References of "Servais, Anne-Catherine"
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See detailCapillary electrophoresis-mass spectrometry of derivatized amino acids for targeted neurometabolomics – pH mediated reversal of diastereomer migration order
Moldovan, Radu-Cristian ULiege; Bodoki, Ede; Servais, Anne-Catherine ULiege et al

in Journal of Chromatography. A (in press)

A targeted CE-MS approach was developed for the chiral analysis of biologically relevant amino acids in artificial cerebrospinal fluid (aCSF). In order to achieve chiral resolution, the five amino acids ... [more ▼]

A targeted CE-MS approach was developed for the chiral analysis of biologically relevant amino acids in artificial cerebrospinal fluid (aCSF). In order to achieve chiral resolution, the five amino acids (Ser, Asn, Asp, Gln and Glu) were derivatized with (+)-1-(9-fluorenyl)ethyl chloroformate ((+)-FLEC). The diastereoselectivity was found to be highly dependent on pH for all analytes and the optimized background electrolyte (BGE) consisted of 150 mM acetic acid, adjusted to pH 3.7 with NH4OH. Furthermore, a reversal of the migration order of Asp derivatives was observed. This phenomenon seems to be caused by intra-molecular interactions affecting the pKa of the second ionizable group (the side chain carboxyl). The applicability of this method was evaluated using aCSF. A solid phase extraction (SPE) protocol was developed for the selective extraction of the FLEC derivatives. A full evaluation of the matrix effect and extraction yield was performed concluding that the matrix effect is marginal and the recoveries are between 46 and 92%. The method offers adequate sensitivity (limits of detection below 1 μM). [less ▲]

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See detailCESI-MS Workflow for Protein Quantification
Nyssen, Laurent ULiege; Fillet, Marianne ULiege; CAVALIER, Etienne ULiege et al

Poster (2018, October)

Introduction: Sheathless CE-MS interfaces allow increase in sensitivity by coupling low-flow electrospray ionization and tandem MS detection. Peak intensity will depend on spray voltage as well as ... [more ▼]

Introduction: Sheathless CE-MS interfaces allow increase in sensitivity by coupling low-flow electrospray ionization and tandem MS detection. Peak intensity will depend on spray voltage as well as migration and injection conditions. Nevertheless, these parameters influence each other and require methodical optimization to get the most of each instrument. In the present work we share our experience with sheathless CE-MS and neutral coating to analyze peptide and protein samples. Methods: Experiments were conducted on a CESI 8000 capillary electrophoresis holding a neutral coated OptiMS cartridge and coupled to a QT 6500 mass spectrometer. Separation buffer and voltage, curtain gas and source temperature were conserved through experiments. Separation pressure and source voltage were optimized while applying voltage and pressure on separation buffer spiked with the peptide used (pI 9.5 marker from the Advance cIEF starter kit by Sciex). A daily reference run was used to compare modifications to the injection despite variable capillary performance. Finally, shifts in spray voltage due to injection parameters were determined using sequences of runs with different spray voltages. Preliminary results: Decreasing separation pressure from 5 to 1.5 psi increased peptide intensity; electrokinetic injection (EKI) increased peak intensity compared to hydrodynamic injection (HDI); the HDI of a water plug before the EKI increased peak intensity further, as well as a high percentage of acetonitrile in the sample medium. Finally, we compared our initial and our final conditions. In both cases, a positive Q1 scan of 1000 Da/s for m/z 300 to 1000 was acquired, and the electropherograms display the extracted ion current for a 1 m/z interval centered on the m/z of the doubly charged peptide. In the initial method, the peptide was diluted in BGE and was introduced by HDI (1 % of total length); 5 psi pressure were applied to both inlet and outlet; source voltage was 1800 V. When analyzing a 1:160 (v/v) dilution of the peptide, the intensity recorded for [M+2H]2+ was 9.3e7 counts. In the final method, the peptide was diluted in 75:25 acetonitrile:water (v/v) and was introduced by EKI (+ 10 kV 100s). Before the EKI, a HDI of water (0.5 % of total length) was performed, and after the EKI, separation buffer was introduced by HDI (0.5 % of total length). The separation pressure was changed to 1.5 psi and the spray voltage adjusted to 1600 V. When analyzing a 1:160000 (v/v) dilution of the peptide, the recorded intensity of [M+2H]2+ was 9e8 counts. Therefore, following these guidelines, we were able to increase intensity by a 10000 factor. Novel aspect: Frequent monitoring of spray voltage and peak intensity in similar conditions allows good inter-run comparison and troubleshooting. [less ▲]

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See detailPartial filling capillary electrophoretic mobility shift competition assay: a versatile and reliable tool for the assessment of weak biomolecular interactions
Farcas, Elena ULiege; Servais, Anne-Catherine ULiege; Hanson, Julien ULiege et al

Conference (2018, September 10)

Fragment-based drug discovery (FBDD) proved its efficacy in the past 20 years, due to its ability to perform efficient and fruitful optimization campaigns, and is now a well recognized strategy for both ... [more ▼]

Fragment-based drug discovery (FBDD) proved its efficacy in the past 20 years, due to its ability to perform efficient and fruitful optimization campaigns, and is now a well recognized strategy for both academia and pharmaceutical industry. FBDD detects low molecular-weight (MW) ligands (fragments) that bind to biologically important targets, then a structure-guided fragment growing or merging approach is performed giving rise to potent molecules with drug-like properties. However, the analytical arsenal able to point out weak interactions is rather expensive, time consuming or unable to reflect the physiological environment. In this framework, we developed a generic, fully automated, microscale electrophoretic mobility shift competition assay that can be used for primary screening of weak biomolecular interactions between fragments and the target of interest. The affinity capillary electrophoresis (ACE) competitive approach is based on the monitoring of the competition of fragments with a known target inhibitor (PL) for the same active site. The consequence of the competition is a modification of PL electrophoretic mobility, modification that can be measured and used for ligand screening and/or IC50 determination. To achieve our goal, particular attention has been paid to the optimization of the binding environment parameters: an optimal buffer was used for the binding measurements, a partial filling approach was considered to gain sensitivity and to reduce protein consumption and a neutral dynamic coating was performed to reduce protein adsorption to the capillary wall. Moreover, the binding partners concentrations and the electrophoretic conditions were carefully optimized. It is noteworthy that the interactions occur in solution, using the protein in its native form, thus mimicking the physiological environment.The accuracy and reliability of the proposed method was demonstrated by monitoring the competition of two known fragments inhibiting thrombin, namely benzamidine and p-aminobenzamidine and a relatively weak inhibitor, nafamostat with a known thrombin inhibitor, pefabloc (PEFA). The measured IC50 were found to be in good accordance with the previously reported ones. Additionally, a small chemical library was built to evaluate the performance of the newly developed screening-bioassay. The optimized method proved to be remarkably reproducible (migration time RSDs < 1.2%) and selective. The results prove the high discriminatory potency of the method and its ability to screen neutral, negatively or positively charged molecules, as well as molecules that have no or low UV-VIS absorbance, significantly expanding the applicability of the assay compared to a direct approach [1]. Finally, the ability of this approach to discriminate between competitive and irreversible thrombin binders was also demonstrated.References [1] E. Farcas, C. Bouckaert, A.-C. Servais, J. Hanson, L. Pochet, M. Fillet, Analytica Chimica Acta, 2017, 984, 211-222 [less ▲]

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See detailComparison of nanofluidic and ultra-high performance liquid chromatography-tandem mass spectrometry for high sensitive pharmacokinetic studies of estrogens starting from whole blood microsampling
Nys, Gwenaël ULiege; COBRAIVILLE, Gaël ULiege; Kok, Miranda ULiege et al

in Journal of Chromatography. A (2017), 1524

Pharmacokinetic (PK) studies on small animals are challenging as only small volumes of samples are available, in which the analyte is present at low concentration in a complex matrix. In this context, the ... [more ▼]

Pharmacokinetic (PK) studies on small animals are challenging as only small volumes of samples are available, in which the analyte is present at low concentration in a complex matrix. In this context, the use of miniaturized analytical techniques may provide undeniable advantages in terms of sensitivity, sample and solvent consumption compared to the reference UHPLC-MS/MS methods In this study, we present the development of a nanofluidic-LC-MS/MS method to analyze two model analytes of therapeutic interest, namely estradiol (E2) and estetrol (E4) after microsampling with volumetric absorptive microsampling (VAMS) devices, an innovative sampling technique to collect small volumes of whole blood. The nanofluidic LC-MS/MS method was developed using an experimental design to find the optimal conditions to analyze both E2 and E4 with the highest sensitivity. Subsequently, the optimized method was validated according to ICH guidelines and compared to a previously developed UHPLC-MS/MS method. A limit of quantitation of 50. pg/ml was reached with the LC-chip method, which is 50 times better than UHPLC-MS/MS. Both methods were then critically evaluated from the analytical and operational points of view. Finally, the quantitation of estrogens after whole blood microsampling was compared with the results obtained with the corresponding plasma samples. © 2017 Elsevier B.V. [less ▲]

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See detailIdentification and quantitation of intact virus-like particles of human papillomavirus (HPV-VLP) using capillary electrophoresis
Bettonville, Virginie ULiege; Nicol, Jérôme; Furst, Tania et al

Conference (2017, September 19)

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See detailChallenges in the determination of amyloid oligomeric species by two electrophoretic techniques
Napp, Aurore ULiege; Houbart, Virginie ULiege; Demelenne, Alice ULiege et al

Poster (2017, September 18)

Parkinson’s disease is a frequent degenerative disorder, and for the moment the diagnosis is mainly clinical. When the first symptoms appear, loss of more than 70% of the dopaminergic cells already ... [more ▼]

Parkinson’s disease is a frequent degenerative disorder, and for the moment the diagnosis is mainly clinical. When the first symptoms appear, loss of more than 70% of the dopaminergic cells already occurred. Knowing that, it is of high interest to have one (or more) reliable biomarker(s) at our disposal to diagnose Parkinson before the first symptoms appear. Alpha-synuclein (aSyn) is a protein physiologically expressed at high level by neuronal cells, under a monomeric form. This protein would play a critical role in the development of the disease because under certain conditions, aSyn is capable of self-assembly to form fibrils like those found in Lewy bodies. Other intermediate soluble forms like dimers and oligomers are also formed. As these forms seems to be the toxic species, they are the center of many attentions. The quantification of each form would be a great help, but for the moment only the total forms (of monomeric or oligomeric) can be quantified. In this study, aSyn oligomers were generated after optimization of incubation conditions (pH, temperature, agitation, …). Then, different approaches were investigated to detect and follow the different species formed during the aggregation. We analyzed the oligomers by capillary gel electrophoresis (CGE) and SDS-PAGE. We found that capillary gel electrophoresis is a promising automated technique to analyze aSyn oligomers, due to the fact that it separates the aggregates according to their size, like the SDS-PAGE, but with more advantages. To gain sensitivity and selectivity by CGE, we used a laser-induced fluorescence detector. As aSyn do not have a native fluorescence, we derivatized it. After careful screening and optimization of various derivatization reagents, we could quantify with high sensitivity aSyn oligomers by CGE-LIF. We realized different calibration curves, and we had promising results that will allow us to quantify the different aSyn oligomeric forms in biological fluids. [less ▲]

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See detailFully automated MEKC-MS method with in-capillary derivatization for the chiral analysis of amino acids
Crommen, Jacques ULiege; Moldovan, Radu-Cristian; Bodoki, Ede et al

Conference (2017, July)

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See detailQuality control of insulin formulations: API , protamine and aggregates follow-up
Demelenne, Alice ULiege; Napp, Aurore ULiege; Lamalle, Caroline et al

Poster (2017, June 21)

The prevalence of diabetes is increasing every year and insulin preparations are mostly prescribed for treatment of both type 1 and type 2 diabetes. Since these biopharmaceutical formulations are ... [more ▼]

The prevalence of diabetes is increasing every year and insulin preparations are mostly prescribed for treatment of both type 1 and type 2 diabetes. Since these biopharmaceutical formulations are expensive and require a prescription, they are an important target for counterfeiting in developing countries. Therefore, there is a need for fast and efficient methods for quality control of biopharmaceutical products such as insulin formulations. A fully validated micellar electrokinetic chromatography (MEKC) method was developed for quantification of human insulin and NPH insulin (insulin combined with protamine) in formulations. The BGE used was made of 50 mM ammonium acetate (pH 9.0), 20 mM Sodium dodecyl sulfate and 13 % (v/v) acetonitrile and samples were introduced at the short capillary end allowing a separation of insulin and two major excipients (phenol and m-cresol) within 3 minutes. This method was compared with HPLC method using a mobile phase composed of water with 0.1% formic acid / acetonitrile with 0.1 % formic acid in gradient mode. Secondly a MEKC method was also optimized to analyze protamine peptides in insulin formulations. The major protamine peptides could be separated using a BGE made of 100 mM phosphate buffer (pH 2) with 50 mM Thesit®. This method was used to check conformity of protamine in commercially available formulations. Finally, different approaches were investigated in order to follow insulin aggregation. Indeed, insulin is prone to unfold when submitted to denaturating factors as temperature, ionic strength, agitation and pH. An accumulation of unfolds protein in bloodstream results in a high tendency to aggregate and form amyloid fibrils. A deposit of those fibrils in the subcutaneous tissue leads to a complication called “insulin-derived amyloidosis”. On the other hand, during its production, insulin is often subjected to extreme conditions making aggregation, as well as protein stability, important parameters to be controlled during its quality control. In this study, insulin aggregates were generated after optimization of incubation conditions (pH, temperature, agitation…). Those aggregates were then analyzed by size-exclusion chromatography (SEC) and capillary electrophoresis (CE). We showed that capillary gel electrophoresis (CGE) is a promising technique to analyze covalent aggregates of insulin due to the fact that it separates the aggregates according to their size and not to their size/charge ratio. The use of a laser-induced fluorescence detector was also found attractive to enhance the sensitivity of the method. [less ▲]

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See detailOptimization of an electrophoretic approach for the screening and the development of new antithrombotic drugs
Farcas, Elena ULiege; Bouckaert, Charlotte; Servais, Anne-Catherine ULiege et al

Conference (2017, June 20)

The discovery of lead compounds that can modulate the activity of a biological target is essential to provide efficient pharmacological tools and to serve as starting points for new drug generations ... [more ▼]

The discovery of lead compounds that can modulate the activity of a biological target is essential to provide efficient pharmacological tools and to serve as starting points for new drug generations. Fragment-based drug discovery (FBDD) approach is an attractive tool for the identification of new selective inhibitors of a target of interest, but its success largely depends on the ability to develop screening bioassays capable to detect and gauge weak affinity binders. To achieve this goal, we investigated capillary electrophoresis (CE) for identifying and ranking fragments from an initial library. Indeed, due to its ability to evaluate weak interactions, CE seems to be promising for fragment-based screening. This technique is a powerful analytical tool with a unique separation mechanism, speed, efficiency and versatility. Its main advantages are low protein consumption, higher throughput compared to NMR and X-ray crystallography and the fact that screening can be carried out using native protein in physiological solution without the need of immobilization. We developed a proof of concept study on thrombin, a serine protease implicated in the coagulation cascade using affinity capillary electrophoresis (ACE) for ranking fragments from an initial library. For this study, we followed a probe ligand, benzamidine, and we investigated interactions with the target by monitoring the changes of its electrophoretic mobility upon binding. The first step of this study consisted in the optimization of the experimental conditions suitable for the CE method (target and probe ligand concentrations, separation buffer composition, voltage, separation effective length, target partial filling…). Then, numerous thrombin inhibitors with a wide range of inhibitory potency (i.e. Ki 200 µM – 5 nM) were tested to validate our system demonstrating the possibility to fish binders in the optimized conditions. We also checked the absence of non-specific binding with the target using the inactivated enzyme at the binding site. It is noteworthy that in this operating system (ACE assay), binding occurs in free solution using physiological buffers, thus preventing artifacts that may result from target immobilization, which is a requirement for some techniques such as SPR. [less ▲]

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See detailOptimiaztion of an electrophoretic approach for the screening and the development of new antithrombotic drugs
Farcas, Elena ULiege; Bouckaert, Charlotte; Servais, Anne-Catherine ULiege et al

Poster (2017, June 19)

The discovery of lead compounds that can modulate the activity of a biological target is essential to provide efficient pharmacological tools and to serve as starting points for new drug generations ... [more ▼]

The discovery of lead compounds that can modulate the activity of a biological target is essential to provide efficient pharmacological tools and to serve as starting points for new drug generations. Fragment-based drug discovery (FBDD) approach is an attractive tool for the identification of new selective inhibitors of a target of interest, but its success largely depends on the ability to develop screening bioassays capable to detect and gauge weak affinity binders. To achieve this goal, we investigated capillary electrophoresis (CE) for identifying and ranking fragments from an initial library. Indeed, due to its ability to evaluate weak interactions, CE seems to be promising for fragment-based screening. This technique is a powerful analytical tool with a unique separation mechanism, speed, efficiency and versatility. Its main advantages are low protein consumption, higher throughput compared to NMR and X-ray crystallography and the fact that screening can be carried out using native protein in physiological solution without the need of immobilization. We developed a proof of concept study on thrombin, a serine protease implicated in the coagulation cascade using affinity capillary electrophoresis (ACE) for ranking fragments from an initial library. For this study, we followed a probe ligand, benzamidine, and we investigated interactions with the target by monitoring the changes of its electrophoretic mobility upon binding. The first step of this study consisted in the optimization of the experimental conditions suitable for the CE method (target and probe ligand concentrations, separation buffer composition, voltage, separation effective length, target partial filling…). Then, numerous thrombin inhibitors with a wide range of inhibitory potency (i.e. Ki 200 µM – 5 nM) were tested to validate our system demonstrating the possibility to fish binders in the optimized conditions. We also checked the absence of non-specific binding with the target using the inactivated enzyme at the binding site. It is noteworthy that in this operating system (ACE assay), binding occurs in free solution using physiological buffers, thus preventing artifacts that may result from target immobilization, which is a requirement for some techniques such as SPR. [less ▲]

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See detailWhole blood microsampling for the quantitation of estetrol without derivatization by liquid chromatography-tandem mass spectrometry
Nys, Gwenaël ULiege; Gallez, Anne ULiege; Kok, Miranda ULiege et al

in Journal of Pharmaceutical and Biomedical Analysis (2017), 140

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See detailSeparation of FLEC diastereomers by CE vs. LC approaches in the context of neurometabolomics
Moldovan, Radu-Cristian ULiege; Bodoki, Ede; Servais, Anne-Catherine ULiege et al

Poster (2017, June)

Some of the D-amino acids (D-Ser, D-Asp, D-Glu) have gained an increasing attention during the last decades, due to the discovery of their role as neurotransmitters and their implication in different ... [more ▼]

Some of the D-amino acids (D-Ser, D-Asp, D-Glu) have gained an increasing attention during the last decades, due to the discovery of their role as neurotransmitters and their implication in different neurological pathologies (Alzheimer’s disease, schizophrenia etc.). Nevertheless, their use as biomarkers is particularly relevant when correlated with the levels of other neurotransmitters. In order to develop a fast and efficient separation method widely accessible for the quantitation of these molecules, we used only common separation tools such as RP-18 stationary phases for reversed phase liquid chromatography (RP-LC) or bare fused capillaries for capillary zone electrophoresis (CZE). For achieving chiral resolution, a derivatization procedure was implemented. (-)-FLEC was the chiral derivatization agent of choice due to its fast and quantitative reaction with primary and secondary amines and the ability of performing in-capillary derivatization. Moreover, the derivatization process implies only a simple mix of the sample and reagent, at room temperature. The separation of the FLEC derivatives of several biologically relevant D- and L- amino acids (Asp, Glu, Ser, Tyr, Trp, Phe, His) together with certain neurotransmitter molecules have been optimized using CZE or RP-LC, chiral resolution being achievable for all amino acids of interest. By the CZE approach the running buffer’s pH turned out to be critical in achieving baseline separation of the targeted analytes. The derivatives of most amino acids could be separated using 60mM acetate buffer at pH 5, while for Asp derivatives the separation could be achieved only at pH 4. Being stronger bases, a third run at a more alkaline pH was needed for the separation of the remainder neurotransmitters. Moreover, the implemented in-capillary derivatization allows a fast and fully automated separation procedure. As for the RP-LC approach 50 mM acetate buffer in combination with an organic modifier (methanol, acetonitrile or tetrahydrofuran (THF)) was tested as mobile phase using gradient elution. Once again, the strong influence of pH on the resolution was observed. The organic modifier nature was of critical importance, where only THF enabled baseline resolution for all amino acid derivatives. [less ▲]

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See detailLc-chip versus UHPLC-tandem mass spectrometry for the quantitation of estetrol and estradiol without derivatization after whole blood microsampling
Nys, Gwenaël ULiege; Servais, Anne-Catherine ULiege; Pequeux, Christel ULiege et al

Conference (2017, March 29)

the aim of this work was to conduct a PK study on mice to select the most appropriate administration route for E2 and E4 formulations. To achieve this goal, a reference method for the quantitation of both ... [more ▼]

the aim of this work was to conduct a PK study on mice to select the most appropriate administration route for E2 and E4 formulations. To achieve this goal, a reference method for the quantitation of both estrogens after whole blood microsampling was developed and validated on a UHPLC-MS/MS system. This reference method was later transferred on LC-chip device and both methods were compared in terms of analytical parameters such as response function, accuracy, precision, trueness and limit of quantification. [less ▲]

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