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Synthesis and characterization of molecularly imprinted polymers for the selective extraction of oxazepam from complex environmental and biological samples
Fanny Varenne, Porkodi Kadhirvel, Pauline Bosman, Loïc Renault, Audrey Combès, Valérie Pichon
Anal. Bioanal. Chem - - doi: 10.1007/s00216-021-03268-w. - 2021
Oxazepam, one of the most frequently prescribed anxiolytic drugs, is not completely removed from wastewater with conventional treatment processes. It can thus be found at trace levels in environmental water, with human urine constituting the major source of contamination. This study focused on the development and characterization of molecularly imprinted polymers (MIPs) for the selective solid-phase extraction of oxazepam at trace levels from environmental water and human urine samples. Two MIPs were synthesized, and their selectivity in pure organic and aqueous media were assayed. After optimizing the extraction procedure adapted to a large sample volume to reach a high enrichment factor, the most promising MIP was applied to the selective extraction of oxazepam from environmental water. Extraction recoveries of 83 ± 12, 92 ± 4 and 89 ± 10% were obtained using the MIP for tap, mineral and river water, respectively, while a recovery close to 40% was obtained on the corresponding non-imprinted polymer (NIP). Thanks to the high enrichment factors, a limit of quantification (LOQ) of 4.5 ng L-1 was obtained for river water. A selective extraction procedure was also developed for urine samples and gave rise to extraction recoveries close to 95% for the MIP and only 23% for the NIP. Using the MIP, a LOQ of 357 ng L-1 was obtained for oxazepam in urine. The use of the MIP also helped to limit the matrix effects encountered for the quantification of oxazepam in environmental samples and in human urine samples after extraction on an Oasis HLB sorbent.

Identification and semi-relative quantification of intact glycoforms of human chorionic gonadotropin alpha and beta subunits by nano liquid chromatography-Orbitrap mass spectrometry
AmiraAl Matari, Anastasia Goumenou, Audrey Combèsa, Thierry Fournier, Valérie Pichon, Nathalie Delaunay
J. Chromatography A - 1640 461945 - - 2021
The human chorionic gonadotropin (hCG) protein belongs to a family of glycoprotein hormones called gonadotropins. It is a heterodimer made of two non-covalently linked subunits. The α-subunit structure, hCGα, has 2 N-glycosylation sites, while the beta subunit, hCGβ, has 2 N- and 4 O-glycosylation sites. This leads to numerous glycoforms. A method based on the analysis of hCG glycoforms at the intact level by nano-reversed phase liquid chromatography coupled to high resolution mass spectrometry (nanoLC-HRMS) with an Orbitrap analyzer was previously developed using a recombinant hCG-based drug, Ovitrelle®, as standard. It allowed the detection of about 30 hCGα glycoforms, but didn't allow the detection of hCGβ glycoforms. This method was thus here significantly modified (addition of a pre-concentration step of the sample to increase the sample volume from 70 nl to 1 µl, optimization of the gradient slope and the nature and content of the acidic additive in the mobile phase). It led to an improvement of the separation of hCGα and hCGβ glycoforms, which allowed for the first time the detection of 33 hCGβ glycoforms at intact level. In addition, a higher number of hCGα glycoforms (42 in total, i.e. a 40% increase) was detected. The figures of merit of this new method were next assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.02 and 0.95% (n = 3), with an average value of 0.36% for the alpha glycoforms and between 0.01 and 1.08% (n = 3) with an average value of 0.23% for the beta glycoforms. The RSDs of the relative peak area measured on the extracted ion chromatogram of each glycoform were below 20% (n = 3), with an average value of 9.8%, thus allowing semi-relative quantification. Therefore, this method has a high potential for rapid quality control aiming for the detection and comparison of glycoforms present in glycoprotein-based pharmaceutical preparations.

Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of tryptic digest of human hemoglobin exposed to sulfur mustard
Florine Hallez, Audrey Combès, Charlotte Desoubries, Anne Bossée, Valérie Pichon
J. Chromatography A - 1163 122518 - - 2021
Sulfur mustard is a highly reactive chemical warfare agent that causes severe damages to the victims exposed by alkylating multiple biomolecules such as proteins. Resulting alkylated products can be used as biomarkers of exposure to this chemical agent. A liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method was thus developed to detect alkylated peptides after the tryptic digestion of hemoglobin (50 mg.mL−1) incubated with sulfur mustard at different concentrations (0.25, 0.5, 1, 10 and 100 µg.mL−1). Five new alkylation sites were accurately identified on the protein (α-His72, α-His87, α-His89, β-His2 and β-Val98) and fifteen adducted peptides were detected, among which eight of them resulted from the alkylation of four peptides, each presenting two potential sites of adduction that could be discriminated by the method specificity. Similarly, it was possible to discriminate the three potential adduction sites of the peptide α-T9. Moreover, the method allowed the quantification of all the alkylated peptides with a satisfying repeatability, with RSD ranging from 0.5 to 9.3% for an exposure of hemoglobin to sulfur mustard at 100 µg.mL−1. The analysis of hemoglobin incubated with different concentrations of sulfur mustard levels led to a linear response for all the alkylated peptides with the studied concentrations (0.25, 0.5, 1, 10 and 100 µg.mL−1). A variation of the alkylation rate was also observed between the different peptides studied, with a preferential adduction of sulfur mustard on the histidine residues but also on the N-terminal valine residues of both globin chains and on the Val98 residue of globin β. Furthermore, the presented method proved to be sensitive, with a theoretical possibility to detect alkylated peptides resulting from in vitro incubation of hemoglobin in deionized water with sulfur mustard at 2.63 ng.mL−1. After further development, this method could potentially be used for the analysis of blood samples in vivo exposed to sulfur mustard.

COVID-19 and Dentistry in 72 Questions: An Overview of the Literature
Stéphane Derruau, Jérôme Bouchet, Ali Nassif, Alexandre Baudet, Kazutoyo Yasukawa, Sandrine Lorimier, Isabelle Prêcheur, Agnès Bloch-Zupan, Bernard Pellat, Hélène Chardin
J. Clin. Med. - 10 122518 - 4 - 2021
The outbreak of Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has significantly affected the dental care sector. Dental professionals are at high risk of being infected, and therefore transmitting SARS-CoV-2, due to the nature of their profession, with close proximity to the patient’s oropharyngeal and nasal regions and the use of aerosol-generating procedures. The aim of this article is to provide an update on different issues regarding SARS-CoV-2 and COVID-19 that may be relevant for dentists. Members of the French National College of Oral Biology Lecturers (“Collège National des EnseignantS en Biologie Orale”; CNESBO-COVID19 Task Force) answered seventy-two questions related to various topics, including epidemiology, virology, immunology, diagnosis and testing, SARS-CoV-2 transmission and oral cavity, COVID-19 clinical presentation, current treatment options, vaccine strategies, as well as infection prevention and control in dental practice. The questions were selected based on their relevance for dental practitioners. Authors independently extracted and gathered scientific data related to COVID-19, SARS-CoV-2 and the specific topics using scientific databases. With this review, the dental practitioners will have a general overview of the COVID-19 pandemic and its impact on their practice. View Full-Text

Molecularly imprinted polymers in miniaturized extraction and separation devices
Thomas Bouvarel, Nathalie Delaunay, Valérie Pichon
First published - 44(8) 1727-1751 - - 2021
Molecularly imprinted polymers are highly selective and cost-effective materials, which have attracted significant interest in various areas such as sample pretreatment and chromatographic and electrophoretic separations. This review aims to present the state of the art concerning the miniaturization of these materials in order to meet the societal demand for reliable, fast, cheap, and solvent/sample saving analyses. The polymerization route specificities for the production of miniaturized molecularly imprinted polymers in capillaries or chip channels, such as open tubular, packed particles, magnetic nanoparticles, and in situ imprinted monoliths, are investigated. Their performances as selective supports in solid phase extraction and as stationary phases in electrochromatography and liquid chromatography, as well as their possible perspectives are discussed.

Using an Untargeted Metabolomics Approach to Identify Salivary Metabolites in Women with Breast Cancer
Daniele Xavier Assad ,Ana Carolina Acevedo, Elisa Cançado Porto Mascarenhas, Ana Gabriela Costa Normando,Valérie Pichon,Helene Chardin,Eliete Neves Silva Guerra and Audrey Combes
Metabolites - 10(12) 1727-1751 - - 2020
Metabolic alterations are a hallmark of the malignant transformation in cancer cells, which is characterized by multiple changes in metabolic pathways that are linked to macromolecule synthesis. This study aimed to explore whether salivary metabolites could help discriminate between breast cancer patients and healthy controls. Saliva samples from 23 breast cancer patients and 35 healthy controls were subjected to untargeted metabolomics using liquid chromatography-quadrupole time-of-flight mass spectrometry and a bioinformatics tool (XCMS Online), which revealed 534 compounds, characterized by their retention time in reverse-phase liquid chromatography and by the m/z ratio detected, that were shared by the two groups. Using the METLIN database, 31 compounds that were upregulated in the breast cancer group (p < 0.05) were identified, including seven oligopeptides and six glycerophospholipids (PG14:2, PA32:1, PS28:0, PS40:6, PI31:1, and PI38:7). In addition, pre-treatment and post-treatment saliva samples were analyzed for 10 patients who experienced at least a partial response to their treatment. In these patients, three peptides and PG14:2 were upregulated before but not after treatment. The area under the curve, sensitivity, and specificity for PG14:2 was 0.7329, 65.22%, and 77.14%, respectively. These results provide new information regarding the salivary metabolite profiles of breast cancer patients, which may be useful biomarkers. View Full-Text

Synthesis and characterization of molecularly imprinted polymers for the selective extraction of oxazepam from complex environmental and biological samples
Daniele Xavier Assad Elisa Cançado Porto Mascarenhas Ana Gabriela Costa Normando Hélène Chardin Gustavo Barcelos Barra Riccardo Pratesi Yanna Karla De Medeiros Nóbrega Ana Carolina Acevedo Eliete Neves Silva Guerra
Molecular and Clinical Oncology - 155-161 - - 2020
The early detection of breast cancer enables the use of less aggressive treatment and increases patient survival. The transmembrane glycoprotein mucin 1, which is also known as cancer antigen 15‑3 (CA15‑3), is aberrantly glycosylated and overexpressed in a variety of epithelial cancers, and serves a crucial role in the progression of the disease. CA15‑3 is currently used as a marker of breast cancer. In the present study, CA15‑3 concentrations in saliva and blood of patients with breast cancer were evaluated to test new assays to detect salivary CA15‑3 in addition to ELISA and its diagnostic value. To the best of our knowledge, there are no previous reports of the use of chemiluminescence assay (CLIA) and electrochemiluminescence assay (ECLIA) in saliva. Saliva and blood were collected on the same day from patients with breast cancer (n=26) and healthy controls (n=28). For each subject, the level of serum CA15‑3 was measured using ECLIA, and the level of salivary CA15‑3 was measured using ECLIA, CLIA and enzyme‑linked immunosorbent assay (ELISA). ELISA and CLIA were able to detect CA15‑3 in saliva; however, ECLIA could not detect salivary CA15‑3. There was no significant difference between the mean serum and salivary CA15‑3 levels in patients with breast cancer or healthy controls. The levels of CA15‑3 were highest for luminal breast cancer subtypes and stage IV cases. A moderate correlation was observed between salivary and serum CA15‑3 levels as measured by ELISA in breast cancer patients (r=0.56; P=0.0047). The results demonstrated that ECLIA was not a good method to detect salivary CA15‑3, although it is the gold standard for detecting serum CA15‑3. The presence of CA15‑3 in saliva was confirmed, and this will be useful in future research. Further investigations are necessary to confirm the ability to detect salivary CA15‑3 and its correlation with serum CA15‑3.
How high resolution mass spectrometry can help for the accurate quantification of difficult fragrance allergens
Pierre‐Alain Remy, C. Pérès, J. Dugay, E. Corbi, Nathalie David, J. Vial
Flavour and Fragrance Journal - 36(1) - DOI:10.1002/ffj.3639 - 2020
Two high‐resolution mass spectrometers (HRMS) with different analyzer technology, Orbitrap and hybrid quadrupole time‐of‐flight (QTOF), were compared with a low‐resolution mass spectrometer, quadrupole, to analyse a set of 35 difficult allergens. These difficult allergens are commonly coeluted fragrance allergens with matrix compounds, using standard gas chromatography‐mass spectrometer conditions, from the extended list of the Scientific Committee on Consumer Safety (SCCS). Although the fundamental role of chromatographic separation has been demonstrated many times, the aim of this work is to demonstrate the benefits of high‐resolution. The added value of high‐resolution was illustrated in both a qualitative and a quantitative way. For qualitative aspect, the high resolution extracted ion signals of these two detectors were compared with the low‐resolution extracted ion signals. About 50% of the coeluted cases observed with the low‐resolution detector are easily resolved by the two high‐resolution detectors. For the quantitative aspect, an accuracy profile methodology and a performance metric were used to propose an overall evaluation. The Orbitrap mass spectrometer demonstrated a better overall performance, while the QTOF presented similar or even lower quantification performances than the quadrupole on the set of analysed fragrance
Impact of the Oil Matrix on Anionic and Nonionic Surfactant Separation Using Ultra-High-Performance Liquid Chromatography Hyphenated to High-Resolution Mass Spectrometry
Alizée Dufour, Didier Thiébaut, Matthieu Loriau, Leticia Ligiero, and Jérôme Vial
American Chemical Society - 34(11) 13943–13953 - - 2020
For the first time, to our knowledge, anionic and nonionic surfactants were analyzed in an oil matrix by ultra-high-performance liquid chromatography hyphenated to high-resolution mass spectrometry (UHPLC-HRMS). The feasibility of this analysis was studied using synthetic mixtures of surfactants prepared in water (quality controls), binary THF/toluene 50/50 v/v (surfactant + THF/toluene), and binary THF/toluene containing 1 and 10% crude oil (Crude1% and Crude10%). These compositions were chosen in order to be as close as possible to petroleum related samples to be investigated in the future. Analyses were carried out by UHPLC methods using both reverse phase and anion-exchange mechanisms with a mixed mode column. Despite the complexity of the oil matrix and the presence of organic solvents used for dilution, the retention times of the surfactants were not affected whatever the concentration of crude oil present in the sample. Nevertheless, a significant matrix effect caused a loss of signal when the concentration of oil reached 10% in mass. For the analysis of samples with this crude oil concentration range, it would be advisable to dilute the sample.

Two-step local functionalization of fluoropolymer Dyneon THV microfluidic materials by scanning electrochemical microscopy combined to click reaction
Kadhirvel P, Combès A, Bordron L, Pichon V
Anal. Bioanal. Chem - 411(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.


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27 publications.