13 results on '"Wouters, Bert"'
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2. An automated online three-phase electro-extraction setup with machine-vision process monitoring hyphenated to LC-MS analysis.
- Author
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He Y, Miggiels P, Drouin N, Lindenburg PW, Wouters B, and Hankemeier T
- Subjects
- Humans, Chromatography, Liquid methods, Indicator Dilution Techniques, Propranolol, Solid Phase Extraction methods, Tandem Mass Spectrometry methods, Robotics
- Abstract
Sample preparation is a labor-intensive and time-consuming procedure, especially for the bioanalysis of small-volume samples with low-abundant analytes. To minimize losses and dilution, sample preparation should ideally be hyphenated to downstream on-line analysis such as liquid chromatography-mass spectrometry (LC-MS). In this study, an automated three-phase electro-extraction (EE) method coupled to machine vision was developed, integrated with a robotic autosampler hyphenated to LC-MS. Eight model compounds, i.e. amitriptyline, clemastine, clomipramine, haloperidol, loperamide, propranolol, oxeladin, and verapamil were utilized for the optimization and evaluation of the automated EE setup. The stability of automated EE was evaluated by monitoring the acceptor droplet size by machine vision and recording the current during EE. A Design of Experiment approach (Box-Behnken design) was utilized to optimize the critical parameters of the EE method, i.e., the ratio of formic acid in the sample to acceptor phase, extraction voltage, and extraction time. The developed quadratic models showed good fitness (p < 0.001, R
2 > 0.95). Automated EE could be achieved in less than 2 min with enrichment factors (EF) up to 387 and extraction recoveries (ER) up to 97% for academic samples. Finally, the optimized EE method was successfully applied to both spiked human urine and plasma samples with low-concentration (50 ng mL-1 ) analytes and a low starting sample volume of 20 μL of plasma and urine in 10-fold diluted samples. The developed automated EE setup is easy to operate, provides a fast extraction method for analytes from volume-limited biological samples, and is hyphenated with on-line LC-MS analysis. Therefore, this method can provide fast and automated sample preparation to solve bottlenecks in high-throughput bioanalysis workflows., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2022
- Full Text
- View/download PDF
3. Development of a fast, online three-phase electroextraction hyphenated to fast liquid chromatography-mass spectrometry for analysis of trace-level acid pharmaceuticals in plasma.
- Author
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He Y, Drouin N, Wouters B, Miggiels P, Hankemeier T, and Lindenburg PW
- Subjects
- Chromatography, Liquid, Mass Spectrometry, Proteins, Acids, Pharmaceutical Preparations
- Abstract
Sample preparation is a challenge for high-throughput analysis, especially for volume-limited samples with low-abundant analytes. Ideally, sample preparation enriches the analytes of interest while removing the interferents to reduce the matrix effect and improve both sensitivity and quantification. In this study, a three-phase electroextraction (EE) method hyphenated to fast online liquid chromatography-mass spectrometry (LC-MS) was developed. Four model acidic drugs of relevance for drug monitoring in plasma, i.e. naproxen, fenoprofen, flurbiprofen, and ibuprofen, were utilized for the optimization and evaluation of the method. A Design of Experiment approach (Box-Behnken design) was used to optimize the critical parameters of the method, i.e., the type of organic solvent, pH of the sample and acceptor phase, and the extraction voltage and time. Good fitness (P < 0.02, R
2 > 0.95) was observed for the developed quadratic model. Extraction could be achieved in less than 2 min (115 s) with enrichment factors (EF) up to 190 and extraction recoveries (ER) up to 38% for academic samples. Additionally, the optimized three-phase EE method was successfully applied to spiked plasma samples with low-abundant (50 ng mL-1 ) analytes and a low sample volume of 15 μL plasma in 10-fold diluted samples. Finally, two crucial contributors to the matrix effect of three-phase EE application on plasma samples were determined. Specifically, the ion-suppression effect in the MS source was reduced by the fast LC separation, and the matrix effect during extraction was negligible for the diluted protein-precipitated plasma samples. The developed three-phase EE method is easy to operate and provides fast and online extraction of trace-level acidic analytes from volume-limited biological samples. Therefore, this method can provide a potential solution for sample-preparation bottlenecks in high-throughput bioanalysis workflows., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)- Published
- 2022
- Full Text
- View/download PDF
4. A high-throughput, ultrafast, and online three-phase electro-extraction method for analysis of trace level pharmaceuticals.
- Author
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He Y, Miggiels P, Wouters B, Drouin N, Guled F, Hankemeier T, and Lindenburg PW
- Subjects
- Humans, Limit of Detection, Mass Spectrometry, Solid Phase Extraction, Flow Injection Analysis, Pharmaceutical Preparations
- Abstract
Sample preparation is often reported as the main bottleneck of analytical processes. To meet the requirements of both high-throughput and high sensitivity, improved sample-preparation methods capable of fast analyte preconcentration are urgently needed. To this end, a new three-phase electroextraction (EE) method is presented that allows for ultrafast electroextraction hyphenated to flow-injection analysis mass spectrometry (FIA-MS). Four model compounds, i.e., propranolol, amitriptyline, bupivacaine, and oxeladin, were used to optimize and evaluate the method. Within only 30 s extraction time, enrichment factors (EF) of 105-569 and extraction recoveries (ER) of 10.2%-55.7% were achieved for these analytes, with limits of detection (LODs) ranging from 0.36 to 3.21 ng mL
-1 , good linear response function (R2 > 0.99), low relative standard deviation (0.6%-17.8%) and acceptable accuracy (73-112%). Finally, the optimized three-phase EE method was successfully applied to human urine and plasma samples. Our three-phase electroextraction method is simple to construct and offers ultrafast, online extraction of trace amounts of analytes from biological samples, and therefore has great potential for high-throughput analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2021
- Full Text
- View/download PDF
5. Experimental and numerical study of band-broadening effects associated with analyte transfer in microfluidic devices for spatial two-dimensional liquid chromatography created by additive manufacturing.
- Author
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Adamopoulou T, Nawada S, Deridder S, Wouters B, Desmet G, and Schoenmakers PJ
- Subjects
- Computer Simulation, Chromatography, Liquid instrumentation, Lab-On-A-Chip Devices
- Abstract
Conventional one-dimensional column-based liquid chromatographic (LC) systems do not offer sufficient separation power for the analysis of complex mixtures. Column-based comprehensive two-dimensional liquid chromatography offers a higher separation power, yet suffers from instrumental complexity and long analysis times. Spatial two-dimensional liquid chromatography can be considered as an alternative to column-based approaches. The peak capacity of the system is ideally the product of the peak capacities of the two dimensions, yet the analysis time remains relatively short due to parallel second-dimension separations. Aspects affecting the separation efficiency of this type of systems include flow distribution to homogeneously distribute the mobile phase for the second-dimension (
2 D) separation, flow confinement during the first-dimension (1 D) separation, and band-broadening effects during analyte transfer from the1 D separation channel to the2 D separation area. In this study, the synergy between computational fluid dynamics (CFD) simulations and rapid prototyping was exploited to address band broadening during the2 D development and analyte transfer from1 D to2 D. Microfluidic devices for spatial two-dimensional liquid chromatography were designed, simulated, 3D-printed and tested. The effects of presence and thickness of spacers in the2 D separation area were addressed and leaving these out proved to be the most efficient solution regarding band broadening reduction. The presence of a stationary-phase material in the1 D channel had a great effect on the analyte transfer from the1 D to the2 D and the resulting band broadening. Finally, pressure limit of the fabricated devices and printability are discussed., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)- Published
- 2019
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6. On-line microfluidic immobilized-enzyme reactors: A new tool for characterizing synthetic polymers.
- Author
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Wouters B, Pirok BWJ, Soulis D, Garmendia Perticarini RC, Fokker S, van den Hurk RS, Skolimowski M, Peters RAH, and Schoenmakers PJ
- Subjects
- Enzymes, Immobilized chemistry, Enzymes, Immobilized metabolism, Lab-On-A-Chip Devices, Polymers chemical synthesis, Polymers chemistry
- Abstract
Biodegradable polymeric materials may eventually replace biostable materials for medical applications, including therapeutic devices, scaffolds for tissue engineering, and drug-delivery vehicles. To further develop such materials, a more fundamental understanding is necessary to correlate parameters including chemical-composition distribution within a macromolecular structure with the final properties of the material, including particle-size. A wide variety of analytical techniques have been applied for the characterization of polymer materials, including hyphenated techniques such as comprehensive two-dimensional liquid chromatography (LC × LC). In this context, we have investigated enzymatic degradation of polyester-based nanoparticles, both in-solution and by the use of an immobilized-enzyme reactor (IMER). We have demonstrated for the first time the implementation of such an IMER in a size-exclusion chromatography system for on-line degradation and subsequent analysis of the polymer degradation products. The effect of residence times ranging from 12 s to 4 min on polymer degradation was assessed. IMER-assisted degradation is much faster compared to in-solution degradation, which requires several hours to days, and opens the possibility to use such reactors in LC × LC modulation interfaces., (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Published
- 2019
- Full Text
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7. Prototyping of thermoplastic microfluidic chips and their application in high-performance liquid chromatography separations of small molecules.
- Author
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Wouters S, De Vos J, Dores-Sousa JL, Wouters B, Desmet G, and Eeltink S
- Subjects
- Polymers chemistry, Porosity, Pressure, Silicon Dioxide chemistry, Small Molecule Libraries isolation & purification, Solvents chemistry, Chemistry Techniques, Analytical instrumentation, Chemistry Techniques, Analytical methods, Chromatography, High Pressure Liquid instrumentation, Microfluidics
- Abstract
The present paper discusses practical aspects of prototyping of microfluidic chips using cyclic olefin copolymer as substrate and the application in high-performance liquid chromatography. The developed chips feature a 60mm long straight separation channel with circular cross section (500μm i.d.) that was created using a micromilling robot. To irreversibly seal the top and bottom chip substrates, a solvent-vapor-assisted bonding approach was optimized, allowing to approximate the ideal circular channel geometry. Four different approaches to establish the micro-to-macro interface were pursued. The average burst pressure of the microfluidic chips in combination with an encasing holder was established at 38MPa and the maximum burst pressure was 47MPa, which is believed to be the highest ever report for these polymer-based microfluidic chips. Porous polymer monolithic frits were synthesized in-situ via UV-initiated polymerization and their locations were spatially controlled by the application of a photomask. Next, high-pressure slurry packing was performed to introduce 3μm silica reversed-phase particles as the stationary phase in the separation channel. Finally, the application of the chip technology is demonstrated for the separation of alkyl phenones in gradient mode yielding baseline peak widths of 6s by applying a steep gradient of 1.8min at a flow rate of 10μL/min., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
- Full Text
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8. A cyclic-olefin-copolymer microfluidic immobilized-enzyme reactor for rapid digestion of proteins from dried blood spots.
- Author
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Wouters B, Dapic I, Valkenburg TS, Wouters S, Niezen L, Eeltink S, Corthals GL, and Schoenmakers PJ
- Subjects
- Humans, Blood Proteins analysis, Cycloparaffins chemistry, Dried Blood Spot Testing instrumentation, Dried Blood Spot Testing methods, Enzymes, Immobilized chemistry, Enzymes, Immobilized metabolism, Microfluidic Analytical Techniques instrumentation, Polymers chemistry
- Abstract
A critical step in the bottom-up characterization of proteomes is the conversion of proteins to peptides, by means of endoprotease digestion. Nowadays this method typically uses overnight digestion and as such represents a considerable bottleneck for high-throughput analysis. This report describes protein digestion using an immobilized-enzyme reactor (IMER), which enables accelerated digestion times that are completed within seconds to minutes. For rapid digestion to occur, a cyclic-olefin-copolymer microfluidic reactor was constructed containing trypsin immobilized on a polymer monolithic material through a 2-vinyl-4,4-dimethylazlactone linker. The IMER was applied for the rapid offline digestion of both singular protein standards and a complex protein mixture prior to liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) analysis. The effects of protein concentration and residence time in the IMER were assessed for protein standards of varying molecular weight between 11 and 240kDa. Compared to traditional in-solution digestion, IMER-facilitated protein digestion at room temperature for 5min yielded similar results in terms of sequence coverage and number of identified peptides. Good repeatability was demonstrated with a relative standard deviation of 6% for protein-sequence coverage. The potential of the IMER was also demonstrated for a complex protein mixture in the analysis of dried blood spots. Compared to a traditional workflow a similar number of proteins could be identified, while reducing the total analysis time from 22.5h to 4h and importantly omitting the sample-pre-treatment steps (denaturation, reduction, and alkylation). The identified proteins from two workflows showed similar distributions in terms of molecular weight and hydrophobic character., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2017
- Full Text
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9. Using contemporary liquid chromatography theory and technology to improve capillary gradient ion-exchange separations.
- Author
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Wouters B, Broeckhoven K, Wouters S, Bruggink C, Agroskin Y, Pohl CA, and Eeltink S
- Subjects
- Anions chemistry, Chromatography, Ion Exchange instrumentation, Kinetics, Limit of Detection, Pressure, Chromatography, Ion Exchange methods
- Abstract
The gradient-performance limits of capillary ion chromatography have been assessed at maximum system pressure (34.5 MPa) using capillary columns packed with 4.1 μm macroporous anion-exchange particles coated with 65 nm positively-charged nanobeads. In analogy to the van-Deemter curve, the gradient performance was assessed applying different flow rates, while decreasing the gradient time inversely proportional to the increase in flow rate in order to maintain the same retention properties. The gradient kinetic-performance limits were determined at maximum system pressure, applying tG/t0=5, 10, and 20. In addition, the effect of retention on peak width was assessed in gradient mode for mono-, di-, and trivalent inorganic anions. The peak width of late-eluting ions can be significantly reduced by using concave gradient, resulting in better detection sensitivity. A signal enhancement factor of 8 was measured for a late-eluting ion when applying a concave instead of a linear gradient. For the analysis of a complex anion mixture, a coupled column with a total length of 1.05 m was operated at the kinetic-performance limit applying a linear 250 min gradient (tG/t0=10). The peak capacity varied between 200 and 380 depending on analyte retention, and hence on charge and size of the ion., (Copyright © 2014 Elsevier B.V. All rights reserved.)
- Published
- 2014
- Full Text
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10. Design and performance evaluation of a microfluidic ion-suppression module for anion-exchange chromatography.
- Author
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Wouters S, Wouters B, Jespers S, Desmet G, Eghbali H, Bruggink C, and Eeltink S
- Subjects
- Anions chemistry, Chromatography, Ion Exchange instrumentation, Electrolysis, Microfluidics instrumentation, Chromatography, Ion Exchange methods, Microfluidics methods
- Abstract
A microfluidic membrane suppressor has been constructed to suppress ions of alkaline mobile-phases via an acid-base reaction across a sulfonated poly(tetrafluoroethylene)-based membrane and was evaluated for anion-exchange separations using conductivity detection. The membrane was clamped between two chip substrates, accommodating rectangular microchannels for the eluent and regenerant flow, respectively. Additionally, a clamp-on chip holder has been constructed which allows the alignment and stacking of different chip modules. The response and efficacy of the microfluidic chip suppressor was assessed for a wide range of eluent (KOH) concentrations, using 127 and 183μm thick membranes, while optimizing the flow rate and concentration of the regenerant solution (H2SO4). The optimal operating eluent flow rate was determined at 5μL/min, corresponding to the optimal van-Deemter flow velocity of commercially-available column technology, i.e. a 0.4mm i.d.×250mm long column packed with 7.5μm anion-exchange particles. When equilibrated at 10mM KOH, a 99% decrease in conductivity signal could be obtained within 5min when applying 10mM H2SO4 regenerant at 75μL/min. A background signal as low as 1.2μS/cm was obtained, which equals the performance of a commercially-available electrolytic hollow-fiber suppressor. When increasing the temperature of the membrane suppressor from 15 to 20°C, ion suppression was significantly improved allowing the application of 75mM KOH. The applicability of the chip suppressor has been demonstrated with an isocratic baseline separation of a mixture of seven inorganic ions, yielding plate numbers between 5300 and 10,600 and with a gradient separation of a complex ion mixture., (Copyright © 2014 Elsevier B.V. All rights reserved.)
- Published
- 2014
- Full Text
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11. Visualization procedures for proteins and peptides on flat-bed monoliths and their effects on matrix-assisted laser-desorption/ionization time-of-flight mass spectrometric detection.
- Author
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Wouters B, Vanhoutte DJ, Aarnoutse P, Visser A, Stassen C, Devreese B, Kok WT, Schoenmakers PJ, and Eeltink S
- Subjects
- Chromatography, Liquid instrumentation, Coloring Agents chemistry, Limit of Detection, Methacrylates chemistry, Molecular Weight, Peptides chemistry, Polymers chemistry, Proteins chemistry, Chromatography, Liquid methods, Peptides analysis, Proteins analysis, Proteomics methods, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods
- Abstract
The present study concerns the application of visualization methods, i.e. coomassie-brilliant-blue-R staining (CBB-R), silver-nitrate staining, and fluorescamine labeling, and subsequent MALDI-MS analysis of intact proteins and peptides on the surface of flat-bed monoliths, intended for spatial two-dimensional chromatographic separations. The use of 100-μm thick macroporous poly(butyl methacrylate-co-ethylene dimethacrylate) flat-bed monoliths renders a fixation step obsolete, so that CBB-R and silver-nitrate staining and destaining could be achieved in 10-15 min as opposed to up to 24h, as is typical on 2D-PAGE gels. The detection limits remained comparable. The compatibility of the monolithic layer with subsequent MALDI-MS analysis of individual proteins and peptide spots was investigated with regards to mass accuracy, mass precision, resolution, and signal intensity. When comparing results from MALDI-MS analysis of proteins and peptides on a flat-bed monolith to results obtained directly on stainless-steel target plates, significant losses in mass precision, signal intensity, and an increased variation in resolution were observed. In addition, a loss in signal intensity up to two orders of magnitude was observed when using monolithic layers. After CCB-R and silver-nitrate staining and destaining to disrupt the protein-dye complexes no MALDI spectra with significant S/N ratios could be achieved. After fluorescamine labeling heterogeneous signals were observed, which resulted from a distribution in the number of fluorescence-labeled lysine groups and from the presence of labeled derivatives that had undergone condensation reactions., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2013
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12. High-resolution separations of protein isoforms with liquid chromatography time-of-flight mass spectrometry using polymer monolithic capillary columns.
- Author
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Eeltink S, Wouters B, Desmet G, Ursem M, Blinco D, Kemp GD, and Treumann A
- Subjects
- Mass Spectrometry instrumentation, Protein Isoforms analysis, Sensitivity and Specificity, Chromatography, High Pressure Liquid methods, Mass Spectrometry methods, Protein Isoforms isolation & purification
- Abstract
The separation of intact proteins, including protein isoforms arising from various amino-acid modifications, employing a poly(styrene-co-divinylbenzene) monolithic capillary column in high-performance liquid chromatography coupled on-line to a time-of-flight mass spectrometer (MS) is described. Using a 250 mm × 0.2 mm monolithic capillary column high-sensitivity separations yielding peak capacities of >600 were achieved with a 2h linear gradient and formic acid added in the mobile phase as ion-pairing agent. The combination of high-resolution chromatography with high-accuracy MS allowed to distinguish protein isoforms that differ only in their oxidation and biotinylation state allowing the separation between structural isoforms. Finally, the potential to separate proteins isoforms due to glycosylation is discussed., (Copyright © 2011 Elsevier B.V. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
13. Parameters affecting the separation of intact proteins in gradient-elution reversed-phase chromatography using poly(styrene-co-divinylbenzene) monolithic capillary columns.
- Author
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Detobel F, Broeckhoven K, Wellens J, Wouters B, Swart R, Ursem M, Desmet G, and Eeltink S
- Subjects
- Animals, Cattle, Chickens, Horses, Chromatography, High Pressure Liquid methods, Chromatography, Reverse-Phase methods, Polystyrenes chemistry, Proteins isolation & purification, Proteomics methods
- Abstract
An experimental study was performed to investigate the effects of column parameters and gradient conditions on the separation of intact proteins using styrene-based monolithic columns. The effect of flow rate on peak width was investigated at constant gradient steepness by normalizing the gradient time for the column hold-up time. When operating the column at a temperature of 60 degrees C a small C-term effect was observed in a flow rate range of 1-4 microL/min. However, the C-term effect on peak width is not as strong as the decrease in peak width due to increasing flow rate. The peak capacity increased according to the square root of the column length. Decreasing the macropore size of the polymer monolith while maintaining the column length constant, resulted in an increase in peak capacity. A trade-off between peak capacity and total analysis time was made for 50, 100, and 250 mm long monolithic columns and a microparticulate column packed with 5 microm porous silica particles while operating at a flow rate of 2 microL/min. The peak capacity per unit time of the 50mm long monolithic column with small pore size was superior when the total analysis time is below 120 min, yielding a maximum peak capacity of 380. For more demanding separations the 250 mm long monolith provided the highest peak capacity in the shortest possible time frame., (2010 Elsevier B.V. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
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