2000), corresponding to the multiple charged intact protein isoforms. For both approaches, the fragmentor and capillary voltage values were optimized. The composition and the flow-rate of the sheath liquid were then optimized for the strategy focusing on the higher m/z values in order to both increase the charge state of the ionized intact isoforms and the signal-to-noise ratio. The final method was used to compare the two hCG-based drugs, demonstrating the potential of the developed CZE-MS method for isoforms fingerprinting.
Development and application of water-compatible molecularly imprinted polymers for the selective extraction of carbamazepine from environmental waters
Porkodi Kadhirvel, Audrey Combès, Louis Bordron, Valérie Pichon
Anal. Bioanal. Chem - 11(8) 1525-1536 - doi: 10.1007/s00216-019-01586-8 - 2019
A molecularly imprinted polymer (MIP) was designed in order to allow the selective solid-phase extraction of carbamazepine (CBZ), an anticonvulsant and mood-stabilizing drug, at ultra-trace level from aqueous environmental samples. A structural analog of CBZ was selected as a dummy template and different synthesis conditions were screened. The selectivity of the resulting imprinted polymers was evaluated by studying the retention of CBZ in a solvent similar to the one used for the synthesis. The presence of imprinted cavities in the polymers was then demonstrated by comparing the elution profiles (obtained by using MIP and a non-imprinted polymer, NIP, as a control) of the template, of CBZ, and of a structural analog of CBZ. Then, the extraction procedure was further optimized for the treatment of aqueous samples on the two most promising MIPs, with special attention being paid to the volume and composition of the percolation and washing solutions. The best MIP provided a highly selective retention in tap water with 81% extraction recovery for CBZ in the elution fraction of the MIP and only 14% for NIP. The repeatability of the extraction procedure was demonstrated for both tap and river waters (RSD below 4% in river water) for the drugs CBZ, oxcarbamazepine, and one metabolite (carbamazepine 10,11-epoxide). A MIP capacity of 1.15 μmol g-1 was determined. Finally, an analytical procedure involving the MIP was developed allowing the detection of CBZ at a concentration level of only a few nanograms per liter in river water. The selectivity provided by the MIP resulted in a 3000-fold increase of the signal-to-noise ratio in LC/MS analysis as compared to the use of conventional sorbent. Graphical abstract.
Nano- and microplastic analysis: Focus on their occurrence in freshwater ecosystems and remediation technologies
Yolanda Pico, Ahmed Alfarhan and Damia Barcelo
TrAC Trends in Analytical Chemistry - 113 409-425 - doi: 10.1016/j.trac.2019.02.016 - 2019
Plastic pollution is a global problem since 2016 when its production reached 322 million tonnes, excluding fibers. Daily discharges of microplastics (MPs, defined as <5 mm in size) are estimated in the range of 50,000 up to 15 million particles, whereas no information on nanoplastic (NP, <100 nm) release is available yet. Different processes further degraded these materials producing more MPs and NPs. This review attempts to fill the void of information on the state-of-art analysis of MPs and NPs (recently identified as emerging contaminants) and provides a critical overview on modern instrumentation, newly developed workflows, and promising techniques for their characterization (Raman and FT-IR spectroscopies and microscopies, pyrolysis and thermal desorption gas chromatography, imaging techniques, etc.). Available analytical methods, validation as well as applications with cells have been taken into account. MP and NP sampling, identification, and characterization are discussed. Finally, recent applications to establish their occurrence in freshwater ecosystems and the effectiveness of the proposed remediation technologies are considered.
Monitoring of the blend monoethanolamine/methyldiethanolamine/water for post-combustion CO2 capture
Lorena Cuccia, José Dugay, Bontemps Domitille, Myriam Louis-Louisy
International Journal of Greenhouse Gas Control - 80 43-53 - DOI: 10.1016/j.ijggc.2018.11.004 - 2019
The blend MEA/MDEA (5/25%wt.) was studied on the LEMEDES-CO2 lab-scale pilot plant, with representative conditions of post-combustion CO2 capture for power generation during 900 h. CO2 loadings were determined and showed average values of 0.12 and 0.40 respectively for the lean and rich solvents. Stability of the two amines, namely MEA and MDEA, was monitored using ionic chromatography; results did not show any significant degradation of MDEA during the campaign, in contrary to MEA which showed a significant degradation in the range of 0.03 points per day. Analytical methods involving GC–MS and IC were developed in order to identify potential degradation products in the liquid phase of the solvent. Study of the gaseous emissions’ composition was also realized using sampling on different solid sorbents followed by thermal desorption and GC–MS analysis. A total of 22 compounds were listed including amines, organic acids, and pyrazines derivatives. 12 degradation products were found in the solvent itself and 11 in the treated flue gas among which MDEA, the constituent amine of the blend. A quantitative monitoring was carried out for formic and oxalic acids. Results showed concentrations reaching 500 mg/L for oxalic acid and 1400 mg/L for formic acid.
Synthesis and Characterization of Molecularly Imprinted Polymers for the Selective Extraction of Carbamazepine and Analogs from Human Urine Samples
287–295
Chromatographia - 82 287–295 - doi:10.1007/s10337-018-3680-4 - 2019
Two molecularly imprinted polymers (MIPs) were synthesized according to a previous work from our group dealing with the extraction of carbamazepine from environmental water. The potential of these MIPs, which differ in the nature of the monomer used for their synthesis, to selectively extract the drugs carbamazepine and oxcarbazepine and the metabolite 10,11-epoxycarbamazepine was first studied in spiked pure water, and high selectivity was obtained with both MIPs for the three target molecules in this pure medium. This selectivity was maintained when applying one of the MIPs to urine samples. Indeed, extraction recoveries were higher than 82% on the MIP and lower than 20% on the corresponding non-imprinted polymer used as a control. The repeatability of the extraction procedure applied to urine was also demonstrated, with relative standard deviation (RSD) below 20% for extraction recoveries of the three targets at a spiking level of 20 ng L−1. Limits of quantification between 1 and 7 ng L−1 were determined for urine samples using the MIP as extraction sorbent combined with LC–MS analysis. The potential of the MIP was compared to that of the Oasis HLB sorbent. This study shows that the MIP constitutes a powerful tool for avoiding matrix effects encountered in the quantification of the target molecules in urine samples extracted on Oasis HLB.
Online coupling of immunoextraction, digestion, and microliquid chromatography-tandem mass spectrometry for the analysis of sarin and soman-butyrylcholinesterase adducts in human plasma
Maud Bonichon, Valentina Valbi, Audrey Combès, Charlotte Desoubries, Anne Bossée & Valérie Pichon
Anal. Bioanal. Chem - 410 pages1039–1051 - doi.org/10.1007/s00216-017-0640-z - 2018
Organophosphorus nerve agent (OPNA) adducts formed with human butyrylcholinesterase (HuBuChE) can be used as biomarker of OPNA exposure. Indeed, intoxication by OPNAs can be confirmed by the LC/MS2 analysis of a specific HuBuChE nonapeptide on which OPNAs covalently bind. A fast, selective, and highly sensitive online method was developed to detect sarin and soman adducts in plasma, including immunoextraction by anti-HuBuChE antibodies, pepsin digestion on immobilized enzyme reactors (IMER), and microLC/MS2 analysis of the OPNA adducts. The potential of three different monoclonal antibodies, covalently grafted on sepharose, was compared for the extraction of HuBuChE. The online method developed with the most promising antibodies allowed the extraction of up to 100% of HuBuChE contained in plasma and the digestion of 45% of it in less than 40 min. Moreover, OPNA-HuBuChE adducts, aged OPNA adducts, and unadducted HuBuChE could be detected (with S/N > 2000), even in plasma spiked with a low concentration of OPNA (10 ng mL−1). Finally, the potential of this method was compared to approaches involving other affinity sorbents, already described for HuBuChE extraction.
Human odor and forensics: Towards Bayesian suspect identification using GC × GC–MS characterization of hand odor
lVincent Cuzuel Roman Leconte Guillaume Cognon Didier Thiébaut Jérôme Vial Charles Sauleau Isabelle Rivals
Journal of Chromatography B - 1092 379-385 - https://doi.org/10.1016/j.jchromb.2018.06.018 - 2018
A new method for identifying people by their odor is proposed. In this approach, subjects are characterized by a GC × GC–MS chromatogram of a sample of their hand odor. The method is based on the definition of a distance between odor chromatograms and the application of Bayesian hypothesis testing. Using a calibration panel of subjects for whom several odor chromatograms are available, the densities of the distance between chromatograms of the same person, and between chromatograms of different persons are estimated. Given the distance between a reference and a query chromatogram, the Bayesian framework provides an estimate of the probability that the corresponding two odor samples come from the same person. We tested the method on a panel that is fully independent from the calibration panel, with promising results for forensic applications.
Active modulation in neat carbon dioxide packed column comprehensive two-dimensional supercritical fluid chromatography
Orjen Petkovic Pierre Guibal Patrick Sassiat Jérôme Vial Didier Thiébaut
J. Chrom. A - 1536 176-184 - https://doi.org/10.1016/j.chroma.2017.08.063 - 2018
After demonstrating in a first paper the feasibility of SFCxSFC without decompression of the mobile phase, a modified interface has been developed in order to perform active modulation between the two SFC dimensions. In this paper, it is shown that the new interface enabled independent control of modulation parameters in SFCxSFC and performed a band compression effect of solutes between the two SFC dimensions. The effectiveness of this new modulation process was studied using a Design of Experiments. The SFCxSFC prototype was applied to the analysis of a real oil sample to demonstrate the benefits of the active modulator; in comparison to our previous results obtained without active modulation, better separation was obtained with the new interface owing to the peak compression occurring in the modulator.

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27 publications.